U.S. patent application number 16/857380 was filed with the patent office on 2020-09-17 for novel methods and compositions to evaluate and determine inactivation of hazardous biological material.
The applicant listed for this patent is NORTH CAROLINA STATE UNIVERSITY, The United States of America, as represented by the Secretary of Agriculture, The United States of America, as represented by the Secretary of Agriculture. Invention is credited to JANE M. CALDWELL, ILENYS M. PEREZ DIAZ.
Application Number | 20200291461 16/857380 |
Document ID | / |
Family ID | 1000004856577 |
Filed Date | 2020-09-17 |
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United States Patent
Application |
20200291461 |
Kind Code |
A1 |
PEREZ DIAZ; ILENYS M. ; et
al. |
September 17, 2020 |
NOVEL METHODS AND COMPOSITIONS TO EVALUATE AND DETERMINE
INACTIVATION OF HAZARDOUS BIOLOGICAL MATERIAL
Abstract
Novel time and temperature integrator (TTI) assays, kits
containing the components of the assays, and the novel components
for those assays are provided herein. These novel TTI assays
evaluate and/or determine the inactivation of biological material
in/on a sample by quantifying the degradation of DNA using qPCR.
The sample can be a food product (e.g., fruits, vegetables, meat
from animals, or eggs) while the item can be any object (e.g.,
medical equipment, especially reusable medical equipment) for which
one needs to determine that the amount of inactivation of specific
hazardous biological material on the object or in a sample is at or
below a pre-determined amount.
Inventors: |
PEREZ DIAZ; ILENYS M.;
(RALEIGH, NC) ; CALDWELL; JANE M.; (STORM LAKE,
IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary of
Agriculture
NORTH CAROLINA STATE UNIVERSITY |
Washington
Raleigh |
DC
NC |
US
US |
|
|
Family ID: |
1000004856577 |
Appl. No.: |
16/857380 |
Filed: |
April 24, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14917830 |
Mar 9, 2016 |
10718032 |
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PCT/US2014/054749 |
Sep 9, 2014 |
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16857380 |
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61969465 |
Mar 24, 2014 |
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61876425 |
Sep 11, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6888 20130101;
C12Q 2600/158 20130101; C12Q 1/689 20130101 |
International
Class: |
C12Q 1/689 20060101
C12Q001/689; C12Q 1/6888 20060101 C12Q001/6888 |
Claims
1. A kit comprising: a pair of primers comprising a first
polynucleotide and a second polynucleotide; a label; optionally
DNase; and optionally instructions on using said first
polynucleotide and said second polynucleotide in a method to
determine the amount of inactivation of a biological material,
wherein said first polynucleotide has the sequence in one of SEQ ID
NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, and wherein
when said first polynucleotide has the sequence in SEQ ID NO: 1,
said second polynucleotide has the sequence in SEQ ID NO: 2; when
said first polynucleotide has the sequence in SEQ ID NO: 3, said
second polynucleotide has the sequence in SEQ ID NO: 4; when said
first polynucleotide has the sequence in SEQ ID NO: 5, then said
second polynucleotide has the sequence in SEQ ID NO: 6; and when
said first polynucleotide has the sequence in SEQ ID NO: 7, then
said second polynucleotide has the sequence in SEQ ID NO: 8.
2. The kit of claim 1 wherein said label is selected from the group
consisting of a fluorescent composition, an intercalating dye, a
spectroscopic label, a photochemical label, a biochemical label, an
immunochemical label, and a chemical label.
3. The kit of claim 1 wherein said label comprises: a probe; a
quencher dye; and a fluorescent dye, wherein said quencher dye and
said fluorescent dye are linked to said probe, wherein when the
sequence of said first polynucleotide is SEQ ID NO: 1, said probe
has a sequence of between approximately 15 contiguous bases and
approximately 45 contiguous bases of SEQ ID NO: 14 or the reverse
complement thereof, wherein when the sequence of said first
polynucleotide is SEQ ID NO: 3, said probe has a sequence of
between approximately 15 contiguous bases and approximately 45
contiguous bases of SEQ ID NO: 15 or the reverse complement
thereof, wherein when the sequence of said first polynucleotide is
SEQ ID NO: 5, said probe has a sequence of between approximately 15
contiguous bases and approximately 45 contiguous bases of SEQ ID
NO: 16 or the reverse complement thereof, and wherein when the
sequence of said first polynucleotide is SEQ ID NO: 7, said probe
has a sequence of between approximately 15 contiguous bases and
approximately 45 contiguous bases of SEQ ID NO: 17 or the reverse
complement thereof.
4. A kit comprising: a pair of primers comprising a first
polynucleotide and a second polynucleotide; a label; optionally
DNase; and instructions on using said first polynucleotide and said
second polynucleotide in a method to determine the amount of
inactivation of a biological material, wherein said first
polynucleotide has the sequence in one of SEQ ID NO: 1, SEQ ID NO:
3, SEQ ID NO: 5, and SEQ ID NO: 7, and wherein when said first
polynucleotide has the sequence in SEQ ID NO: 1, said second
polynucleotide has the sequence in SEQ ID NO: 2; when said first
polynucleotide has the sequence in SEQ ID NO: 3, said second
polynucleotide has the sequence in SEQ ID NO: 4; when said first
polynucleotide has the sequence in SEQ ID NO: 5, then said second
polynucleotide has the sequence in SEQ ID NO: 6; and when said
first polynucleotide has the sequence in SEQ ID NO: 7, then said
second polynucleotide has the sequence in SEQ ID NO: 8.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. patent application
Ser. No. 14/917,830 filed Mar. 9, 2016, which itself is a National
Phase Application filed under 35 U.S.C. .sctn. 371 as a national
stage of PCT/US2014/054749, filed on Sep. 9, 2014 and claims
priority from both U.S. Provisional Patent Application Ser. No.
61/876,425 filed Sep. 11, 2013 and U.S. Provisional Patent
Application Ser. No. 61/969,465 filed Mar. 24, 2014, all of which
are incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] This invention relates to assays to evaluate and/or
determine the inactivation of biological material in/on a sample by
quantifying the level of integrity, or alternatively, the
degradation of, DNA and the materials necessary to perform the
assays. The sample can be a food product (e.g., fruits, vegetables,
meat from animals, or eggs) while the item can be any object (e.g.,
medical equipment, especially reusable medical equipment) for which
one needs to determine that the amount of inactivation of specific
hazardous biological material on the object or in a sample is at or
below a pre-determined amount. Non-limiting examples of hazardous
biological material are toxins, viruses, parasites, fungi,
bacteria, spores (bacterial, fungal or parasitical) and cancer
cells. This invention further relates to the tools used in the
assays. One assay uses quantitative PCR and polynucleotide primers
to assay the fragmentation of mitochondrial DNA found intrinsically
in the food matrix. Another assay examines the size and
fragmentation of total DNA present in the food matrix using any
device which measures DNA fragment size globally. A third assay
involves use of an extrinsic source of mitochondrial DNA added to
the processing run in a recoverable container.
SEQUENCE LISTING
[0003] The official copy of the sequence listing is submitted
electronically via EFS-Web as an ASCII formatted sequence listing
with a file name "SequenceListing", created on Apr. 24, 2020, and
having a size of 6 kilobytes and is filed concurrently with the
specification. The sequence listing contained in this ASCII
formatted document is part of the specification and is herein
incorporated by reference in its entirety.
DESCRIPTION OF THE PRIOR ART
[0004] In order to render food products (both processed and fresh
food products) safe and to prevent them from spoiling under the
usual conditions of storage, some form of commercial sterilization
is required. Control of food-borne bacterial pathogens such as
Salmonella spp., Shigella spp., pathogenic Escherichia coli,
Campylobacter spp., and Yersinia spp. has traditionally been
achieved using heat treatments such as pasteurization or pressure
sterilization. Heat treatments higher than pasteurization are used
to provide Clostridium botulinum safety in low-acid canned foods
and canned cured meats. Low-acid products have a pH of
approximately 4.6 and above and include most meat and marine
products, corn, peas, lima beans, asparagus and spinach. More
recently, inhibition of surviving Clostridium botulinum spores has
been achieved by treating food with NaNO.sub.2 and/or potassium
sorbate. Failure to adequately inactivate Clostridium botulinum
spores can result in the production of neurotoxins by the bacteria.
Of course, failure to destroy other disease causing bacteria (e.g.,
Salmonella spp., Shigella spp., pathogenic E. coli, Campylobacter
spp., and Yersinia spp.) in food products can result in food
poisoning and severe disease after ingesting the contaminated food
products. In addition, butchered meats and raw eggs can become
contaminated with pathogenic bacteria during the handling and/or
processing of the meats and eggs. It is vital to reduce or
eliminate the bacteria on the surface of these items so that the
workers and people that consume the food are not sickened by the
bacteria.
[0005] Food spoilage and food poisoning from contamination of food
by bacteria is a major problem throughout the world, including
developed countries such as the United States, Canada, Japan, U.K.,
France, Germany, and Russia. In the U.S. alone, illness from
food-borne bacteria costs several billion dollars annually in
morbidity and mortality. Gram-positive and Gram-negative food-borne
bacteria account for many of the pathogens causing food
poisoning.
[0006] Traditional safety guidelines have traditionally used
destruction of bacterial surrogates as indicators of processing
safety and efficacy. Culturing samples of food or items for
viability of bacteria and/or spores is a current method for
assaying the destruction of bacterial surrogates. Two bacterial
surrogates are Geobacillus stearothermophilus and Bacillus subtilis
spores which are placed in or on samples prior to inactivation and
which are cultured after treatment to determine if the bacteria
and/or the bacterial spores have been inactivated. Problems with
the culture approach include tracking and recovering indicator
spores and/or bacteria, excessive time required to culture (48
hours or more), and molecular methods unable to differentiate
between live and dead surrogates. Other devices and methods have
been created to assay for live or killed bacteria and bacterial
spores in food and devices using various culturing conditions
(e.g., US Pub. 2007-0249040; U.S. Pat. Nos. 5,486,459; 5,830,683;
5,989,852; and WO 1995/008639), yet these methods and devices lack
the ability to timely and accurately determine the amount of
reduction of viable hazardous biological materials in or on a food
or item.
[0007] To overcome the limitation of the long incubation times
required for the proliferation of microbes and/or bacterial spores,
time and temperature integrator (TTI) technologies have been
developed. An example of such technologies is the application of
enzymes derived from microbes that can naturally tolerate high
temperatures and need such high temperatures to proliferate. For
example, .alpha.-amylase from Bacillus licheniformis (Cordt, et
al., J. Chem. Tech. and Biotech. 59:193-199 (1994)), lipase (U.S.
Pat. No. 4,284,719), and .beta.-glucosidase (Adams and Langley,
Food Chemistry, 62:65-68 (1998)) have been used as TTIs. These
enzymes can be produced in high concentrations using molecular
biology techniques. The pure enzyme is then encapsulated in plastic
tubing with 1 mm diameter, and the ends are sealed by melting. The
encapsulated enzyme can be incorporated in a food matrix and
recovered after thermal treatment (e.g., pasteurization) of the
food matrix is applied. The activity of the post-thermal treated,
heat resistant enzyme is determined and correlated with the
effectiveness of the heating step. This rapid method allows for
evaluation of thermal treatments uniformity and effectiveness.
However, production of enzymatic TTI is complex. Also, TTI's
enzymatic activity is difficult to maintain during long storage
periods and may vary within multiple production batches.
[0008] Alternative approaches to culturing bacteria and enzymatic
TTI, include assays for bacterial DNA or mRNA in samples of food
using PCR and PCR-related techniques. See, e.g., U.S. Patent App.
Pub. 2010-0167956 in which polynucleotide probes specific for E.
coli are placed on a chip for assaying for the presence of E. coli;
and U.S. Patent App. Pub. 2012-0288864 in which S. enterica, a
food-borne pathogen, is detected by PCR using primers specific for
an S. enterica gene.
[0009] The effect of high temperature on DNA degradation is well
described. Above 100.degree. C. denaturation, depurination,
deamination and loss of secondary structure occurs (Gryson, N.,
Anal. Bioanal. Chem. 396:2003-2022 (2010)). Although autoclaving a
foodstuff at 121.degree. C. for fifteen minutes does not destroy
all DNA available for PCR (Lipp, et al., J. AOAC Int. 82(4):923-928
(1999)), recovery of reduced DNA concentrations via quantitative
PCR (qPCR) from cornmeal and water cooked for sixty minutes at
100.degree. C. have been reported (Murray, et al., J. of Agric.
& Food Chem. 55:2231-2239 (2007)). Increased Ct (threshold
cycles) values occurred in DNA from heat-treated corn grits and
corn flour when compared to untreated corn and resulted in
distortions of qPCR assays for detection of genetically modified
organisms (GMO) (Moreano, et al., J. of Agric. & Food Chem.
53:9971-9979 (2005)).
[0010] However, these prior art methods for detecting bacteria via
PCR are unable to determine if the bacteria or their spores are
still viable or not because PCR often is able to detect a certain
fragment of DNA from the bacteria or spores, despite their death or
inactivation. In a study by Stam (Doctoral Thesis, NCSU Food
Science; Raleigh, N.C. (2008), available at
http://www.lib.ncsu.edu/resolver/1840.16/3192), Clostridium
sporogenes spores were heat-treated to 121.degree. C. in two
minutes intervals for eighteen minutes, and bacterial DNA
degradation over time was determined. It was noted that
heat-treating the spores for only two minutes resulted in the
absence of DNA bands using agarose gel electrophoresis. However,
the autoclaved spore DNA was still detectable by qPCR, having a
reduced Ct value of 35 compared to a Ct of 12 for viable spores
(Stam (2008)). Therefore, bacterial or spore DNA is degraded but
still detectable by qPCR, when using thermal processing techniques
such as heat or microwave suitable for preserving vegetables and
fruits. Obviously, the repercussions of being wrong about viability
of biological material in or on food or other items are serious and
could result in serious morbidity and possible mortality in humans.
It can also lead to false positives, which have a negative
financial impact on the food industry. Also, excessively heating
food matrices to destroy bacterial or bacterial spores DNA is also
problematic because the quality of the texture, flavor and
appearance of the finish good is reduced.
[0011] In addition to food substances, many other items need to be
rendered sterile or have a reduction in the viability of biological
material on the items prior to usage. For example, reusable medical
and dental devices (such as, but not limited to, endoscopes,
catheters, sponges, clamps, scalpels, drills, and suction tubes)
need to be cleaned (biological material inactivated) after being
used on one subject, prior to use on another subject. Biological
material present on robust medical equipment is often inactivated
by subjecting the contaminated equipment to high temperatures and
pressure via a steam autoclave. While such inactivation methods are
very effective for more durable medical instruments, advanced
medical instruments formed of rubber and plastic components with
adhesives are delicate and wholly unsuited to the high temperatures
and pressures associated with a conventional steam autoclave. Thus,
some steam autoclaves have been modified to operate under low
pressure cycling programs to increase the rate of steam penetration
into the medical devices or associated packages of medical devices
undergoing cleaning. However, highly complex instruments which are
often formed and assembled with very precise dimensions, close
assembly tolerances, and sensitive optical components, such as
endoscopes, may be destroyed or have their useful lives severely
curtailed by harsh inactivation methods employing high temperatures
and high or low pressures. Endoscopes, in particular, present
problems in that such devices typically have numerous exterior
crevices and interior lumens which can harbor microbes and thus be
difficult to clean using ordinary techniques. The employment of a
fast-acting yet gentle inactivation method is desirable for
reprocessing sensitive instruments. Other medical or dental
instruments which comprise lumens are also in need of methods of
cleaning which employ an effective reprocessing system which will
not harm sensitive components and materials. Further, the need
exists for a reprocessing system having a shorter reprocessing
cycle time. Regardless of how these devices are cleaned, failure to
inactivate a substantial proportion of the biological material that
may be present on the item after usage could result in the
dangerous illness in the next subject on which the item is
used.
[0012] The Food Safety Modernization Act (FSMA) mandates that
companies document risk-based preventive controls for all
pre-requisite programs as part of their Food Safety program. The
Food and Drug Administration proposed guidelines under FSMA include
the application of prevention standards to sanitation and
environmental controls and monitoring. For example, one of the
modifications under consideration involves sterilization of food
packaging containers prior to filling the containers. Glass jars
used for packaging of finished acidified and acid foods for the
retail market are currently rinse with hot water, filled with the
product, and subjected to a validated pasteurization step, often
considered as a critical control point to render the finished food
safe for consumption. New guidelines would require such containers
to be subjected to a sterilization treatment prior to filling. The
sterilization step for glass containers could be applied as a
pasteurization, ultraviolet light, high pressure, or radiation
treatment. The effectiveness of such treatments to eradicate
pathogens of public health significance would have to be
demonstrated after the treatments are applied.
[0013] As such, there remains a need for one or more assays that
can evaluate and/or determine the amount of inactivation of
biological material on/in food and/or an object in a timely and
accurate manner. There is also a need for assays that can evaluate
inactivation protocols and for evaluating deviations in processing
to reduce the amount of viable biological material in/on items. The
assays described herein use quantitative PCR. PCR and real-time PCR
are well-known laboratory techniques and are accepted by AOAC
International for clinical detection assays, including assays to
detect BRCA1 and BRCA2 mutations.
[0014] Mitochondrial DNA (mtDNA) is used as identifiers in many
scientific disciplines. They have been adopted for barcoding almost
all groups of higher animals (http://www.barcoding.si.edu/). MtDNA
is also used in human typing for forensic analysis (Hopwood, et
al., Int. J. Legal Med. 108(5):237-243 (1996); Andreasson, et al.,
Biotechniques 33(2):402-411 (2002); Budowle, et al., Annu. Rev.
Genomics Hum. Genet. 4:119-141 (2003)) using tissues such as bones,
teeth, and hair shafts for DNA extraction. MtDNA primers or probes
have been developed for source tracking fecal contaminates in
wastewater influents and effluents using multiplex qPCR (Caldwell,
et al., Environ. Sci. & Technol., 41:3277-83 (2007); Caldwell
and Levine, J. Microbiol. Methods 77:17-22 (2009); Caldwell, et
al., "Mitochondrial DNA as source tracking markers of fecal
contamination", In Microbial Source Tracking: Methods,
Applications, and Case Studies, eds. Harwood, Hagedorn and Blanch
(Springer Science and Business Media, NY) 229-250 (2011)). In the
food industry, PCR-based mtDNA analyses are used in the
authentication of food, and to trace contamination of other animals
in the food products (Meyer and Candrian, Lebensm.-Wiss.
u.-Technol. 29:1-9 (1996); Lahiff, et al., Mol. Cell Probes
15(1):27-35 (2001); Zhang, et al., Food Control 18:1149-1158
(2007); Fujimura, et al., Biosci. Biotechnol. Biochem. 72:909-913
(2008)). The development of those molecular tools improved the
monitoring of food quality by preventing fraudulent description of
food content, and identifying adulterants.
SUMMARY OF THE INVENTION
[0015] It is an object of this invention to have a synthetic
polynucleotide which has a nucleic acid sequence that is a
consensus sequence to a portion of a gene that is highly conserved
in plants and/or animals. It is a further object of this invention
that this synthetic polynucleotide is between approximately 80 bp
and approximately 250 bp long. It is a further object of this
invention that this synthetic polynucleotide is between
approximately 100 bp and approximately 200 bp long. It is a further
object of this invention that this synthetic polynucleotide is
between approximately 125 bp and approximately 175 bp long. It is a
further object of this invention that the highly conserved gene be
a mitochondrial gene. It is a further object of this invention that
the mitochondrial gene be atp1. It is a further object of this
invention that the sequence of this synthetic polynucleotide be at
least 95% identical to the sequence of a portion of atp1. It is
another object of this invention that this synthetic polynucleotide
has the sequence selected from the group consisting of SEQ ID NO:
14, 15, 16, and 17, or a sequence that is the reverse complement
thereof.
[0016] It is an object of this invention to have a synthesized
polynucleotide which a consensus sequence to a portion of a gene
that is highly conserved in plants and/or animals. It is a further
object of this invention that the highly conserved gene be a
mitochondrial gene. It is a further object of this invention that
the mitochondrial gene be atp1. It is a further object of this
invention that the sequence of this synthetic polynucleotide be at
least 95% identical to the sequence of a portion of atp1. It is
another object of this invention that the polynucleotide has a
sequence selected from the group consisting of SEQ ID NO: 1, 2, 3,
4, 5, 6, 7, and 8.
[0017] It is an object of this invention to have a composition
useful for determining the integrity of a mitochondrial DNA gene,
the composition containing a probe, a quencher dye, and a
fluorescent dye. It is another object of this invention that the
quencher dye and fluorescent dye are linked to the probe. It is a
further object of this invention that the probe has a nucleotide
sequence of between approximately any 15 contiguous bases and
approximately any 45 contiguous bases selected from the sequences
in SEQ ID NO: 14, 15, 16, and 17, and the reverse complement
thereof.
[0018] It is an object of this invention to have a kit or
composition that is useful for determining the integrity of a
mitochondrial DNA gene, the kit or composition containing a first
polynucleotide and a second polynucleotide and, optionally,
instructions for using the first polynucleotide and second
polynucleotide, and optionally, a DNA polymerase. It is another
object of this invention that the first polynucleotide and second
polynucleotide have sequences which are at least 95% identical to a
portion of a mitochondrial DNA gene. It is another object of this
invention that the sequences of the first polynucleotide and second
polynucleotide are at least 95% identical to a portion of the atp1.
It is a further object of this invention that the first
polynucleotide and the second polynucleotide have the sequences as
follows: the first polynucleotide has SEQ ID NO: 1 and the second
polynucleotide has SEQ ID NO: 2; the first polynucleotide has SEQ
ID NO: 3 and the second polynucleotide has SEQ ID NO: 4; the first
polynucleotide has SEQ ID NO: 5 and the second polynucleotide has
SEQ ID NO: 6; and the first polynucleotide has SEQ ID NO: 7 and the
second polynucleotide has SEQ ID NO: 8. It is a further object of
this invention that the kit or composition optionally contain a
fluorescent composition that can be either an intercalating dye or
a composition of a fluorescent dye, a quencher dye and a probe such
that the fluorescent dye and quencher dye are linked to the probe.
It is another object of this invention that the probe have a
sequence of between approximately fifteen contiguous bases and
approximately forty-five contiguous bases of SEQ ID NO: 14 or the
reverse complement thereof when the first polynucleotide has SEQ ID
NO: 1 and the second polynucleotide has SEQ ID NO: 2; or a sequence
of between approximately fifteen contiguous bases and approximately
forty-five contiguous bases SEQ ID NO: 15 or the reverse complement
thereof when the first polynucleotide has SEQ ID NO: 3 and the
second polynucleotide has SEQ ID NO: 4; or a sequence of between
approximately fifteen contiguous bases and approximately forty-five
contiguous bases of SEQ ID NO: 16 or the reverse complement thereof
when the first polynucleotide has SEQ ID NO: 5 and the second
polynucleotide has SEQ ID NO: 6; or a sequence of between
approximately fifteen contiguous bases and approximately forty-five
contiguous bases of SEQ ID NO: 17 or the reverse complement thereof
when the first polynucleotide has SEQ ID NO: 7 and the second
polynucleotide has SEQ ID NO: 8.
[0019] It is an object of this invention to have a method for
determining the amount of inactivation of hazardous biological
material in a food matrix, the method having the steps of exposing
the food matrix's intrinsic DNA to a first polynucleotide, a second
polynucleotide, and optionally a fluorescent composition;
amplifying the intrinsic DNA using a DNA amplification method to
produce an amplicon; determining the threshold cycle of the
amplified intrinsic DNA; and comparing the obtained threshold cycle
value to a known threshold cycle value of food material intrinsic
DNA that is equivalent to the desired amount of inactivation of
said hazardous biological material. It is a further object of this
invention that the DNA amplification method use DNA polymerase to
generate an amplicon that has the sequence of the first
polynucleotide at the amplicon's 5' end and the sequence of the
second polynucleotide at the amplicon's 3' end. It is another
object of the invention that the food matrix is plant material,
animal material, or a combination thereof. It is another object of
this invention that the fluorescent composition can be an
intercalating dye or a composition of a fluorescent dye, a quencher
dye and a probe such that the fluorescent dye and quencher dye are
linked to the probe and such that the probe has a sequence of
between approximately 15 contiguous bases and approximately 45
contiguous bases of the amplicon or the reverse complement
thereof.
[0020] It is an object of this invention to have a method for
determining the amount of inactivation of hazardous biological
material in a food matrix, the method having the steps of exposing
the food matrix's intrinsic DNA to a first polynucleotide, a second
polynucleotide, and optionally a fluorescent composition;
amplifying the intrinsic DNA using a DNA amplification method to
produce an amplicon; determining the threshold cycle of the
amplified intrinsic DNA; and comparing the obtained threshold cycle
value to a known threshold cycle value of food material intrinsic
DNA that is equivalent to the desired amount of inactivation of
said hazardous biological material. It is a further object of this
invention that the DNA amplification method use DNA polymerase to
generate an amplicon that has the sequence of the first
polynucleotide at the amplicon's 5' end and the sequence of the
second polynucleotide at the amplicon's 3' end. It is another
object of this invention that the first polynucleotide has a
sequence of between approximately 15 contiguous bases and
approximately 45 contiguous bases of SEQ ID NO: 9, and the second
polynucleotide has a sequence of between approximately 15
contiguous bases and approximately 45 contiguous bases of the
reverse complement of SEQ ID NO: 9, and the amplicon generated is
between approximately 80 bp and 250 bp long, is between
approximately 100 bp and approximately 200 bp long, or is between
approximately 125 bp and approximately 175 bp long. It is another
object of this invention that the fluorescent composition be an
intercalating dye or a composition containing a fluorescent dye, a
quencher dye and a probe such that the fluorescent dye and quencher
dye are linked to the probe and such that the probe has a sequence
of between approximately 15 contiguous bases and approximately 45
contiguous bases of SEQ ID NO: 9 or the reverse complement thereof.
It is another object of this invention to have the step of
isolating the intrinsic DNA from the food matrix prior to exposing
the intrinsic DNA to the first polynucleotide and second
polynucleotide. It is another object of the invention that the food
matrix is plant material, animal material, or a combination
thereof.
[0021] It is an object of this invention to have a method for
determining the amount of inactivation of hazardous biological
material in a food matrix, the method having the steps of exposing
the food matrix's intrinsic DNA to a first polynucleotide, a second
polynucleotide, and optionally to a fluorescent composition;
amplifying the intrinsic DNA using a DNA amplification method to
produce an amplicon; determining the threshold cycle of the
amplified intrinsic DNA; and comparing the obtained threshold cycle
value to a known threshold cycle value of food material intrinsic
DNA that is equivalent to the desired amount of inactivation of
said hazardous biological material. It is a further object of this
invention that the DNA amplification method use DNA polymerase to
generate an amplicon that has the sequence of SEQ ID NO: 14. It is
another object of this invention that the first polynucleotide has
the sequence of SEQ ID NO: 1, and the second polynucleotide has a
sequence of SEQ ID NO: 2 or the reverse complement thereof. It is
another object of this invention to have the step of isolating the
intrinsic DNA from the food matrix prior to exposing the intrinsic
DNA to the first polynucleotide and second polynucleotide. It is a
further object of this invention that the fluorescent composition
be an intercalating dye or a composition containing a fluorescent
dye, a quencher dye and a probe such that the fluorescent dye and
quencher dye are linked to the probe and such that the probe has a
sequence of between approximately 15 contiguous bases and
approximately 45 contiguous bases of SEQ ID NO: 14 or the reverse
complement thereof. It is another object of the invention that the
food matrix is plant material, animal material, or a combination
thereof.
[0022] It is an object of this invention to have a method for
determining the amount of inactivation of hazardous biological
material in a food matrix, the method having the steps of exposing
the food matrix's intrinsic DNA to a first polynucleotide, a second
polynucleotide, and optionally a fluorescent composition;
amplifying the intrinsic DNA using a DNA amplification method to
produce an amplicon; determining the threshold cycle of the
amplified intrinsic DNA; and comparing the obtained threshold cycle
value to a known threshold cycle value of food material intrinsic
DNA that is equivalent to the desired amount of inactivation of
said hazardous biological material. It is a further object of this
invention that the DNA amplification method use DNA polymerase to
generate an amplicon that has the sequence of SEQ ID NO: 15. It is
another object of this invention that the first polynucleotide has
the sequence of SEQ ID NO: 3, and the second polynucleotide has a
sequence of SEQ ID NO: 4 or the reverse complement thereof. It is
another object of this invention to have the step of isolating the
intrinsic DNA from the food matrix prior to exposing the intrinsic
DNA to the first polynucleotide and second polynucleotide. It is a
further object of this invention that the fluorescent composition
be an intercalating dye or a composition containing a fluorescent
dye, a quencher dye and a probe such that the fluorescent dye and
quencher dye are linked to the probe and such that the probe has a
sequence of between approximately 15 contiguous bases and
approximately 45 contiguous bases of SEQ ID NO: 15 or the reverse
complement thereof. It is another object of the invention that the
food matrix is plant material, animal material, or a combination
thereof.
[0023] It is an object of this invention to have a method for
determining the amount of inactivation of hazardous biological
material in a food matrix, the method having the steps of exposing
the food matrix's intrinsic DNA to a first polynucleotide, a second
polynucleotide, and optionally a fluorescent composition;
amplifying the intrinsic DNA using a DNA amplification method to
produce an amplicon; determining the threshold cycle of the
amplified intrinsic DNA; and comparing the obtained threshold cycle
value to a known threshold cycle value of food material intrinsic
DNA that is equivalent to the desired amount of inactivation of
said hazardous biological material. It is a further object of this
invention that the DNA amplification method use DNA polymerase to
generate an amplicon that has the sequence of SEQ ID NO: 16. It is
another object of this invention that the first polynucleotide has
the sequence of SEQ ID NO: 5, and the second polynucleotide has a
sequence of SEQ ID NO: 6 or the reverse complement thereof. It is
another object of this invention to have the step of isolating the
intrinsic DNA from the food matrix prior to exposing the intrinsic
DNA to the first polynucleotide and second polynucleotide. It is a
further object of this invention that the fluorescent composition
be an intercalating dye or a composition containing a fluorescent
dye, a quencher dye and a probe such that the fluorescent dye and
quencher dye are linked to the probe and such that the probe has a
sequence of between approximately 15 contiguous bases and
approximately 45 contiguous bases of SEQ ID NO: 16 or the reverse
complement thereof. It is another object of the invention that the
food matrix is plant material, animal material, or a combination
thereof.
[0024] It is an object of this invention to have a method for
determining the amount of inactivation of hazardous biological
material in a food matrix, the method having the steps of exposing
the food matrix's intrinsic DNA to a first polynucleotide, a second
polynucleotide, and optionally a fluorescent composition;
amplifying the intrinsic DNA using a DNA amplification method to
produce an amplicon; determining the threshold cycle of the
amplified intrinsic DNA; and comparing the obtained threshold cycle
value to a known threshold cycle value of food material intrinsic
DNA that is equivalent to the desired amount of inactivation of
said hazardous biological material. It is a further object of this
invention that the DNA amplification method use DNA polymerase to
generate an amplicon that has the sequence of SEQ ID NO: 17. It is
another object of this invention that the first polynucleotide has
the sequence of SEQ ID NO: 7, and the second polynucleotide has a
sequence of SEQ ID NO: 8 or the reverse complement thereof. It is
another object of this invention to have the step of isolating the
intrinsic DNA from the food matrix prior to exposing the intrinsic
DNA to the first polynucleotide and second polynucleotide. It is a
further object of this invention that the fluorescent composition
be an intercalating dye or a composition containing a fluorescent
dye, a quencher dye and a probe such that the fluorescent dye and
quencher dye are linked to the probe and such that the probe has a
sequence of between approximately 15 contiguous bases and
approximately 45 contiguous bases of SEQ ID NO: 17 or the reverse
complement thereof. It is another object of the invention that the
food matrix is plant material, animal material, or a combination
thereof.
[0025] It is an object of this invention to have a method for
determining the efficacy of a protocol to inactivate hazardous
biological material in a food matrix having the steps of,
optionally processing a food matrix sample according to the
inactivation protocol; exposing the intrinsic DNA of the food
matrix processed according to the inactivation protocol to a first
polynucleotide, a second polynucleotide, and optionally a
fluorescent composition; amplifying the intrinsic DNA using an
amplification process to produce an amplicon; determining the
threshold cycle of the amplified intrinsic DNA; and comparing the
obtained threshold cycle value to a known threshold cycle value for
the intrinsic DNA of the food material that has been processed
according to a second inactivation method known to achieve the
desired amount of inactivation of the hazardous biological
material. It is a further object of this invention that the DNA
amplification method use DNA polymerase to generate an amplicon
that has the sequence of the first polynucleotide at the amplicon's
5' end and the sequence of the second polynucleotide at the
amplicon's 3' end. It is another object of the invention that the
food matrix is plant material, animal material, or a combination
thereof. It is another object of this invention that the first
polynucleotide and second polynucleotide bind to mtDNA. It is
another object of this invention that the first polynucleotide and
second polynucleotide bind to atp1. It is another object of this
invention to optionally have the step of isolating the intrinsic
DNA from the processed food matrix prior to exposing the intrinsic
DNA to the first polynucleotide and second polynucleotide. It is a
further object of this invention that the fluorescent composition
be an intercalating dye or a composition containing a fluorescent
dye, a quencher dye and a probe such that the fluorescent dye and
quencher dye are linked to the probe and such that the probe has a
sequence of between approximately 15 contiguous bases and
approximately 45 contiguous bases of the amplicon or the reverse
complement thereof.
[0026] It is an object of this invention to have a method for
determining the efficacy of a protocol to inactivate hazardous
biological material in a food matrix having the steps of,
optionally processing a food matrix sample according to the
inactivation protocol; exposing the intrinsic DNA of the food
matrix processed according to the inactivation protocol to a first
polynucleotide, a second polynucleotide, and optionally a
fluorescent composition; amplifying the intrinsic DNA using an
amplification process to produce an amplicon; determining the
threshold cycle of the amplified intrinsic DNA; and comparing the
obtained threshold cycle value to a known threshold cycle value for
the intrinsic DNA of the food material that has been processed
according to a second inactivation method known to achieve the
desired amount of inactivation of the hazardous biological
material. It is another object of this invention that the first
polynucleotide has a sequence of between approximately 15
contiguous bases and approximately 45 contiguous bases of SEQ ID
NO: 9, and the second polynucleotide has a sequence of between
approximately 15 contiguous bases and approximately 45 contiguous
bases of the reverse complement of SEQ ID NO: 9, and the amplicon
generated is between approximately 80 bp and 250 bp long, is
between approximately 100 bp and approximately 200 bp long, or is
between approximately 125 bp and approximately 175 bp long. It is a
further object of this invention that the DNA amplification method
use DNA polymerase to generate the amplicon. It is another object
of this invention to optionally have the step of isolating the
intrinsic DNA from the processed food matrix prior to exposing the
intrinsic DNA to the first polynucleotide and second
polynucleotide. It is a further object of this invention that the
fluorescent composition be an intercalating dye or a composition
containing a fluorescent dye, a quencher dye and a probe such that
the fluorescent dye and quencher dye are linked to the probe and
such that the probe has a sequence of between approximately 15
contiguous bases and approximately 45 contiguous bases of the
amplicon or the reverse complement thereof. It is another object of
the invention that the food matrix is plant material, animal
material, or a combination thereof.
[0027] It is an object of this invention to have a method for
determining the efficacy of a protocol to inactivate hazardous
biological material in a food matrix having the steps of,
optionally processing a food matrix sample according to the
inactivation protocol; optionally isolating the intrinsic DNA of
the processed food matrix; exposing the intrinsic DNA of the food
matrix processed according to the inactivation protocol to a first
polynucleotide, a second polynucleotide, and optionally a
fluorescent composition; amplifying the intrinsic DNA using a DNA
amplification method to produce an amplicon; determining the
threshold cycle of the amplified intrinsic DNA; and comparing the
obtained threshold cycle value to a known threshold cycle value for
the intrinsic DNA of the food material that has been processed
according to a second inactivation method known to achieve the
desired amount of inactivation of the hazardous biological
material. It is a further object of this invention that the DNA
amplification method use DNA polymerase to generate an amplicon
that has the sequence of SEQ ID NO: 14. It is another object of
this invention that the first polynucleotide has the sequence of
SEQ ID NO: 1, and the second polynucleotide has a sequence of SEQ
ID NO: 2 or the reverse complement thereof. It is a further object
of this invention that the fluorescent composition be an
intercalating dye or a composition containing a fluorescent dye, a
quencher dye and a probe such that the fluorescent dye and quencher
dye are linked to the probe and such that the probe has a sequence
of between approximately 15 contiguous bases and approximately 45
contiguous bases of SEQ ID NO: 14 or the reverse complement
thereof. It is another object of the invention that the food matrix
is plant material, animal material, or a combination thereof.
[0028] It is an object of this invention to have a method for
determining the efficacy of a protocol to inactivate hazardous
biological material in a food matrix having the steps of,
optionally processing a food matrix sample according to the
inactivation protocol; optionally isolating the intrinsic DNA of
the processed food matrix; exposing the intrinsic DNA of the food
matrix processed according to the inactivation protocol to a first
polynucleotide, a second polynucleotide, and optionally a
fluorescent composition; amplifying the intrinsic DNA using a DNA
amplification method to produce an amplicon; determining the
threshold cycle of the amplified intrinsic DNA; and comparing the
obtained threshold cycle value to a known threshold cycle value for
the intrinsic DNA of the food material that has been processed
according to a second inactivation method known to achieve the
desired amount of inactivation of the hazardous biological
material. It is a further object of this invention that the DNA
amplification method use DNA polymerase to generate an amplicon
that has the sequence of SEQ ID NO: 15. It is another object of
this invention that the first polynucleotide has the sequence of
SEQ ID NO: 3, and the second polynucleotide has a sequence of SEQ
ID NO: 4 or the reverse complement thereof. It is a further object
of this invention that the fluorescent composition be an
intercalating dye or a composition containing a fluorescent dye, a
quencher dye and a probe such that the fluorescent dye and quencher
dye are linked to the probe and such that the probe has a sequence
of between approximately 15 contiguous bases and approximately 45
contiguous bases of SEQ ID NO: 15 or the reverse complement
thereof. It is another object of the invention that the food matrix
is plant material, animal material, or a combination thereof.
[0029] It is an object of this invention to have a method for
determining the efficacy of a protocol to inactivate hazardous
biological material in a food matrix having the steps of,
optionally processing a food matrix sample according to the
inactivation protocol; optionally isolating the intrinsic DNA of
the processed food matrix; exposing the intrinsic DNA of the food
matrix processed according to the inactivation protocol to a first
polynucleotide, a second polynucleotide, and optionally a
fluorescent composition; amplifying the intrinsic DNA using a DNA
amplification method to produce an amplicon; determining the
threshold cycle of the amplified intrinsic DNA; and comparing the
obtained threshold cycle value to a known threshold cycle value for
the intrinsic DNA of the food material that has been processed
according to a second inactivation method known to achieve the
desired amount of inactivation of the hazardous biological
material. It is a further object of this invention that the DNA
amplification method use DNA polymerase to generate an amplicon
that has the sequence of SEQ ID NO: 16. It is another object of
this invention that the first polynucleotide has the sequence of
SEQ ID NO: 5, and the second polynucleotide has a sequence of SEQ
ID NO: 6 or the reverse complement thereof. It is a further object
of this invention that the fluorescent composition be an
intercalating dye or a composition containing a fluorescent dye, a
quencher dye and a probe such that the fluorescent dye and quencher
dye are linked to the probe and such that the probe has a sequence
of between approximately 15 contiguous bases and approximately 45
contiguous bases of SEQ ID NO: 16 or the reverse complement
thereof. It is another object of the invention that the food matrix
is plant material, animal material, or a combination thereof.
[0030] It is an object of this invention to have a method for
determining the efficacy of a protocol to inactivate hazardous
biological material in a food matrix having the steps of,
optionally processing a food matrix sample according to the
inactivation protocol; optionally isolating the intrinsic DNA of
the processed food matrix; exposing the intrinsic DNA of the food
matrix processed according to the inactivation protocol to a first
polynucleotide, a second polynucleotide, and optionally a
fluorescent composition; amplifying the intrinsic DNA using a DNA
amplification method to produce an amplicon; determining the
threshold cycle of the amplified intrinsic DNA; and comparing the
obtained threshold cycle value to a known threshold cycle value for
the intrinsic DNA of the food material that has been processed
according to a second inactivation method known to achieve the
desired amount of inactivation of the hazardous biological
material. It is a further object of this invention that the DNA
amplification method use DNA polymerase to generate an amplicon
that has the sequence of SEQ ID NO: 17. It is another object of
this invention that the first polynucleotide has the sequence of
SEQ ID NO: 7, and the second polynucleotide has a sequence of SEQ
ID NO: 8 or the reverse complement thereof. It is a further object
of this invention that the fluorescent composition be an
intercalating dye or a composition containing a fluorescent dye, a
quencher dye and a probe such that the fluorescent dye and quencher
dye are linked to the probe and such that the probe has a sequence
of between approximately 15 contiguous bases and approximately 45
contiguous bases of SEQ ID NO: 17 or the reverse complement
thereof. It is another object of the invention that the food matrix
is plant material, animal material, or a combination thereof.
[0031] It is an object of this invention to have a method for
assessing the efficacy of a protocol for inactivation of hazardous
material in or on an item, the method having the steps of
optionally processing a sample of extrinsic DNA according to the
protocol; optionally isolating the processed extrinsic DNA;
exposing the extrinsic DNA to a first polynucleotide, to a second
polynucleotide, and optionally a fluorescent composition;
amplifying the extrinsic DNA using a DNA amplification method to
produce an amplicon; determining the threshold cycle of the
extrinsic DNA; and comparing the obtained threshold cycle value to
a known threshold cycle value for extrinsic DNA that has been
processed according to a second inactivation method known to
achieve the desired amount of inactivation of the hazardous
biological material. It is a further object of this invention that
the DNA amplification method use DNA polymerase to generate an
amplicon that has the sequence of the first polynucleotide at the
amplicon's 5' end and the sequence of the second polynucleotide at
the amplicon's 3' end. It is a further object of this invention
that the fluorescent composition be an intercalating dye or a
composition containing a fluorescent dye, a quencher dye and a
probe such that the fluorescent dye and quencher dye are linked to
the probe and such that the probe has a sequence of between
approximately 15 contiguous bases and approximately 45 contiguous
bases of the amplicon or the reverse complement thereof. It is a
further object of this invention that the item be a food matrix, a
container, equipment, or a medical device.
[0032] It is an object of this invention to have a method for
assessing the efficacy of a protocol for inactivation of hazardous
material in or on an item, the method having the steps of
optionally processing a sample of extrinsic DNA according to the
protocol; optionally isolating the processed extrinsic DNA;
exposing the extrinsic DNA to a first polynucleotide, to a second
polynucleotide, and optionally to a fluorescent composition;
amplifying the extrinsic DNA using a DNA amplification method to
produce an amplicon; determining the threshold cycle of the
extrinsic DNA; and comparing the obtained threshold cycle value to
a known threshold cycle value for extrinsic DNA that has been
processed according to a second inactivation method known to
achieve the desired amount of inactivation of the hazardous
biological material. It is a further object of this invention that
the DNA amplification method use DNA polymerase to generate an
amplicon that has the sequence of the first polynucleotide at the
amplicon's 5' end and the sequence of the second polynucleotide at
the amplicon's 3' end. It is another object of this invention that
the first polynucleotide has a sequence of between approximately 15
contiguous bases and approximately 45 contiguous bases of any mtDNA
gene, and the second polynucleotide has a sequence of between
approximately 15 contiguous bases and approximately 45 contiguous
bases of the reverse complement of the same mtDNA gene, and the
amplicon generated is between approximately 80 bp and 250 bp long,
is between approximately 100 bp and approximately 200 bp long, or
is between approximately 125 bp and approximately 175 bp long. It
is a further object of this invention that the fluorescent
composition be an intercalating dye or a composition containing a
fluorescent dye, a quencher dye and a probe such that the
fluorescent dye and quencher dye are linked to the probe and such
that the probe has a sequence of between approximately 15
contiguous bases and approximately 45 contiguous bases of the
amplicon or the reverse complement thereof. It is a further object
of this invention that the item be a food matrix, a container,
equipment, or a medical device.
[0033] It is an object of this invention to have a method for
assessing the efficacy of a protocol for inactivation of hazardous
material in or on an item, the method having the steps of
optionally processing a sample of extrinsic DNA according to the
protocol; optionally isolating the processed extrinsic DNA;
exposing the extrinsic DNA to a first polynucleotide, a second
polynucleotide, and optionally a fluorescent composition;
amplifying the extrinsic DNA using a DNA amplification method to
produce an amplicon; determining the threshold cycle of the
extrinsic DNA; and comparing the obtained threshold cycle value to
a known threshold cycle value for extrinsic DNA that has been
processed according to a second inactivation method known to
achieve the desired amount of inactivation of the hazardous
biological material. It is a further object of this invention that
the DNA amplification method use DNA polymerase to generate an
amplicon that has the sequence of the first polynucleotide at the
amplicon's 5' end and the sequence of the second polynucleotide at
the amplicon's 3' end. It is another object of this invention that
the first polynucleotide has a sequence of between approximately 15
contiguous bases and approximately 45 contiguous bases of SEQ ID
NO: 9, and the second polynucleotide has a sequence of between
approximately 15 contiguous bases and approximately 45 contiguous
bases of the reverse complement of SEQ ID NO: 9, and the amplicon
generated is between approximately 80 bp and 250 bp long, is
between approximately 100 bp and approximately 200 bp long, or is
between approximately 125 bp and approximately 175 bp long. It is a
further object of this invention that the fluorescent composition
be an intercalating dye or a composition containing a fluorescent
dye, a quencher dye and a probe such that the fluorescent dye and
quencher dye are linked to the probe and such that the probe has a
sequence of between approximately 15 contiguous bases and
approximately 45 contiguous bases of the amplicon or the reverse
complement thereof. It is a further object of this invention that
the item be a food matrix, a container, equipment, or a medical
device.
[0034] It is an object of this invention to have a method for
assessing the efficacy of a protocol for inactivation of hazardous
material in or on an item, the method having the steps of
optionally processing a sample of extrinsic DNA according to the
protocol; optionally isolating the processed extrinsic DNA;
exposing the extrinsic DNA to a first polynucleotide, a second
polynucleotide, and optionally a fluorescent composition;
amplifying the extrinsic DNA using a DNA amplification method to
produce an amplicon; determining the threshold cycle of the
extrinsic DNA; and comparing the obtained threshold cycle value to
a known threshold cycle value for extrinsic DNA that has been
processed according to a second inactivation method known to
achieve the desired amount of inactivation of the hazardous
biological material. It is a further object of this invention that
the DNA amplification method use DNA polymerase to generate an
amplicon that has the sequence of SEQ ID NO: 14; that the first
polynucleotide has the sequence of SEQ ID NO: 1, and the second
polynucleotide has the sequence of SEQ ID NO: 2. It is a further
object of this invention that the fluorescent composition be an
intercalating dye or a composition containing a fluorescent dye, a
quencher dye and a probe such that the fluorescent dye and quencher
dye are linked to the probe and such that the probe has a sequence
of between approximately 15 contiguous bases and approximately 45
contiguous bases of SEQ ID NO: 14 or the reverse complement
thereof. It is a further object of this invention that the item be
a food matrix, a container, equipment, or a medical device.
[0035] It is an object of this invention to have a method for
assessing the efficacy of a protocol for inactivation of hazardous
material in or on an item, the method having the steps of
optionally processing a sample of extrinsic DNA according to the
protocol; optionally isolating the processed extrinsic DNA;
exposing the extrinsic DNA to a first polynucleotide, a second
polynucleotide, and optionally a fluorescent composition;
amplifying the extrinsic DNA using a DNA amplification method to
produce an amplicon; determining the threshold cycle of the
extrinsic DNA; and comparing the obtained threshold cycle value to
a known threshold cycle value for extrinsic DNA that has been
processed according to a second inactivation method known to
achieve the desired amount of inactivation of the hazardous
biological material. It is a further object of this invention that
the DNA amplification method use DNA polymerase to generate an
amplicon that has the sequence of SEQ ID NO: 15; that the first
polynucleotide has the sequence of SEQ ID NO: 3, and the second
polynucleotide has the sequence of SEQ ID NO: 4. It is a further
object of this invention that the fluorescent composition be an
intercalating dye or a composition containing a fluorescent dye, a
quencher dye and a probe such that the fluorescent dye and quencher
dye are linked to the probe and such that the probe has a sequence
of between approximately 15 contiguous bases and approximately 45
contiguous bases of SEQ ID NO: 15 or the reverse complement
thereof. It is a further object of this invention that the item be
a food matrix, a container, equipment, or a medical device.
[0036] It is an object of this invention to have a method for
assessing the efficacy of a protocol for inactivation of hazardous
material in or on an item, the method having the steps of
optionally processing a sample of extrinsic DNA according to the
protocol; optionally isolating the processed extrinsic DNA;
exposing the extrinsic DNA to a first polynucleotide, a second
polynucleotide, and optionally a fluorescent composition;
amplifying the extrinsic DNA using a DNA amplification method to
produce an amplicon; determining the threshold cycle of the
extrinsic DNA; and comparing the obtained threshold cycle value to
a known threshold cycle value for extrinsic DNA that has been
processed according to a second inactivation method known to
achieve the desired amount of inactivation of the hazardous
biological material. It is a further object of this invention that
the DNA amplification method use DNA polymerase to generate an
amplicon that has the sequence of SEQ ID NO: 16; that the first
polynucleotide has the sequence of SEQ ID NO: 5, and the second
polynucleotide has the sequence of SEQ ID NO: 6. It is a further
object of this invention that the fluorescent composition be an
intercalating dye or a composition containing a fluorescent dye, a
quencher dye and a probe such that the fluorescent dye and quencher
dye are linked to the probe and such that the probe has a sequence
of between approximately 15 contiguous bases and approximately 45
contiguous bases of SEQ ID NO: 16 or the reverse complement
thereof. It is a further object of this invention that the item be
a food matrix, a container, equipment, or a medical device.
[0037] It is an object of this invention to have a method for
assessing the efficacy of a protocol for inactivation of hazardous
material in or on an item, the method having the steps of
optionally processing a sample of extrinsic DNA according to the
protocol; optionally isolating the processed extrinsic DNA;
exposing the extrinsic DNA to a first polynucleotide, a second
polynucleotide, and optionally a fluorescent composition;
amplifying the extrinsic DNA using a DNA amplification method to
produce an amplicon; determining the threshold cycle of the
extrinsic DNA; and comparing the obtained threshold cycle value to
a known threshold cycle value for extrinsic DNA that has been
processed according to a second inactivation method known to
achieve the desired amount of inactivation of the hazardous
biological material. It is a further object of this invention that
the DNA amplification method use DNA polymerase to generate an
amplicon that has the sequence of SEQ ID NO: 17; that the first
polynucleotide has the sequence of SEQ ID NO: 7, and the second
polynucleotide has the sequence of SEQ ID NO: 8. It is a further
object of this invention that the fluorescent composition be an
intercalating dye or a composition containing a fluorescent dye, a
quencher dye and a probe such that the fluorescent dye and quencher
dye are linked to the probe and such that the probe has a sequence
of between approximately 15 contiguous bases and approximately 45
contiguous bases of SEQ ID NO: 17 or the reverse complement
thereof. It is a further object of this invention that the item be
a food matrix, a container, equipment, or a medical device.
[0038] It is an object of this invention to have a quality control
method for determining the amount of inactivation of a hazardous
material in or on an item, the method having the steps of
optionally processing a sample containing either intrinsic DNA or
extrinsic DNA according to the pre-determined inactivation
protocol; optionally isolating the processed intrinsic DNA or
extrinsic DNA; exposing the intrinsic DNA or extrinsic DNA to a
first polynucleotide, a second polynucleotide, and optionally a
fluorescent composition; amplifying the intrinsic DNA or extrinsic
DNA using a DNA amplification method to produce an amplicon;
determining the threshold cycle of the intrinsic DNA or extrinsic
DNA; and comparing the obtained threshold cycle value to a known
threshold cycle value for intrinsic DNA or extrinsic DNA that is
equivalent to the desired amount of inactivation of the hazardous
biological material. It is a further object of this invention that
the DNA amplification method use DNA polymerase to generate an
amplicon that is between approximately 80 bp and approximately 250
bp long and that has the sequence of the first polynucleotide at
the amplicon's 5' end and the sequence of the second polynucleotide
at the amplicon's 3' end. It is a further object of this invention
that the fluorescent composition be an intercalating dye or a
composition containing a fluorescent dye, a quencher dye and a
probe such that the fluorescent dye and quencher dye are linked to
the probe and such that the probe has a sequence of between
approximately 15 contiguous bases and approximately 45 contiguous
bases of the amplicon or the reverse complement thereof. It is a
further object of this invention that the item be a food matrix, a
container, equipment, or a medical device.
[0039] It is an object of this invention to have a quality control
method for determining the amount of inactivation of a hazardous
material in or on an item, the method having the steps of
optionally processing a sample containing either intrinsic DNA or
extrinsic DNA according to the pre-determined inactivation
protocol; optionally isolating the processed intrinsic DNA or
extrinsic DNA; exposing the intrinsic DNA or extrinsic DNA to a
first polynucleotide, a second polynucleotide, and optionally a
fluorescent composition; amplifying the intrinsic DNA or extrinsic
DNA using a DNA amplification method to produce an amplicon;
determining the threshold cycle of the intrinsic DNA or extrinsic
DNA; and comparing the obtained threshold cycle value to a known
threshold cycle value for intrinsic DNA or extrinsic DNA that is
equivalent to the desired amount of inactivation of the hazardous
biological material. It is a further object of this invention that
the DNA amplification method use DNA polymerase to generate an
amplicon that has the sequence of the first polynucleotide at the
amplicon's 5' end and the sequence of the second polynucleotide at
the amplicon's 3' end. It is another object of this invention that
the first polynucleotide has a sequence of between approximately 15
contiguous bases and approximately 45 contiguous bases of SEQ ID
NO: 9, and the second polynucleotide has a sequence of between
approximately 15 contiguous bases and approximately 45 contiguous
bases of the reverse complement of SEQ ID NO: 9, and the amplicon
generated is between approximately 80 bp and 250 bp long, is
between approximately 100 bp and approximately 200 bp long, or is
between approximately 125 bp and approximately 175 bp long. It is a
further object of this invention that the fluorescent composition
be an intercalating dye or a composition containing a fluorescent
dye, a quencher dye and a probe such that the fluorescent dye and
quencher dye are linked to the probe and such that the probe has a
sequence of between approximately 15 contiguous bases and
approximately 45 contiguous bases of the amplicon or the reverse
complement thereof. It is a further object of this invention that
the item be a food matrix, a container, equipment, or a medical
device.
[0040] It is an object of this invention to have a quality control
method for determining the amount of inactivation of a hazardous
material in or on an item, the method having the steps of
optionally processing a sample containing either intrinsic DNA or
extrinsic DNA according to the pre-determined inactivation
protocol; optionally isolating the processed intrinsic DNA or
extrinsic DNA; exposing the intrinsic DNA or extrinsic DNA to a
first polynucleotide, a second polynucleotide, and optionally a
fluorescent composition; amplifying the intrinsic DNA or extrinsic
DNA using a DNA amplification method to produce an amplicon;
determining the threshold cycle of the intrinsic DNA or extrinsic
DNA; and comparing the obtained threshold cycle value to a known
threshold cycle value for intrinsic DNA or extrinsic DNA that is
equivalent to the desired amount of inactivation of the hazardous
biological material. It is a further object of this invention that
the DNA amplification method use DNA polymerase to generate an
amplicon that has the sequence of SEQ ID NO: 14; that the first
polynucleotide has the sequence of SEQ ID NO: 1, and the second
polynucleotide has the sequence of SEQ ID NO: 2. It is a further
object of this invention that the fluorescent composition be an
intercalating dye or a composition containing a fluorescent dye, a
quencher dye and a probe such that the fluorescent dye and quencher
dye are linked to the probe and such that the probe has a sequence
of between approximately 15 contiguous bases and approximately 45
contiguous bases of SEQ ID NO: 14 or the reverse complement
thereof. It is a further object of this invention that the item be
a food matrix, a container, equipment, or a medical device.
[0041] It is an object of this invention to have a quality control
method for determining the amount of inactivation of a hazardous
material in or on an item, the method having the steps of
optionally processing a sample containing either intrinsic DNA or
extrinsic DNA according to the pre-determined inactivation
protocol; optionally isolating the processed intrinsic DNA or
extrinsic DNA; exposing the intrinsic DNA or extrinsic DNA to a
first polynucleotide, a second polynucleotide, and optionally a
fluorescent composition; amplifying the intrinsic DNA or extrinsic
DNA using a DNA amplification method to produce an amplicon;
determining the threshold cycle of the intrinsic DNA or extrinsic
DNA; and comparing the obtained threshold cycle value to a known
threshold cycle value for intrinsic DNA or extrinsic DNA that is
equivalent to the desired amount of inactivation of the hazardous
biological material. It is a further object of this invention that
the DNA amplification method use DNA polymerase to generate an
amplicon that has the sequence of SEQ ID NO: 15; that the first
polynucleotide has the sequence of SEQ ID NO: 3, and the second
polynucleotide has the sequence of SEQ ID NO: 4. It is a further
object of this invention that the fluorescent composition be an
intercalating dye or a composition containing a fluorescent dye, a
quencher dye and a probe such that the fluorescent dye and quencher
dye are linked to the probe and such that the probe has a sequence
of between approximately 15 contiguous bases and approximately 45
contiguous bases of SEQ ID NO: 15 or the reverse complement
thereof. It is a further object of this invention that the item be
a food matrix, a container, equipment, or a medical device.
[0042] It is an object of this invention to have a quality control
method for determining the amount of inactivation of a hazardous
material in or on an item, the method having the steps of
optionally processing a sample containing either intrinsic DNA or
extrinsic DNA according to the pre-determined inactivation
protocol; optionally isolating the processed intrinsic DNA or
extrinsic DNA; exposing the intrinsic DNA or extrinsic DNA to a
first polynucleotide, a second polynucleotide, and optionally a
fluorescent composition; amplifying the intrinsic DNA or extrinsic
DNA using a DNA amplification method to produce an amplicon;
determining the threshold cycle of the intrinsic DNA or extrinsic
DNA; and comparing the obtained threshold cycle value to a known
threshold cycle value for intrinsic DNA or extrinsic DNA that is
equivalent to the desired amount of inactivation of the hazardous
biological material. It is a further object of this invention that
the DNA amplification method use DNA polymerase to generate an
amplicon that has the sequence of SEQ ID NO: 16; that the first
polynucleotide has the sequence of SEQ ID NO: 5, and the second
polynucleotide has the sequence of SEQ ID NO: 6. It is a further
object of this invention that the fluorescent composition be an
intercalating dye or a composition containing a fluorescent dye, a
quencher dye and a probe such that the fluorescent dye and quencher
dye are linked to the probe and such that the probe has a sequence
of between approximately 15 contiguous bases and approximately 45
contiguous bases of SEQ ID NO: 16 or the reverse complement
thereof. It is a further object of this invention that the item be
a food matrix, a container, equipment, or a medical device.
[0043] It is an object of this invention to have a quality control
method for determining the amount of inactivation of a hazardous
material in or on an item, the method having the steps of
optionally processing a sample containing either intrinsic DNA or
extrinsic DNA according to the pre-determined inactivation
protocol; optionally isolating the processed intrinsic DNA or
extrinsic DNA; exposing the intrinsic DNA or extrinsic DNA to a
first polynucleotide, a second polynucleotide, and optionally a
fluorescent composition; amplifying the intrinsic DNA or extrinsic
DNA using a DNA amplification method to produce an amplicon;
determining the threshold cycle of the intrinsic DNA or extrinsic
DNA; and comparing the obtained threshold cycle value to a known
threshold cycle value for intrinsic DNA or extrinsic DNA that is
equivalent to the desired amount of inactivation of the hazardous
biological material. It is a further object of this invention that
the DNA amplification method use DNA polymerase to generate an
amplicon that has the sequence of SEQ ID NO: 17; that the first
polynucleotide has the sequence of SEQ ID NO: 7, and the second
polynucleotide has the sequence of SEQ ID NO: 8. It is a further
object of this invention that the fluorescent composition be an
intercalating dye or a composition containing a fluorescent dye, a
quencher dye and a probe such that the fluorescent dye and quencher
dye are linked to the probe and such that the probe has a sequence
of between approximately 15 contiguous bases and approximately 45
contiguous bases of SEQ ID NO: 17 or the reverse complement
thereof. It is a further object of this invention that the item be
a food matrix, a container, equipment, or a medical device.
[0044] It is an object of this invention to have a quality control
method for determining the amount of inactivation of a hazardous
material in or on an item, the method having the steps of
optionally processing a sample containing either intrinsic DNA or
extrinsic DNA according to the pre-determined inactivation
protocol; optionally isolating the processed intrinsic DNA or
extrinsic DNA; running the intrinsic DNA or extrinsic DNA on a
electrophoretic gel; determining the amount of DNA fragmentation
for DNA sizes ranging from approximately 35 bp to approximately
10,380 bp; determining the DNA integrity number; and comparing the
obtained DNA integrity number to a known DNA integrity number that
is equivalent to the desired amount of inactivation of the
hazardous biological material. It is a further object of this
invention that the intrinsic DNA or extrinsic DNA be exposed to a
fluorescent composition prior to or after running the intrinsic DNA
or extrinsic DNA on the electrophoretic gel; such that the
fluorescent composition is a fluorescent dye for imaging DNA. It is
a further object of this invention that the item be a food matrix,
a container, equipment, or a medical device.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 illustrates the standard curve for qPCR for mtDNA
copy number. The standard curve is also used to determine
amplification efficiency of the protocol (106%), the limit of
detection (10 copies) and the linear dynamic range (7 orders of
magnitude).
[0046] FIG. 2 illustrates the effect of hot oil bath (at
121.degree. C.) on sweet potato puree mtDNA fragmentation (increase
in Ct value) over time.
[0047] FIG. 3 illustrates the high correlation between surrogate GS
spore destruction and increase in Ct value of sweet potato puree
mtDNA over time for the hot oil assay (121.degree. C.).
[0048] FIG. 4 illustrates the high correlation (R.sup.2=0.87)
between surrogate G. stearothermophilus spore destruction and
increase in Ct value over time when the sweet potato puree and G.
stearothermophilus spore are autoclaved (121.degree. C.).
[0049] FIG. 5 illustrates the change in Ct value indicating mtDNA
fragmentation of cucumbers during pasteurization (75.degree. C. for
15 minutes), fermentation and storage compared to Ct values of
cucumbers in autoclave treatment (121.degree. C.).
[0050] FIG. 6 illustrates the mtDNA degradation (increase in Ct
value) of peanuts dry roasted (167.degree. C.) at the indicated
time intervals.
[0051] FIG. 7 illustrates the correlation between mtDNA degradation
(increase in Ct value) in dry roast treatment of peanuts
(167.degree. C.) and survival of a Salmonella surrogate, E. faecium
(log.sub.10 CFU/ml).
[0052] FIG. 8 compares mtDNA degradation (increase in Ct value) of
dry roasted peanuts (167.degree. C.) with the Hunter L color, a
roasting quality indicator.
[0053] FIG. 9 shows the sequence of I. batatas atp1, the location
of the primers used, and the sequences of the amplicons
generated.
[0054] FIG. 10 shows the extrinsic DNA thermometer at three
different pH levels from 0-24 minutes at 96.degree. C.
[0055] FIG. 11 shows the extrinsic DNA thermometer at three
different pH levels from 0-5 minutes at 96.degree. C.
[0056] FIG. 12 illustrates the fragmentation and reduction of total
DNA integrity caused by autoclave treatment by comparing size and
concentrations of cucumber DNA globally.
[0057] FIG. 13 is a flow chart illustrating the invention described
herein.
[0058] FIG. 14A illustrates the correlation between Ct values of
sweet potato mtDNA and F values of inactivation of G.
stearothermophilus in canned sweet potato using substandard 12-D
protocol (protocol 00). FIG. 14B illustrates the correlation
between Ct values of sweet potato mtDNA and F values of
inactivation of G. stearothermophilus in canned sweet potato using
standard 12-D protocol (protocol 01).
[0059] FIG. 15 illustrates the slopes used to calculate D values
for timed oil bath treatments for G. stearothermophilus spores at
116.degree. C., 121.degree. C., 123.degree. C., and 126.degree. C.
for the indicated times.
[0060] FIG. 16 illustrates the slope used to calculate the z-value
for timed oil bath treatments for G. stearothermophilus spores at
116.degree. C., 121.degree. C., 123.degree. C., and 126.degree. C.
for the indicated times.
[0061] FIG. 17 illustrates sweet potato puree mtDNA fragmentation
and D values in hot oil bath at 116.degree. C., 121.degree. C.,
123.degree. C., and 126.degree. C. for the indicated times.
[0062] FIG. 18 illustrates the slope used to calculate z-value for
sweet potato puree mtDNA fragmentation in hot oil bath at
116.degree. C., 121.degree. C., 123.degree. C., and 126.degree. C.
for the indicated times.
[0063] FIG. 19 illustrates the linear relationship between sweet
potato puree mtDNA fragmentation in hot oil bath at 121.degree. C.
versus G. stearothermophilus spore counts in hot oil bath at
121.degree. C.
DETAILED DESCRIPTION OF THE INVENTION
[0064] This invention determines inactivation of biological
material, primarily hazardous biological material such as in or on
food or an item by quantifying the amount of degradation in a food
matrix's intrinsic DNA or in extrinsic DNA added to food or an
item. The amount of DNA degradation is correlated to inactivation
of hazardous biological material such as disease causing pathogens,
and/or widely accepted surrogates for bacterial pathogen's spores.
More specifically, the quantified DNA degradation is a
time/temperature indicator which is a surrogate for inactivation of
hazardous biological material. When DNA degradation is at or after
specified values, the inactivation of hazardous biological material
in or on a food or item is assured. Because this process uses
quantitative PCR, some commercially available reagents, and/or
apparatuses, the assay is inexpensive and simple to use. Further,
it provides an answer regarding inactivation of the hazardous
biological material of interest significantly faster than prior art
methods of culturing for biological material, and more accurately
over prior art methods of using PCR to detect the presence of a
pathogen's DNA. One method for determining DNA degradation is by
performing quantitative PCR (qPCR) on specific genes contained in
mitochondrial DNA (mtDNA) of the food matrix. A second method is to
examine DNA integrity of a food matrix. A third method is to add
mtDNA (extrinsic DNA) as an extrinsic source in a recoverable
container. All three methods are used as time/temperature
integrators. These assays serve as presumptive verification of
processing efficacy. Using the tool box approach, these assays
provide rapid results that a processor can use to evaluate the
reduction in amount of viable hazardous biological material in or
on an item (food, device, etc.) prior to shipping or using the
item. These assays can also be used to evaluate deviations in
normal processing of items and to evaluate the efficacy of new
processing methods under consideration. These assays use
mitochondrial DNA (mtDNA) fragmentation or DNA integrity to
determine the degradation of DNA over the range of time,
temperature, and other inactivation process' conditions such as,
but not limited to, high pressure or ultraviolet light. MtDNA is a
surrogate for the inactivation of hazardous biological material in
or on the item being treated (food, device, etc.). DNA integrity,
whether measuring intrinsic DNA integrity or extrinsic DNA
integrity, is a surrogate for the inactivation of hazardous
biological material in or on the item being treated (food, device,
etc.). These methods can be used to determine if an appropriate
reduction in viable hazardous biological material (bacteria,
bacterial spores, viruses, fungi, parasites, cancer cells, etc.)
occurs after the processing of the food matrix or device. An
appropriate reduction in viable hazardous biological material could
be a 5 log reduction of a particular pathogen or any other amount
desired or required by regulations governing food safety or medical
device safety. These methods can also be used as quality control
for a particular inactivation process. These methods can also be
used to assay the survival of hazardous biological material after
processing.
[0065] Polymerase chain reaction (or PCR) is a technique to copy
(or amplify) a small quantity of DNA. Using PCR, one can generate
greater than 100,000,000 or even one billion copies of the desired
DNA within a couple of hours. To amplify a segment of DNA using
PCR, the sample is first heated so the DNA denatures (separates
into two pieces of single-stranded DNA). Next, the sample is cooled
to a temperature lower than the melting (or denaturing) temperature
of the DNA but still substantially higher than room temperature. At
this temperature primers bind specific, pre-determined sites. Taq
polymerase (a DNA polymerase active at high temperatures)
synthesizes two new strands of DNA, using the original strands as
templates and primers that bind to the original strands of DNA as
initiation points for DNA extension by Taq polymerase. Of course,
sufficient amounts of free nucleic acids are added to the reaction
mixture for use by Taq polymerase to generate the new DNA. This
process results in the duplication of a section of the original DNA
based on the binding location of the primers. Each new DNA segment
(also referred to as an amplicon) contains one old and one new
strand of DNA. The sample is heated again to denature the DNA again
and allowed to cool so that Taq polymerase can generate new
amplicons. The cycle of denaturing and synthesizing new DNA is
repeated as many as thirty or forty times, leading to more than one
billion amplicons. A thermocycler is a programmable apparatus that
automates the temperature changes utilized in PCR, controlling DNA
denaturation and synthesis. PCR can be completed in a few hours.
Some early U.S. patents on PCR include U.S. Pat. Nos. 4,683,195;
4,683,202; and 4,800,159.
[0066] Quantitative PCR (qPCR), also called real-time PCR, involves
monitoring DNA amplification during each cycle of PCR using
fluorescent label. When the DNA is in the log linear phase of
amplification, the amount of fluorescence increases above the
background. The point at which the fluorescence becomes measurable
is called the Threshold Cycle (Ct) or crossing point. By using
multiple dilutions of a known amount of standard DNA, a standard
curve can be generated of log concentration against Ct (see FIG.
1). The amount of DNA or cDNA in an unknown sample can then be
calculated from its Ct value. Two types of fluorescent labels are
used with qPCR. One label is an intercalating dye that incorporates
into double-stranded DNA, such as, but not limited to, SYBR.RTM.
Green. An intercalating dye is appropriate when a single amplicon
is being studied. The second type of fluorescent label is a probe
that binds specifically to the target DNA, such as TaqMan.RTM.
probes, Molecular Beacons.TM., or Scorpion primers. The probe is
labeled with a fluorescent dye (such as, but not limited to Texas
Red.RTM., FAM, TET, HEX, TAMRA, JOE, and ROX) and a quencher (such
as, but not limited to Dabcyl and Dabsyl). The oligonucleotide
itself has no significant fluorescence, but fluoresces either when
annealed to the template (as in Molecular Beacons.TM.) or when the
dye is clipped from the oligonucleotide during extension (as in
TaqMan.RTM. probes). Multiplex PCR is possible by using dyes with
different fluorescent emissions for each probe. The fluorescent
compositions described herein are simply examples of compositions
for imaging, identifying, and/or quantifying DNA. Instead of the
fluorescent compositions described herein, one can label DNA with
compositions that are known in the art (some of which are described
infra) or that are developed in the future. These labels can be
used to image, identify, and/or quantify DNA using similar methods
as described herein. The fluorescent compositions are simply one
well-known and well-accepted compositions for imaging, identifying,
and/or quantifying DNA for the methods described herein.
[0067] The term "nucleic acid" as used herein, refers to a polymer
of ribonucleotides or deoxyribonucleotides. Typically, "nucleic
acid" polymers occur in either single- or double-stranded form, but
are also known to form structures comprising three or more strands.
The term "nucleic acid" includes naturally occurring nucleic acid
polymers as well as nucleic acids comprising known nucleotide
analogs or modified backbone residues or linkages, which are
synthetic, naturally occurring, and non-naturally occurring, which
have similar binding properties as the reference nucleic acid, and
which are metabolized in a manner similar to the reference
nucleotides. Exemplary analogs include, without limitation,
phosphorothioates, phosphoramidates, methyl phosphonates,
chiral-methyl phosphonates, 2-O-methyl ribonucleotides,
peptide-nucleic acids (PNAs) and locked nucleic acids (RNA monomers
with a modified backbone). "DNA", "RNA", "polynucleotides",
"polynucleotide sequence", "oligonucleotide", "nucleotide",
"nucleic acid", "nucleic acid molecule", "nucleic acid sequence",
"nucleic acid fragment", and "isolated nucleic acid fragment" are
used interchangeably herein.
[0068] The term "label" as used herein, refers to a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. Exemplary labels include
.sup.32P, fluorescent dyes, electron-dense reagents, enzymes (e.g.,
as commonly used in an ELISA), biotin, digoxigenin, or haptens and
proteins for which antisera or monoclonal antibodies are
available.
[0069] As used herein a nucleic acid "probe", oligonucleotide
"probe", or simply a "probe" refers to a nucleic acid capable of
binding to a target nucleic acid of complementary sequence through
one or more types of chemical bonds, usually through complementary
base pairing, usually through hydrogen bond formation. As used
herein, a probe may include natural (i.e., A, G, C, or T) or
modified bases (e.g., 7-deazaguanosine, inosine, etc.). In
addition, the bases in a probe may be joined by a linkage other
than a phosphodiester bond, so long as it does not interfere with
hybridization. Thus, for example, probes may be peptide nucleic
acids in which the constituent bases are joined by peptide bonds
rather than phosphodiester linkages. It will be understood by one
of skill in the art that probes may bind target sequences lacking
complete complementarity with the probe sequence depending upon the
stringency of the hybridization conditions. In one exemplary
embodiment, probes are directly labeled as with isotopes,
chromophores, lumiphores, chromogens etc. In other exemplary
embodiments probes are indirectly labeled e.g., with biotin to
which a streptavidin complex may later bind. By assaying for the
presence or absence of the probe, one can detect the presence or
absence of the select sequence or subsequence.
[0070] Thus, the term "probe" as used herein refers to a probe that
is bound, either covalently, through a linker or a chemical bond,
or noncovalently, through ionic, van der Waals, electrostatic, or
hydrogen bonds to a label such that the presence of the probe may
be detected by detecting the presence of the label bound to the
probe.
[0071] The term "primer" as used herein, refers to short nucleic
acids, typically a DNA oligonucleotide of at least about 15
nucleotides in length. In an exemplary embodiment, primers are
annealed to a complementary target DNA strand by nucleic acid
hybridization to form a hybrid between the primer and the target
DNA strand. Annealed primers are then extended along the target DNA
strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification of a nucleic acid sequence, e.g., by the polymerase
chain reaction (PCR) or other nucleic-acid amplification methods
known in the art.
[0072] PCR primer pairs are typically derived from a known
sequence, for example, by using computer programs intended for that
purpose such as Primer (Version 0.5 .COPYRGT.1991, Whitehead
Institute for Biomedical Research, Cambridge, Mass.). One of
ordinary skill in the art will appreciate that the specificity of a
particular probe or primer increases with its length. Thus, for
example, a primer comprising 20 consecutive nucleotides of a
promoter complex sequence will anneal to a related target sequence
with a higher specificity than a corresponding primer of only 15
nucleotides. Thus, in an exemplary embodiment, greater specificity
of a nucleic acid primer or probe is attained with probes and
primers selected to comprise 20, 25, 30, 35, 40, 50 or more
consecutive nucleotides of a selected sequence. When discussing
primer pairs, one provides the sequence of both the forward and
reverse primers in the 5' to 3' direction and is the sequence of
the positive strand of DNA. However, when performing PCR, the
sequence of the reverse primer actually used is the reverse
complement of the sequence of the reverse primer. Thus for 174R
primer, the actual sequence used is in SEQ ID NO: 10; for 108R
primer, the actual sequence uses is in SEQ ID NO: 11; for 81R
primer, the actual sequence used is in SEQ ID NO: 12; and for 141R
primer, the actual sequence used is in SEQ ID NO: 13.
[0073] Nucleic acid probes and primers are readily prepared based
on the nucleic acid sequences disclosed herein. Methods for
preparing and using probes and primers and for labeling and
guidance in the choice of labels appropriate for various purposes
are discussed, e.g., in Sambrook et al., Molecular Cloning, A
Laboratory Manual 2nd ed. 1989, Cold Spring Harbor Laboratory; and
Current Protocols in Molecular Biology, Ausubel et al., eds., 1994,
John Wiley & Sons).
[0074] The term "capable of hybridizing under stringent
hybridization conditions" as used herein, refers to annealing a
first nucleic acid to a second nucleic acid under stringent
hybridization conditions (defined below). In an exemplary
embodiment, the first nucleic acid is a test sample, and the second
nucleic acid is the sense or antisense strand of a nucleic acid of
interest. Hybridization of the first and second nucleic acids is
conducted under standard stringent conditions, e.g., high
temperature and/or low salt content, which tend to disfavor
hybridization of dissimilar nucleotide sequences.
[0075] The terms "identical" or percent "identity", in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (e.g., 85% identity, 90% identity, 99%, or 100% identity),
when compared and aligned for maximum correspondence over a
comparison window, or designated region as measured using a
sequence comparison algorithm or by manual alignment and visual
inspection.
[0076] The phrase "substantially identical", in the context of two
nucleic acids or polypeptides, refers to two or more sequences or
subsequences that have at least about 85%, identity, at least about
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100% nucleotide or amino acid residue identity, when
compared and aligned for maximum correspondence, as measured using
a sequence comparison algorithm or by visual inspection. In an
exemplary embodiment, the substantial identity exists over a region
of the sequences that is at least about 50 residues in length. In
another exemplary embodiment, the substantial identity exists over
a region of the sequences that is at least about 100 residues in
length. In still another exemplary embodiment, the substantial
identity exists over a region of the sequences that is at least
about 150 residues or more, in length. In one exemplary embodiment,
the sequences are substantially identical over the entire length of
nucleic acid or protein sequence.
[0077] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0078] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from about 20 to about 600, usually
about 50 to about 200, more usually about 100 to about 150 in which
a sequence may be compared to a reference sequence of the same
number of contiguous positions after the two sequences are
optimally aligned. Methods of alignment of sequences for comparison
are well-known in the art. Optimal alignment of sequences for
comparison can be conducted, e.g., by the local homology algorithm
of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the
homology alignment algorithm of Needleman & Wunsch, J. Mol.
Biol. 48:443 (1970), by the search for similarity method of Pearson
& Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
manual alignment and visual inspection (see, e.g., Current
Protocols in Molecular Biology (Ausubel et al., eds. 1995
supplement)).
[0079] An exemplary algorithm for sequence comparison is PILEUP.
PILEUP creates a multiple sequence alignment from a group of
related sequences using progressive, pairwise alignments to show
relationship and percent sequence identity. It also plots a tree or
dendogram showing the clustering relationships used to create the
alignment. PILEUP uses a simplification of the progressive
alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360
(1987). The method used is similar to the method described by
Higgins & Sharp, CABIOS 5:151-153 (1989). The program can align
up to 300 sequences, each of a maximum length of 5,000 nucleotides
or amino acids. The multiple alignment procedure begins with the
pairwise alignment of the two most similar sequences, producing a
cluster of two aligned sequences. This cluster is then aligned to
the next most related sequence or cluster of aligned sequences. Two
clusters of sequences are aligned by a simple extension of the
pairwise alignment of two individual sequences. The final alignment
is achieved by a series of progressive, pairwise alignments. The
program is run by designating specific sequences and their amino
acid or nucleotide coordinates for regions of sequence comparison
and by designating the program parameters. Using PILEUP, a
reference sequence is compared to other test sequences to determine
the percent sequence identity relationship using the following
parameters: default gap weight (3.00), default gap length weight
(0.10), and weighted end gaps. PILEUP can be obtained from the GCG
sequence analysis software package, e.g., version 7.0 (Devereaux et
al., 1984. Nuc. Acids Res. 12:387-395.
[0080] The phrase "selectively hybridizes to" or "specifically
hybridizes to" refers to the binding, duplexing, or hybridizing of
a molecule only to a particular nucleotide sequence under stringent
hybridization conditions when that sequence is present in a complex
mixture (e.g., total cellular or library DNA or RNA). In general,
two nucleic acid sequences are said to be "substantially identical"
when the two molecules or their complements selectively or
specifically hybridize to each other under stringent
conditions.
[0081] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology-Hybridization with
Nucleic Probes, "Overview of principles of hybridization and the
strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions will be those in which the salt concentration
is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. For high stringency hybridization, a positive signal
is at least two times background, preferably 10 times background
hybridization. Exemplary high stringency or stringent hybridization
conditions include: 50% formamide, 5.times.SSC and 1% SDS incubated
at 42.degree. C. or 5.times.SSC and 1% SDS incubated at 65.degree.
C., with a wash in 0.2.times.SSC and 0.1% SDS at 65.degree. C.
However, other high stringency hybridization conditions known in
the art can be used.
[0082] Intrinsic DNA means nucleic acids (DNA or RNA) that are
present naturally in a sample, including nucleic acids from
hazardous biological material present in the sample. For example,
intrinsic DNA for a food matrix is the DNA and RNA in the cells of
the plant or animal ingredients in the food matrix. Intrinsic DNA
includes mitochondrial DNA (mtDNA), but is not limited to
mtDNA.
[0083] Extrinsic DNA means any nucleic acids (DNA or RNA) added to
a system for inactivating hazardous biological material. Usually,
the extrinsic DNA is double-stranded polynucleotide which can range
from between approximately 80 bp to approximately 250 bp long.
Extrinsic DNA does not need to be from any particular gene or
non-coding region. However, one must have primers, and optionally a
labeled probe, which can be used while performing qPCR on the
extrinsic DNA. The extrinsic DNA is used when one does not have or
does not want to perform qPCR on intrinsic DNA to determine the
efficacy of an inactivation process. For example, when determining
if a certain process is sufficient robust to destroy hazardous
biological material present on an item (e.g., a jar or medical
device), one subjects a test sample of extrinsic DNA in a low pH
solution or high pH solution to the inactivation process. An item,
optionally, may or may not be subjected to the inactivation process
with the extrinsic DNA. Then one performs the qPCR assays described
herein on the extrinsic DNA to determine the amount of nucleic acid
degradation and correlate that degradation to the
destruction/inactivation of hazardous biological material on/in the
item.
[0084] DNA degradation, RNA degradation, nucleic acid degradation
are the breaking of the chemical bonds with the nucleic acids so
that the organism containing those degraded nucleic acids is not
viable. Nucleic acid degradation can occur when nucleic acids are
exposed to certain conditions, such as, elevated temperature,
acidity, alkalinity, salt, preservatives, UV light, high pressure,
to name a few.
[0085] DNA integrity is the wholeness or completeness of a cell's
genomic and organelle-based DNA. After heat treatments, such as
autoclaving, or other types of conditions that degrades a cell's
nucleic acids, the cell's DNA integrity is reduced. The amount
fragmentation (reduction in integrity) can be measured globally and
is used as a time/temperature integrator. One can measure the
integrity of either intrinsic DNA or extrinsic DNA to determine the
amount of inactivation of the hazardous biological material.
[0086] Hazardous biological material is any biological material
that could harm an animal if the animal ingests the biological
material or, if placed on or inside the animal. Hazardous
biological material include, but are not limited to, toxins,
viruses, parasites, fungi, bacteria, spores (bacterial, fungal or
parasitical), other types of pathogens, and cancer cells.
[0087] Mitochondria are "power-house" organelles found in multiple
numbers in all cells of eukaryotes, and each mitochondrion
possesses its own genome in multiple copies. Mitochondrial DNA
contains polynucleotide sequences that are species-specific or
family-specific. In addition, the mitochondrial genome also
contains polynucleotide sequences that are highly conserved in many
eukaryotic plants or animals. These properties make mtDNA sequences
excellent targets for amplification in terms of specificity,
sensitivity and robustness in addition to the fact that multiple
copies per cell (>1,000) exist. Therefore, the advantages of
targeting mtDNA with qPCR are substantial.
[0088] All mtDNA qPCR assays for this invention meet MIQE
Guidelines (Bustin, et al., Clinical Chem. 55:611-622 (2009)) or
Minimum Information for publication of Quantitative real-time PCR
Experiments which features a quality control checklist on sample
processing, nucleic acid extraction, target amplicon
specifications, reaction optimization, specificity of reaction,
internal amplification controls (IAC), calibration curves with
calculated PCR efficiency, linear dynamic range and data analysis
including repeatability and statistical methods. This approach to
monitoring food safety and cleaning of objects, in general,
represents a paradigm shift by using qPCR to quantify the
disappearance of mtDNA over time caused by thermal or microwave
processing and correlating the degradation rates closely to the
thermal death time (D) of spore-forming bacteria. This method also
involves correlating the thermal death time (D) of the bacteria, as
determined by culture methods, to the destruction of mtDNA of the
thermally- or microwave-processed foodstuff. Assessing mtDNA
decomposition over time can also be used to assess shelf life of a
food and the level of inactivation of biological material on or in
medical equipment, empty or filled food containers, or other
items.
[0089] The mtDNA qPCR assays of this invention are adjusted to
highly correlate to D values (time required for 1 log reduction of
pathogens at a certain temperature) according to the length of the
amplicon, secondary DNA structure, annealing temperature, use of
locked nucleic acids and primer/probe efficiencies.
[0090] As used herein, the term "about" and "approximately" refers
to a quantity, level, value or amount that varies by as much as
30%, preferably by as much as 20%, and more preferably by as much
as 10% to a reference quantity, level, value or amount. All cited
prior art documents are incorporated by reference.
Example 1 Primers for Plant mtDNA Selection and Generation of
Standardized Curves
[0091] Primers are designed which use consensus sequences to target
a wide variety of plant foods. Four sets of qPCR primers shown in
Table 1 are designed with Primer Quest software
(http://scitools.idtdna.com/Primerquest/) from Integrated DNA
Technologies, Inc. (IDT) (Coralville, Iowa) targeting the Ipomoea
batatas F1-ATPase alpha subunit (atp1) mitochondrial gene (GenBank
AY596672.1). Amplicon length for each primer set is indicated by
the number in the primer sets' name, 81 bp, 108 bp, 141 bp, and 174
bp. FIG. 9 shows the sequence of I. batatas atp1 (SEQ ID NO: 9),
the location of the primers used, and the sequences of the
amplicons generated. All primer sets match the Ipomoea batatas atp1
gene with 100% identity, and between approximately 95% and 100%
identity for a wide range of common fruits, vegetables, and nuts
(see Table 2 infra) when subjected to NCBI BLAST searches. Primers
are purchased from IDT (Coralville, Iowa). Oligonucleotide primers
are reconstituted in TE buffer (pH 7.5) and stored at -20.degree.
C. prior to use.
TABLE-US-00001 TABLE 1 Primer Start name Position Sequence 174F 698
5'-TTTCCGCGATAATGGAATGCACGC-3' (forward) (SEQ ID NO: 1) 174R 871
5'-TCCGATCGTTTAGCCGCTCTTTCT-3' (reverse) (SEQ ID NO: 2) 108F 1133
5'-CGCCTTTGCTCAATTTGGCTCAGA-3' (forward) (SEQ ID NO: 3) 108R 1240
5'-GGCAGTGGTGCATATTGTGGTTGT-3' (reverse) (SEQ ID NO: 4) 81F 1133
5'-CGCCTTTGCTCAATTTGGCTCAGA-3' (forward) (SEQ ID NO: 5) 81R 1213
5'-AGTACTTCTGTCAGCCTTGCACCT-3' (reverse) (SEQ ID NO: 6) 141F 87
5'-GAATTTGCCAGCGGTGTGAAAGGA-3' (forward) (SEQ ID NO: 7) 141R 227
5'-TCCCGCAGGAACATCCACAATAGA-3' (reverse) (SEQ ID NO: 8)
[0092] A test comparing autoclaved (steamed at 121.degree. C. for
20 minutes) versus non-autoclaved sweet potato puree DNA is run
with each primer set. Puree is generated prior to treatment by
grinding the sweet potato and water in Waring blender until puree
is formed. Sweet potato DNA is then isolated after treatment using
MO BIO PowerSoil.RTM. DNA isolation kit (Carlsbad, Calif.)
according to manufacturer's directions. qPCR is performed in 25
.mu.l total volume with 2.times.IQ SYBR Green supermix (SYBR Green
I dye, 50 U/ml iTaq DNA polymerase, 0.4 mM each of dATP, dCTP, dGTP
and dTTP, 6 mM MgCl.sub.2, 40 mM Tris-HCl, pH 8.4, 100 mM KCl, and
20 nM fluorescein (BioRad, Hercules, Calif.)), 300 nM final
concentration each for forward and reverse primers listed in Table
1, sweet potato total DNA (5-10 ng/reaction), and qPCR water
(Ambion, Austin, Tex.) to final volume. qPCR amplifications are
performed in a MyiQ (BioRad, Hercules, Calif.) thermal cycler with
the following conditions: 95.0.degree. C. for 3 minutes; 40 cycles
of 95.0.degree. C. for 30 seconds, 60.0.degree. C. for 30 seconds,
72.0.degree. C. for 30 seconds; with FAM channel optics "on" during
annealing stage. Negative control is performed without any DNA ("no
template control" or "NTC"), substituting same volume of molecular
grade water for DNA. Positive and normalizing controls are used for
all assays. For a sample to be considered positive, its threshold
cycle (Ct) value must be less than all negative control reactions,
and the corresponding amplification curve has to exhibit the three
distinct phases of real-time PCR: lag, linear and plateau.
[0093] All four primer sets produced amplicons of expected lengths
(81 bp, 108 bp, 141 bp, and 174 bp) when run in 1% agarose gels.
All amplicons are isolated and sequenced, and each amplicon
exhibits 100% identity to the Ipomoea batatas atp1 mitochondrial
gene under NCBI BLAST analysis as well as atp1 in many other
plants. The sequence of the 174 bp amplicon generated by 174F and
174R primers is SEQ ID NO: 14. The sequence of the 108 bp amplicon
generated by 108F and 108R primers is SEQ ID NO: 15. The sequence
of the 81 bp amplicon generated by 81F and 81R primers is SEQ ID
NO: 16. The sequence of the 141F and 141R primers is SEQ ID NO: 17.
Of the four primer pairs assayed, primer set 174 exhibits the
greatest difference in Ct values between the two samples,
autoclaved sweet potato and unautoclaved control sweet potato (9 Ct
difference versus 8, 5 & 5 for amplicon lengths of 141, 108,
and 81 base pairs, respectively). This result is understandable as
longer amplicons are statistically more likely to experience
degradation than shorter ones. As such, of these four sets of
primer pairs, 174F and 174R, provides better results compared to
the other three primer sets and, thus, is used in the other
examples described infra. However, this invention is not limited to
the above listed primers. Any other primer set that has high
identity to atp1 and which yields an amplicon of between
approximately 80 bp to approximately 250 bp can be used for fruits,
vegetable, and nuts. In another embodiment, the primer set for
assaying fruits, vegetable and nuts generates an amplicon within
atp1 ranging from approximately 100 bp to approximately 200 bp. In
yet another embodiment, the primer set for assaying fruits,
vegetable and nuts generates an amplicon within atp1 ranging from
approximately 125 bp to approximately 175 bp. It is also helpful
that the primer set used for any food matrix or medical device
generates a Ct difference of approximately 9 or greater between the
negative control (unprocessed sample) and processed sample.
[0094] Standard curves are generated using double-stranded,
sequence-verified oligonucleotides of the I. batatas atp1, having
the same sequence as the amplicon generated by the 174F and 174R
primers (SEQ ID NO: 14) (gBlocks.RTM. gene fragments purchased from
IDT (Coralville, Iowa)). Ten-fold serially dilutions of atp1
amplicon copies (10.sup.7 to 10.sup.1) are performed, qPCR is
performed using the protocol above with 174F and 174R primers. PCR
amplification efficiency (E) is determined using the slope of the
standard curve: E=(10.sup.-1/slope)-1. Data analysis of the qPCR
standard curve is performed using goodness-of-fit linear regression
correlation coefficient (R.sup.2). The slope value is used to
assess the robustness of the assay using the efficiency value
above. The amplification efficiency is calculated as 106%,
R.sup.2=0.9884 (see FIG. 1).
[0095] Bustin, et al. (2009) published the MIQE guidelines (Minimum
Information for publication of Quantitative real-time PCR
Experiments) to facilitate assessment and evaluation of new,
clinical qPCR assays. The guidelines include a checklist for
authors, reviewers and editors to help them ensure the integrity of
scientific literature and promote consistency between laboratories
(Bustin, et al. (2009)). According to the guidelines, essential
information includes experimental design, sample description and
processing, nucleic acid extraction, qPCR target information, qPCR
oligonucleotides, qPCR protocol, qPCR validation, and data
analysis. The present invention meets all essential information
requirements.
Example 2 Hot Oil Bath as Substitute for Industrial Microwave
System
[0096] A hot oil bath (EW-111, Neslab Instruments, Newington, N.H.)
filled with 8 L white mineral oil (Therminol XP, Solutia, Inc, St.
Louis, Mo.) is used to maintain a temperature of 121.1.degree. C.
for substances placed in a thermal death tube (TDT). A thermal
death tube allows one to replicate the conditions of an industrial
microwave system yet still obtain samples of the substance at
various time points. 100 .mu.l sweet potato (SP) puree in 1:4
dilution with 0.9% saline or 100 .mu.l of Geobacillus
stearothermophilus (GS) spores (ca. 10.sup.8 CFU/ml) are inserted
into separate TDTs and are sealed according to instructions.
Samples are heated for 0, 0.5, 1, 2, 4, 8, 16 and 20 minutes at
121.1.degree. C., taking into account the 30 seconds come up time
(CUT). Three repetitions are run per time point; three TDTs are
placed in a metal tea strainer to facilitate removal of samples
from hot oil. Strainers containing TDTs are taken out of oil bath
and immediately placed in an ice slurry for 30 seconds to quickly
cool them. Strainers are stored at room temperature until ready for
DNA extraction or culture plating. Total amount of sweet potato
puree recovered from hot oil bath treatment ranges between
approximately 50 .mu.l to approximately 75 .mu.l from an initial
sample of 100 .mu.l (see FIG. 2).
[0097] GS spores are serially diluted with 0.9% saline solution and
plated with a spiral plater (Spiral Biotech, Inc., Norwood, Mass.)
on BHI agar (Becton Dickinson, Sparks, Md.). After 24 hours
incubation at 55.degree. C., colonies are enumerated with an
automated spiral plate counter (Q-count, Spiral Biotech Inc.
Norwood, Mass.). The lower detection limit is 10.sup.2 CFU/ml.
Another G. stearothermophilus indicator system, the Prospore
ampoule (Mesa Laboratories, Inc, Lakewood, Colo.) are incubated at
55.degree. C. for 48 hours, and then checked for color change as
directed.
[0098] Treated sweet potato puree is removed from TDT and placed
directly into a MO BIO bead beater tube (Carlsbad, Calif.). The MO
BIO PowerSoil.RTM. DNA isolation kit (Carlsbad, Calif.) is used
according to manufacturer's recommendations to extract sweet potato
DNA from the treated puree. Total DNA samples are analyzed by
spectrophotometer (Nanodrop, Wilmington, Del.) for quantity and
quality. For qPCR, DNA is normalized by concentration: between 5-10
ng/.mu.l per reaction.
[0099] qPCR is performed in 25 .mu.l total volume with 2.times.IQ
SYBR Green supermix (SYBR Green I dye, 50 U/ml iTaq DNA polymerase,
0.4 mM each of dATP, dCTP, dGTP and dTTP, 6 mM MgCl.sub.2, 40 mM
Tris-HCl, pH 8.4, 100 mM KCl, and 20 nM fluorescein (BioRad,
Hercules, Calif.)), 300 nM final concentration each for 174F and
174R primers, sweet potato DNA (5-10 ng/reaction) and qPCR water
(Ambion, Austin, Tex.) to final volume. qPCR amplifications are
performed in a MyiQ (BioRad, Hercules, Calif.) thermal cycler with
the following conditions: 95.0.degree. C. for 3 minutes; 40 cycles
of 95.0.degree. C. for 30 seconds, 60.0.degree. C. for 30 seconds,
72.0.degree. C. for 30 seconds; with FAM channel optics "on" during
annealing stage. No template control (NTC) and positive controls
are used for all assays. A positive control is used to normalize
data between assays. For a sample to be considered positive, its
threshold cycle (Ct) value must be less than all negative control
reactions, and the corresponding amplification curve has to exhibit
the three distinct phases of real-time PCR: lag, linear and
plateau.
[0100] FIG. 2 illustrates the effect of hot oil bath (at
121.degree. C.) on sweet potato puree mtDNA fragmentation (increase
in Ct value) over time. In the hot oil bath, Ct value increased
from approximately 24 at time zero to between approximately 30 and
approximately 32, a 6-8 unit increase. This hot oil bath assay
exhibits a high correlation (R.sup.2=0.97) between surrogate GS
spore destruction and increase in Ct value over time (see FIG.
3).
Example 3 Autoclave Degradation of Sweet Potato mtDNA and G.
stearothermophilus Spores
[0101] To determine if mtDNA also fragments during the high heat
and pressure of autoclaving, and assess the level of degradation of
the DNA, sweet potato puree and G. stearothermophilus spores are
assayed in a laboratory autoclave (Amsco Eagle SG-3021 Scientific
Gravity Sterilizer, Steris Corp., Mentor, Ohio). The autoclave is
programmed to run liquid sterilizing times of 2, 4, 8 and 20
minutes at 121.degree. C. Come up times (initial CUT=5 minutes, all
others=1 minute) and come down times (Exhaust=11:53 to 13:25
minutes) are similar for all runs. In a plastic micro-centrifuge
holder three samples are included per run: 300 mg sweet potato
puree (prepared as described supra), 250 .mu.l G.
stearothermophilus spores (log 8 CFU/ml) (prepared as described
supra) both samples in 1.5 ml micro-centrifuge tubes containing a
small hole in the top to vent water vapor, and one commercial G.
stearothermophilus vial (Prospore, Mesa Laboratories, Inc,
Lakewood, Colo.). Vent holes are then covered with parafilm. After
autoclave treatment, sample tubes are placed on ice until they cool
to room temperature. DNA is extracted from each sample using the
PowerSoil.RTM. DNA isolation kit (MO BIO, Carlsbad, Calif.) using
manufacturer's recommendations. DNA is quantified and qualified via
spectrophotometry (Nanodrop, Wilmington, Del.). G.
stearothermophilus spores are serially diluted as described supra
and are plated with a spiral plater (Spiral Biotech, Inc., Norwood,
Mass.) on BHI agar (Becton Dickinson, Sparks, Md.). After 24 hours
incubation at 55.degree. C., colonies are enumerated with an
automated spiral plate counter (Q-count, Spiral Biotech, Inc.,
Norwood, Mass.). The lower detection limit is 10.sup.2 CFU/ml.
Prospore ampoules (Mesa Laboratories, Inc, Lakewood, Colo.) are
incubated at 55.degree. C. for 48 hours, and then checked for
indicator colors. qPCR is performed using the above described
methods.
[0102] In the autoclave treatment, Ct value for sweet potato puree
mtDNA increases from approximately 25 at time zero to 32 after 20
minutes. Ct value increase is similar in the autoclave treatment
which is conducted using the same time/temperature profile as the
oil bath. The autoclave assay exhibits a high correlation
(R.sup.2=0.87) between surrogate G. stearothermophilus spore
destruction and increase in Ct value over time (see FIG. 4)
Example 4 Assaying mtDNA Degradation on Various Heat Processed Food
Substances
[0103] The atp1 gene is found to be highly conserved among plant
species. As described above, BLAST analysis of the 174 forward and
reverse primers reveals that they exhibit 100% identity with a wide
variety of fruits, nuts and vegetables. To assess if the 174
primers can be used universally to test plant-based foods, both
singly and in mixtures such as soups, the following experiment is
performed. Fresh, uncooked fruits, vegetables and nuts (see Table 2
for items) are purchased from a retail grocery store. The samples
are processed immediately by grinding in a Hamilton Beach coffee
mill. The coffee mill is thorough cleaned with distilled water and
70% ethanol between samples and repetitions to prevent DNA
cross-contamination. For each variety of plant tested, three
separate samples of that plant are used. Six repetitions are tested
in all, three uncooked controls and three autoclave treatments (20
minutes at 121.degree. C. using procedure described above). The
tissue culture protocol of the MasterPure DNA purification kit
(Epicentre, Madison, Wis.) is used according to manufacturer's
recommendations. DNA is quantified and qualified using a
spectrophotometer (Nanodrop, Wilmington, Del.). DNA is normalized
to 5-10 ng/well and undergo qPCR assays using the 174F and 174R
primers and the protocol described above. Each sample is run in
duplicate wells. DNA is saved at -20.degree. C. in case amplicon
sequencing is required. Mean Ct values for uncooked and autoclaved
plant materials are recorded as well as the increase of Ct caused
by autoclave treatment and the slope of the line formed by the
graph of the two values. The results are presented in Table 2.
TABLE-US-00002 TABLE 2 Mean Mean uncooked autoclaved Sample Ct Ct
Difference Slope Vegetables White potato 19.73 32.77 13.04 0.65
Sweet potato 24.06 33.00 8.90 0.45 Tomato 18.86 32.27 13.41 0.67
Green pepper 19.45 35.66 16.21 0.81 Red pepper 18.99 34.63 15.65
0.78 Jalapeno 19.96 35.66 15.70 0.79 pepper Carrot 15.71 32.45
16.74 0.84 Green bean 22.45 32.38 9.93 0.50 Corn 22.40 27.24 4.84
0.24 Cucumber 18.47 29.88 11.40 0.57 Biofuels Switch grass 28.26
34.69 6.43 0.32 Fruits Apple 22.95 36.27 13.32 0.67 Blueberry 25.88
35.51 9.63 0.48 Peach 20.93 37.52 16.60 0.83 Strawberry 23.27 33.17
9.90 0.50 Pineapple 22.97 33.31 10.35 0.52 Grape 27.95 32.11 4.16
0.21 Nuts Peanut* 17.00 23.10 6.10 0.32 Almond 18.31 27.25 8.94
0.45 Pecan 25.86 31.43 5.57 0.28 *Roasted at 167.degree. C. for 19
min. All others autoclaved at 121.degree. C. for 20 min.
[0104] Using the 174F and 174R primers, a significant, detectable
increase in Ct value (approximately 4 to approximately 17 units) is
found between uncooked and autoclaved samples (121.degree. C. for
20 minutes) for the vegetables, fruits and nuts listed in Table 2.
Therefore, the 174F and 174R primers are be suitable for
quantifying heat treatment efficacy and quality in a wide variety
of plant foods, in complex mixtures such as soups, and plant
precursors for biofuels. Not wishing to be bound to any particular
hypothesis, Ct differences that occur between different foods may
result from differences in DNA extraction because some plant foods
are high in sugars or fats, while other plant foods are high in
fiber.
Example 5 mtDNA Degradation in Acidified Food Substances
[0105] Determining mtDNA fragmentation can assess shelf life in
vegetable products. Cucumbers and hamburger dill chips are assayed
during processing and storage (FIG. 5). Cucumbers are fermented in
NaCl for 8 months, are pasteurized at 75.degree. C. for 15 minutes
and then are stored at room temperature. Using the qPCR protocol
and the 174F and 174R primers, both described supra, mtDNA
fragmentation at pre-fermentation, immediately after
pasteurization, at 2 months, and at 20 months is assayed. mtDNA
fragmentation of autoclaved cucumbers is performed for comparison
using the protocols provided above. Threshold cycle (Ct) values are
significantly different between all treatments, except between a
fermented and pasteurized cucumber stored for 2 months and an
autoclaved cucumber. There is a significant difference (student
t-test, P<0.05) between the pickles stored at 2 and 20 months.
The fermented, pasteurized pickle has a similar Ct value as the
autoclaved cucumber. The results demonstrate that lower thermal
processes (75.degree. C. for 15 minutes) under acidified conditions
(pH=3.8) yield similar mtDNA fragmentation results to more elevated
temperature conditions (121.degree. C.) and that thermal processes
under 100.degree. C. with high acid products can be monitored for
the reliability of the heat treatment, acidification and/or
fermentation using qPCR of mtDNA.
Example 6 mtDNA Degradation in Dry Roasted Peanuts
[0106] To demonstrate that mtDNA degradation is an effective
time/temperature integrator for roasted solid foodstuffs such as
nuts, Virginia green runner peanuts are spiked with 10.sup.8 CFU/g
Enterococcus faecium (ATCC 8459; a Salmonella surrogate) and
compared with Ct values of the same peanut samples (not spiked with
E. faecium) during dry roasting at 167.degree. C. E. faecium is
inoculated into BHI broth (Remel, Lenexa, Kans.) from freshly
plated colonies and incubated statically overnight at 35.degree. C.
Cultures are concentrated 2.times. by centrifugation (5810R,
Eppendorf, Hamburg, Germany) at 6,000 rpm for 10 minutes at
4.degree. C. and resuspended in 0.5.times. initial volume with
sterile 0.9% saline. Target concentration is 10.sup.8 CFU/ml.
Initial culture concentration is determined by spectrometry at A600
and a simplified agar plate technique (Jett, et al., BioTechniques
23:648-650 (1997)) utilizing square petri dishes and the
track-dilution method. E. faecium-inoculated saline is added to the
total weight of peanuts to be tested in a large plastic zipper bag,
diluting the culture 1:20. The bag is closed and secured. Contents
are mixed thoroughly, and then sit for 5 minutes to absorb the
liquid. Inoculated peanuts are poured onto wire racks in 100 g
aliquots and allowed to air dry for 20 minutes.
[0107] A convection oven (Despatch Model LXD1-42-2; Minneapolis,
Minn.) is set to 167.degree. C. and is allowed to equilibrate for
30 minutes. A metal rack is inserted in the oven and is brought to
temperature. This rack holds smaller racks made of hardware cloth
through which peanuts do not pass. Each batch consists of 100 g of
peanuts laid out on the small hardware cloth racks. When the
smaller racks are inserted into the larger rack, the peanuts are
essentially suspended in the moving air inside the oven. For each
roasting batch, the oven door is quickly opened, the tray with the
peanuts is slid into the large rack, and the door of the oven
quickly closed. There is a drop in the oven temperature caused by
the door opening. The lowest temperature reached and the number of
seconds required for the oven to return to set point are recorded
for each batch. At the appropriate time point, the oven is opened
quickly, and peanuts are removed in the small rack. This rack is
placed over a homemade cooler with sufficient flow to cool the
peanuts to room temperature in 30 seconds. To prevent cross
contamination between repetitions, a clean gloves are used for
loading each batch into the small rack and into the oven, and clean
gloves are used to remove each batch. Between runs, the small rack
and the cooler are sprayed with 70% ethanol and allowed to dry
thoroughly before the next batch comes into contact with them.
Cooled peanuts are placed in plastic bags and stored until ready
for mtDNA analysis and E. faecium plate count analysis. It is
acknowledged that while the oven used for this experiment has
convective airflow, the oven is not comparable to industrial-scale,
commercial ovens used for peanuts. However, it is assumed that the
results would be similar when an industrial-scale, commercial oven
is used.
[0108] Three replicate samples are taken from the following time
points: 0, 3, 6, 9, 12, 15, 18, and 21 minutes. Ten grams are taken
from each 100 g replicate and placed in a stomacher bag
(Filtra-bag, Fisher, Pittsburgh, Pa.) with 10 ml sterile 0.9%
saline (1:1 dilution). Peanuts are stomached in a Seward Stomacher
400 (Tekmar, Cincinnati, Ohio) for 2 minutes at normal speed.
Filtrate is aseptically removed from the stomacher bag, serially
diluted and plated as described above using the simplified agar
plate technique (Jett, et al. (1997)) with BHI agar (BD, Sparks,
Md.). Plates are incubated at 35.degree. C. over-night. Plates are
counted manually, and CFU/g peanuts are calculated, taking into
account concentration and dilution factors.
[0109] Three peanuts from each replicate are ground under liquid
nitrogen in a mortar and pestle. The mortar and pestle are
thoroughly cleaned between samples with 70% ethanol to prevent
cross contamination. DNA is extracted using ca. 2.5 mg or one
inoculation loop of ground peanut in the MasterPure DNA
purification kit (Epicentre, Madison, Wis.) using the tissue sample
portion of the protocol. DNA is quantified and qualified with a
spectrophotometer (Nanodrop, Wilmington, Del.).
[0110] qPCR is performed in 25 .mu.l total volume with 2.times. IQ
SYBR Green supermix (SYBR Green I dye, 50 U/ml iTaq DNA polymerase,
0.4 mM each of dATP, dCTP, dGTP and dTTP, 6 mM MgCl.sub.2, 40 mM
Tris-HCl, pH 8.4, 100 mM KCl, and 20 nM fluorescein (BioRad,
Hercules, Calif.)), 300 nM final concentration each for 174 forward
and 174 reverse primers, peanut DNA (5-10 ng/reaction) and qPCR
water (Ambion, Austin, Tex.) to final volume. Amplifications are
performed in a MyiQ (BioRad, Hercules, Calif.) thermal cycler with
the following conditions: 95.0.degree. C. for 3 minutes; 40 cycles
of 95.0.degree. C. for 30 seconds, 60.0.degree. C. for 30 seconds,
72.0.degree. C. for 30 seconds; with FAM channel optics "on" during
annealing stage. No template control (NTC) and positive controls
are used for all assays. The positive control is used to normalize
data between assays. For a sample to be considered positive, its
threshold cycle (Ct) value must be less than all negative control
reactions, and its corresponding amplification curve must exhibit
the three distinct phases of real-time PCR: lag, linear and
plateau. Ct values are initially steady, then increase after 12
minutes of roasting to a mean value approximately 22 units after 21
minutes (see FIG. 6). Peanut mtDNA fragmentation is correlated to
E. faecium death because R.sup.2=0.67 (see FIG. 7).
[0111] The remainder of each batch of peanuts is used to determine
the Hunter L value color. The skins are removed from approximately
40 g peanuts which are placed in a glass petri dish and inserted
above a calibrated HunterLab DP9000 with D25 sensor (Hunter
Associates Laboratory, Reston, Va.) utilizing the Lab scale (a
standardized color scale). Readouts are recorded three times per
sample with the peanuts removed and resorted in the petri dish
between readings with the peanuts placed outer-side down, if
broken. The color is expressed as the mean of the three
replications for each peanut sample presented to the colorimeter.
The scale of the readings range from 1 to 100 with 1 representing
black and 100 representing white. Peanut mtDNA fragmentation is a
good correlation to Hunter L color (see FIG. 8), a quality
parameter used to determine roasting endpoint because
R.sup.2=0.95.
Experiment 7 Using of Fluorescent Probe with Hot Oil Bath as
Surrogate for Industrial Microwave System
[0112] The protocol of Experiment 2 is repeated except instead of
using SYBR Green I dye (an intercalater of DNA), a nucleic acid
probe such as TaqMan.RTM. is used to measure the amount of mtDNA
degradation. TaqMan.RTM. probes contain a fluorescent reporter dye
(e.g., 6-carboxyfluorescein (6-FAM.TM.) or
tetrachloro-6-carboxy-fluorescein (TET)) at the 5' end and a
quencher dye at the 3' end (e.g., Iowa Black FQ or Black Hole
Quencher (BHQ-2) quenchers). For TaqMan.RTM. detection, during each
amplification cycle the probe attaches along with the primers to
the target sequence of DNA to be copied. As the DNA strand is
copied, the reporter dye is released from the probe sequence, and
its fluorescent signal is measurable because it is no longer near
the quencher dye. The amount of fluorescence increases with each
PCR cycle in proportion to the amount of target DNA amplified,
thereby allowing direct detection and quantification of the target
DNA sequence with a high degree of specificity, accuracy, and
sensitivity. The probe's sequence can range from approximately 15
bp to approximately 30 bp and is the sequence of the coding or
non-coding strand (reverse complement of the coding strand
sequence) of the amplicon.
[0113] The GS spores and sweet potato are processed according to
the protocol in Experiment 2. The fluorescence of the samples is
measured, and the Ct values obtained are almost identical to the
values obtained in Experiment 2.
[0114] While the above experiments examined the Ct value of atp1
from mtDNA, one can determine the Ct value of any mtDNA. One can
pick any primers that generate a desired amplicon of approximately
250 bp or less during the DNA amplification step.
Example 8 Extrinsic DNA Thermometer Testing the Efficacy of
Pasteurization and Other Thermal Processes
[0115] This experiment demonstrates the use of extrinsic DNA as a
time/temperature indicator for inactivation of hazardous biological
material when one does not want or cannot perform qPCR on intrinsic
DNA. One example of using this type of assay is to assess the
efficacy of inactivation of hazardous biological material on a food
container (e.g., a jar) or a medical device. Extrinsic DNA
thermometers (described infra) are time/temperature indicators
which are added and recovered from thermal processing or other
inactivation processing methods for foods, packaging, and medical
equipment to test the efficacy of the inactivation system and/or
method. A solution is prepared containing 0.5% citric acid and
divided into three aliquots. Each aliquot is adjusted to either pH
3.6, 4.0 or 4.4 using 1M NaOH. All solutions are filter sterilized
by passage through 0.45 um filters. An extrinsic DNA thermometer is
created by combining 2 .mu.l of 10.sup.8 copies/.mu.l of the 174
primer amplicon for atp1 (SEQ ID NO: 14) (gBlock.RTM. gene fragment
purchased from IDT, Coralville, Iowa) (see FIG. 1) with 18 .mu.l of
a citric acid solution in a 200 .mu.l domed thermal cycler tube.
Final concentration of extrinsic DNA in each tube is 10.sup.7
copies/.mu.l. Tubes are placed in a thermal cycler (MyiQ, BioRad,
Hercules, Calif.) when the temperature reaches 96.degree. C.
Samples are removed at time points 0, 4, 8, 16, 24 minutes in assay
1; and 0, 1, 2, 3, 4, 5 minutes in assay 2; with three reps at each
time point using three different pH levels. Total number of samples
is 45 each for each assay. After heat treatment, samples are placed
in ice water slurry until cool, approximately 10 minutes. Atp1 qPCR
protocol is run on each sample as described supra using 174F and
174R primers, being careful to segregate amplicon from reagents and
pipettemen. FIGS. 10 and 11 show Ct values of extrinsic DNA
thermometer, consisting of gBlock.RTM. of atp1 amplicon (SEQ ID NO:
14) in 0.5% citric acid, versus time at 96.degree. C. FIG. 10 runs
from 0-24 minutes, FIG. 11 from 0-5 minutes. For longer time
courses, pH 4.4 had the best goodness-of-fit value (FIG. 10:
R.sup.2=0.90). For shorter thermal processes, 5 minutes or less at
96.degree. C., pH 4.4 had the best goodness-of-fit (FIG. 11;
R.sup.2=0.67). Extrinsic DNA thermometers can be used in low
temperature/low acid thermal processes, medical or container
applications, or any process where intrinsic DNA is difficult to
obtain.
[0116] While a citric acid solution to hold the extrinsic DNA is
used in this example, one can use any organic acid or inorganic
acids to generate a low pH solution into which extrinsic DNA is
placed. A non-limiting example of an organic acid is malic acid.
Non-limiting examples of inorganic acids are HCl and phosphoric
acid. Further, while this example used atp1 amplicon having SEQ ID
NO: 14, any double stranded DNA of between approximately 80 bp and
approximately 250 bp (contiguous bp) of atp1 or any mitochondrial
DNA can be used with the appropriate primers to generate an
amplicon of the sequence used. Also a labeled probe can be used
into of an intercalating dye as per the above protocol.
[0117] One can use extrinsic DNA to determine the efficacy of
inactivation of a hazardous biological material in a food matrix,
instead of analyzing intrinsic DNA. One simply needs to submit an
amount of extrinsic DNA to the processing methods of the food
matrix and then determine the Ct of the extrinsic DNA. The
extrinsic DNA may need to be placed in a container.
Example 9 Using Total DNA Fragmentation of Plant Food Matrix During
Thermal Processing as a Measure of Processing Efficacy
[0118] DNA samples from previous described assays and similar
concentrations (approximately 200 ng/.mu.l) are loaded into a mini
electrophoretic unit containing a global DNA analyzer (Agilent
Bioanalyzer 2100, Santa Clara, Calif.). The analyzer either
contains a fluorescent composition that binds to the DNA within the
electrophoretic gel or one adds it to the analyzer. A graph
comparing DNA fragment size (ranging from approximately 35 to
approximately 10,380 bp) with fragment concentration is generated.
Graphs of total DNA size from fresh and autoclaved cucumber DNA are
compared. FIG. 12 illustrates the global measurement of total
cucumber DNA before and after autoclave treatment. Number of base
pairs (size) versus concentration (pg/ul) are compared. Fresh
cucumber DNA ranges from <1,000 base pairs (bp) to approximately
10,000 bp. After autoclave treatment, total DNA is degraded and
fragmented (<3,500 bp), sizes clustering between approximately
35 bp and approximately 400 bp. DNA integrity is the wholeness or
completeness of a cell's genomic and organelle-based DNA. After
heat treatments such as autoclaving, a cell's DNA integrity is
reduced and this fragmentation can be measured globally. Based on
the results, an algorithm which predicts time/temperature treatment
based on global DNA fragmentation is generated. One uses can use
this measurement of DNA integrity as a time/temperature integrator
for inactivation of hazardous biological material in/on food
matrices and/or medical devices.
[0119] Again, one can use extrinsic DNA or intrinsic DNA with this
protocol for analyzing the DNA fragmentation.
[0120] A flow chart illustrating the methods of these novel
time/temperature integrators of these inventions is in FIG. 13.
Example 10 Comparison of Ct and F Values of Sweet Potato Puree with
12D-Retort Protocol
[0121] Sweet potato puree is produced as described supra and placed
in 68.3.times.101.6 mm cans outfitted with T-type C-2 tube and rod
thermocouples (Ecklund-Harrison Technologies, Fort Myers, Fla.).
Colorimetric G. stearothermophilus ampoules (Raven ProSpore; Mesa
Laboratories, Inc., Lakewood, Colo.) are placed in the center of
each can, adjacent to the thermocouple probes. Cans are sealed with
a double seam using an automated can sealer (Dixie Canner Co.,
Athens, Ga.). Total weights of puree and size of head space are
similar between all cans in each run. Canned sweet potato puree is
loaded into a Model PR-900 pilot retort (Stock
sterilisationstechnik, Hermanstock Maschf.; Neumunster, West
Germany) with thermocouples attached to a recording device and run
in one of two full water immersion protocols listed in Tables 3 and
4 infra. Protocol 00 (Table 3), sub-12D full water immersion, is a
substandard treatment not meant to kill spores. Protocol 01 (Table
4), 12D full water immersion, is a 12D protocol meant to eliminate
all G. stearothermophilus test spores. Each protocol is run in
triplicate using three cans per run. Puree is sampled from the
center of each can, the DNA is extracted, and the atp1 qPCR
protocol performed as described supra using primers 174F and 174R.
ProSpore ampoules are incubated at 55.degree. C. for 48 hours as
recommended by the supplier, and then assessed for colorimetric
change. F values are determined from the thermal couple
time-temperature data collected. F value is calculated as follows:
F=10.sup.(T-121.1/10) .DELTA.t; where T is temperature in .degree.
C. and t is time in minutes. Ct values are correlated to F values
of all 12D and sub-12D runs and are shown in FIG. 14A (protocol 00
which is shown in Table 3) and FIG. 14B (protocol 01 which is shown
in Table 4). The protocols presented in Table 3 and Table 4 contain
information provided by the manufacturer (Hermanstock Maschf.;
Neumunster, West Germany). The results support the use of qPCR of
mtDNA of a food product to assess bacterial spore inactivation,
because the Ct values are highly correlated to F values.
TABLE-US-00003 TABLE 3 Temperature Pressure Temp Pressure Step (F.)
(psi) Time Gradient Gradient Heating 180 20 -- -- -- Storage Vessel
Sterilization I 180 20 20 sec -- -- (Vent) Sterilization II 242 20
10 min 10 -- (Come up) Sterilization III 242 20 35 min -- -- (Hold)
Cooling 1 -- 20 10 min -- -- Cooling 2 90 10 20 min -- 0.6 Drain 90
-- 4 min -- --
TABLE-US-00004 TABLE 4 Temperature Pressure Temp Pressure Step (F.)
(psi) Time Gradient Gradient Heating 200 20 -- -- -- Storage Vessel
Sterilization I 200 20 20 sec -- (Vent) Sterilization II 260 20 12
min 10 -- (Come up) Sterilization III 260 20 35 min -- -- (Hold)
Cooling 1 -- 20 10 min -- -- Cooling 2 90 10 12 min -- 0.6 Drain 90
-- 4 min -- --
Example 11 Comparison of Ct Value with D- and z-Values
[0122] In an effort to mimic and quantify values in a 12D thermal
process, the kill curve of G. stearothermophilus (a C. botulinum
surrogate) spores with resulting D- and z-values are compared to Ct
values of sweet potato puree in a hot oil bath at the following
temperatures: 116.degree. C., 121.degree. C., 123.degree. C., and
126.degree. C. A hot oil bath (Neslab Instruments, Newington, N.H.)
filled with 8 L white mineral oil (Solutia, Inc, St. Louis, Mo.) is
used to maintain each target temperature for substances placed in a
thermal death tube (TDT). This system replicates conditions in an
industrial retort, heat exchanger or microwave thermal process. The
TDT is composed of a 3/4 inch aluminum screw post (Screwpost.com,
Muskegon, Mich.) cut to size and filed for smoothness, 1/4 inch
nylon machine screws, Viton fluoroelastomer O-ring gaskets (screw
size #6) and Viton flat washers size #6 (McMaster-Carr, Atlanta,
Ga.). Temperature is monitored using a type J-K-T microprocessor
thermometer thermocouple (Omega Corp., Stamford, Conn.). Come up
time (CUT) for TDTs is determined for all target temperatures. In
each TDT, 100 .mu.l of 1:4 diluted puree or 100 .mu.l of G.
stearothermophilus (GS) spores (approximately 10.sup.8 CFU/ml) are
inserted and sealed. For GS spores, samples are heated for 0, 4, 8
and 12 minutes at 116.degree. C.; for 0, 0.5, 1, 2, 4, 8, 16 and 20
minutes at 121.degree. C.; for 0, 1, 2 and 4 minutes at 123.degree.
C.; and for 0, 0.5, 1, 2 and 4 minutes at 126.degree. C., with all
heat treatments beginning after come up time. Samples of diluted
sweet potato puree are heated for 0, 12, 24, 48 and 60 minutes at
116.degree. C.; for 0, 4, 8, 12 and 18 minutes at 121.degree. C.;
for 0, 4, 8, 12 and 18 minutes at 123.degree. C.; and for 0, 4, 8,
12 and 18 minutes at 126.degree. C.; with all heat treatments
beginning after come up time. Three replications are run per time
point; 3 TDTs are placed in a metal tea strainer to facilitate
removal of samples from hot oil. Strainers containing TDTs are
taken out of oil bath and immediately placed in an ice slurry for
30 second to quickly cool them. Strainers containing TDTs are
stored at room temperature until ready for DNA extraction or
culture plating. Total amount of sweet potato puree recovered from
hot oil bath treatment is determined from an initial sample of 100
.mu.l.
[0123] The D value (decimal reduction time) is defined as the time
in minutes at a given temperature that results in a one log
reduction in microbial count (Pflug, Irving, Microbiology and
Engineering of Sterilization Processes. 7th Ed., published by
Environmental Sterilization Laboratory; Minneapolis, Minn. (1990)).
Using the equation:
N=N.sub.010.sup.-t/D.sub.T
where N.sub.0 and N are the initial and final number of
microorganisms, respectively; the D value at a given temperature
(D.sub.T) is calculated by graphing the log.sub.10 number of
microorganisms over time (minutes) and determining the slope:
slope=-1/D.sub.T.
[0124] The z-value is the temperature change required for a one log
change in the D value of a microorganism (Pflug, Irvine supra
(1990)). Using the equation
D.sub.T=D.sub.ref10.sup.Tref-T/z
the z-value is calculated by graphing log D value (seconds) versus
temperature and determining the slope: slope=-1/z.
[0125] G. stearothermophilus spores are serially diluted and plated
with on BHI agar (Becton Dickinson, Sparks, Md.) using a spiral
plater (Spiral Biotech Inc., Norwood, Mass.) or a simplified agar
plate technique (Jett, et al., BioTechniques 23:648-650 (1997)).
After 24 hours incubation at 55.degree. C., colonies are enumerated
with an automated spiral plate counter (Spiral Biotech Inc.,
Norwood, Mass.) or counted manually. The lower detection limits are
4.times.10.sup.2 and 1.times.10.sup.3 CFU/ml for the spiral plate
and simplified agar technique, respectively.
[0126] Ct values are converted to log.sub.10 copy numbers using the
linear relationship determined empirically from the standard curve
of the 174 bp amplicon:
y=-3.1909x+38.091
where y is the Ct value and x is the log.sub.10 copy number.
[0127] D.sub.121 and z-values determined in hot oil bath for G.
stearothermophilus spores are 2.71 minutes and 11.0.degree. C. (see
FIGS. 15 and 16), respectively. These values are slightly higher
than values obtained using a commercial product with the same GS
strain spores for autoclave validation (Prospore, Mesa Labs,
Lakewood, Colo.) which cite a D.sub.121 value of 1.8 minutes and a
z-value of 7.4.degree. C. under saturated steam. Prior art D and
z-values for GS spores are Duo from 1.5 to 3 minutes with z-value
of greater than or equal to 6.degree. C. and D.sub.121 of
approximately 2 minutes in water (Lundahl, G., PDA J. Pharm. Sci.
Technol. 57:249-262 (2003)). Both of these cited values are based
on an initial population of 10.sup.6 spores. Head, et al. (J. Appl.
Microbiol. 104:1213-1220 (2007)) determined that D and z-values
vary widely based on the initial concentration of spores (10.sup.3
versus 10.sup.6) when treated with superheated steam. While the TDT
employed herein is a pressurized container, one would not expect
the same time-temperature treatment in a hot oil bath as
pressurized, saturated stream in an autoclave. Based on
precautionary notes in commercial spore technical data sheets
(Prospore, Namsa, Northwood, Ohio) and values in the literature,
spore D and z-values can vary widely because of the type of heat
treatment (wet versus dry), initial concentration of spores, and
spore carrier or media (Head, et al. (2007)). As an added
precaution, a safety factor is added to empirically derived data,
i.e. total death time is rounded up, to ensure complete destruction
of spores (Tucker, et al., History of the minimum botulinum cook
for low-acid canned foods. Campden & Chorleywood Food Research
Association Group. R & D report no. 260. Doc
Ref:FMT/REP/90194/1 (2008)).
[0128] D.sub.121 and z-values for Ct values from a 174-bp universal
plant amplicon are 11.3 minutes and 17.8.degree. C. (see FIGS. 17
and 18), respectively, for mtDNA from sweet potato puree heated in
a hot oil bath. The conversion of Ct to log.sub.10 copy number of
amplicon results in the Ct-D.sub.121 value (11.3 minutes) being
much higher than the G. stearothermophilus D121 (2.71 minutes). G.
stearothermophilus spores have a D121 value approximately 10.times.
greater than C. botulinum (D.sub.121=0.21 minutes; Esty &
Meyer, J. Infectious Dis. 31:650-663 (1922); Townsend, et al., Food
Res. 3:323-346 (1938); Stumbo, C. R., Thermobacteriology in Food
Processing. 1.sup.st Ed. Academic Press 111 Fifth Ave, New York,
N.Y. (1965)), the spore of concern in low acid, canned or
aseptically-packaged foods. The Ct-D.sub.121 value of sweet potato
puree mtDNA is approximately 4.times. greater than the G.
stearothermophilus indicator spore. Because of its higher D121
value, it might be difficult to predict the FDA recommended F value
for sterilization (F.sub.0=5 minutes) using a log function of Ct
value. However, sterilization in the pharmaceutical industry
requires higher values (F.sub.0>12 minutes) where GS spores
leave no measurable outcome (Lundahl, G. (2003)).
[0129] When compared directly, the increase in Ct value has nearly
a 1:1 ratio with G. stearothermophilus destruction at 121.degree.
C. in hot oil bath treatments (ratio=0.875) (FIG. 19). The
destruction of mtDNA as measured by log.sub.10 copy number is not a
first order relationship but a simple inverse relationship with
time-temperature. Therefore, the use of Ct values directly will
have greater utility than conversion to log values.
[0130] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation. All documents cited
herein are incorporated by reference.
Sequence CWU 1
1
21124DNAIpomoea batatas 1tttccgcgat aatggaatgc acgc 24224DNAIpomoea
batatas 2tccgatcgtt tagccgctct ttct 24324DNAIpomoea batatas
3cgcctttgct caatttggct caga 24424DNAIpomoea batatas 4ggcagtggtg
catattgtgg ttgt 24524DNAIpomoea batatas 5cgcctttgct caatttggct caga
24624DNAIpomoea batatas 6agtacttctg tcagccttgc acct 24724DNAIpomoea
batatas 7gaatttgcca gcggtgtgaa agga 24824DNAIpomoea batatas
8tcccgcagga acatccacaa taga 2491293DNAIpomoea batatas 9tggatgagat
cggtcgagtg gtctcagttg gagatgggat tgcacgtgtt tatggattga 60acgagattca
agctggggaa atggtggaat ttgccagcgg tgtgaaagga atagccttga
120atcttgagaa tgagaatgta gggattgttg tctttggtag tgatactgct
attaaggaag 180gagatcttgt caagcgcact ggatctattg tggatgttcc
tgcgggaaag gctatgctag 240ggcgtgtggt cgacgccttg ggagtaccta
ttgatggaag aggggctcta agcgatcacg 300agcgaagacg tgtcgaagtg
aaagcccctg ggattattga acgtaaatct gtgcacgagc 360ctatgcaaac
agggttaaaa gcggtagata gcctggttcc tataggtcgt ggtcaacgag
420aacttataat cggagaccga caaactggaa aaacagctat tgctatcgat
accatattaa 480accaaaagca actgaactca agggcctcct ctgagagtga
gacattgtat tgtgtctatg 540tagcgattgg acagaaacgc tcaactgtgg
cacaattagt tcaaattctt tcagaagcga 600atgctttgga atattccatt
cttgtagcag ccaccgcttc ggatcctgct cctctgcaat 660ttttggcccc
atattctggg tgtgccatgg gggaatattt ccgcgataat ggaatgcacg
720cattaataat ctatgatgat cttagtaaac aggcggtagc atatcgacaa
atgtcattat 780tgttacgccg accaccaggt cgtgaggctt tcccagggga
tgttttttat ttacattccc 840gtctcttaga aagagcggct aaacgatcgg
accagacagg cgcaggtagc ttgaccgcct 900tacccgtcat tgaaacacaa
gctggagacg tatcggccta tattcccacc aatgtgatcc 960ccattactga
tggacaaatc tgtttggaaa cagagctctt ttatcgcgga attagacctg
1020ctattaacgt cggcttatct gtcagtcgcg tcgggtctgc cgctcagttg
aaagctatga 1080aacaagtctg cggtagttta aaactggaat tggcacaata
tcgcgaagtg gccgcctttg 1140ctcaatttgg ctcagacctt gatgcagcga
ctcaggcatt actcaataga ggtgcaaggc 1200tgacagaagt actgaaacaa
ccacaatatg caccactgcc aattgaaaaa caaattctag 1260taatttatgc
agctgtcaat ggattctgtg atc 12931024DNAIpomoea batatas 10agaaagagcg
gctaaacgat cgga 241124DNAIpomoea batatas 11acaaccacaa tatgcaccac
tgcc 241224DNAIpomoea batatas 12aggtgcaagg ctgacagaag tact
241324DNAIpomoea batatas 13tctattgtgg atgttcctgc ggga
2414174DNAIpomoea batatas 14tttccgcgat aatggaatgc acgcattaat
aatctatgat gatcttagta aacaggcggt 60agcatatcga caaatgtcat tattgttacg
ccgaccacca ggtcgtgagg ctttcccagg 120ggatgttttt tatttacatt
cccgtctctt agaaagagcg gctaaacgat cgga 17415108DNAIpomoea batatas
15cgcctttgct caatttggct cagaccttga tgcagcgact caggcattac tcaatagagg
60tgcaaggctg acagaagtac tgaaacaacc acaatatgca ccactgcc
1081681DNAIpomoea batatas 16cgcctttgct caatttggct cagaccttga
tgcagcgact caggcattac tcaatagagg 60tgcaaggctg acagaagtac t
8117141DNAIpomoea batatas 17gaatttgcca gcggtgtgaa aggaatagcc
ttgaatcttg agaatgagaa tgtagggatt 60gttgtctttg gtagtgatac tgctattaag
gaaggagatc ttgtcaagcg cactggatct 120attgtggatg ttcctgcggg a
1411824DNAIpomoea batatas 18tctattgtgg atgttcctgc ggga
241924DNAIpomoea batatas 19agaaagagcg gctaaacgat cgga
242024DNAIpomoea batatas 20aggtgcaagg ctgacagaag tact
242124DNAIpomoea batatas 21acaaccacaa tatgcaccac tgcc 24
* * * * *
References