U.S. patent application number 15/666042 was filed with the patent office on 2018-03-01 for selective ultrasonic lysis of blood and other biological fluids and tissues.
This patent application is currently assigned to AdvanDx, Inc.. The applicant listed for this patent is AdvanDx, Inc.. Invention is credited to Martin Fuchs, Michelle Meltzer.
Application Number | 20180057856 15/666042 |
Document ID | / |
Family ID | 47217646 |
Filed Date | 2018-03-01 |
United States Patent
Application |
20180057856 |
Kind Code |
A1 |
Fuchs; Martin ; et
al. |
March 1, 2018 |
SELECTIVE ULTRASONIC LYSIS OF BLOOD AND OTHER BIOLOGICAL FLUIDS AND
TISSUES
Abstract
The present invention features methods for selective lysis of
endogenous cells in a biological sample. In preferred embodiments,
the methods of the invention comprise contacting the biological
sample with lysis solution, and subjecting the mixture to
ultrasound, thereby selectively lysing the endogenous cells in the
biological sample. The invention also features a lysis solution
comprising Saponin and Proteinase.
Inventors: |
Fuchs; Martin; (Uxbridge,
MA) ; Meltzer; Michelle; (Chelmsford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AdvanDx, Inc. |
Woburn |
MA |
US |
|
|
Assignee: |
AdvanDx, Inc.
Woburn
MA
|
Family ID: |
47217646 |
Appl. No.: |
15/666042 |
Filed: |
August 1, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14084026 |
Nov 19, 2013 |
9719128 |
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15666042 |
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PCT/US2012/038535 |
May 18, 2012 |
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14084026 |
|
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61488434 |
May 20, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 13/00 20130101;
C12Q 1/04 20130101; C12Q 1/6806 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12Q 1/04 20060101 C12Q001/04; C12N 13/00 20060101
C12N013/00 |
Claims
1-65. (canceled)
66. A lysis solution for selective lysis of endogenous cells in a
biological sample comprising: a detergent; and a proteinase.
67. The lysis solution of claim 66, further comprising a Sodium
Phosphate buffer, pH 8.
68. The lysis solution of claim 67, wherein the Sodium Phosphate
buffer is at pH 8.
69. The lysis solution of claim 66, wherein the detergent is
selected from the group consisting of Saponin, nonionic surfactant
and polysorbate surfactant.
70. The lysis solution of claim 69, wherein the nonionic surfactant
comprises Triton X-100.
71. The lysis solution of claim 69, wherein the polysorbate
surfactant comprises Tween 20.
72. The lysis solution of claim 66, wherein the detergent is
Saponin.
73. The lysis solution of claim 72, wherein the Saponin is from
Quillaja bark.
74. The lysis solution of claim 66, wherein the proteinase is
derived from Aspergillus.
75. The lysis solution of claim 74, wherein the proteinase is
derived from Aspergillus melleus.
76. The lysis solution of claim 72, comprising 0.1%-10%
Saponin.
77. The lysis solution of claim 76, comprising 1.15% Saponin.
78. The lysis solution of claim 66, comprising 5.0-37.5 Units
Proteinase.
79. The lysis solution of claim 78, comprising 11.25 Units
Proteinase.
80. The lysis solution of claim 67, comprising 0.01-0.1M Sodium
Phosphate buffer, pH 8.
81. The lysis solution of claim 80, comprising 0.1M Sodium
Phosphate buffer, pH 8.
82. The lysis solution of claim 66, further comprises an enzyme
selected from the group consisting of cholesterol esterase, lipase
and DNase.
83. The lysis solution of claim 66, further comprises a reducing
agent.
84. The lysis solution of claim 83, wherein the reducing agent
comprises TCEP.
85. The lysis solution of claim 66, further comprises a chaotropic
agent.
86. The lysis solution of claim 85, wherein the chaotropic agent
comprises guanidinium chloride.
87. The lysis solution of claim 66, further comprising a hypotonic
salt solution.
88. The lysis solution of claim 66, wherein the biological sample
is a body fluid.
89. The lysis solution of claim 88, wherein the body fluid is
selected from the group consisting of: blood or blood fractions,
respiratory secretions, cerebrospinal fluid, urine, stool, wound
exudates (pus), and naso-pharyngeal fluid/mucus.
90. The lysis solution of claim 66, where the biological sample is
selected from the group consisting of: platelets, platelet
concentrate and a mammalian cell culture.
91. A lysis solution for increasing filterability of
bronchoalveolar lavage comprising: Triton X-100 or Tween 20;
Protease; and TCEP.
92. The lysis solution of claim 91, further comprises Sodium
Phosphate Buffer, pH 8.
93. A method for selective lysis of endogenous cells in a
bronchoalveolar lavage sample and increasing filterability of the
sample comprising: contacting the bronchoalveolar lavage sample
with the lysis solution of claim 26; and subjecting the mixture of
the bronchoalveolar lavage sample and lysis solution to
high-frequency ultrasound; thereby selectively lysing the
endogenous cells in the bronchoalveolar lavage sample.
94. The method of claim 93, wherein the endogenous cells are
mammalian cells.
95. The method of claim 93, wherein the bronchoalveolar lavage
sample comprises microorganisms.
96. The method of claim 95, wherein the microorganisms are selected
from the group consisting of: bacteria, yeast and fungi.
97. The method of claim 93, further comprising filtering or
centrifuging the lysed sample; and detecting, identifying,
characterizing or quantifying microorganisms in the biological
sample; thereby detecting, identifying, characterizing or
quantifying microorganisms in the biological sample.
98. The method of claim 97, wherein the detecting, identifying,
characterizing or quantifying is carried out using peptide nucleic
acid (PNA) fluorescent in situ hybridization (FISH).
Description
PRIORITY
[0001] This application is a continuation application of U.S.
application Ser. No. 14/084,026, filed Nov. 19, 2013, which is a
continuation application of International Application No.
PCT/US2012/038535, filed on May 18, 2012, which claims the benefit
of the filing date, under 35 U.S.C. .sctn. 119(e), of U.S.
Provisional Application No. 61/488,434, filed on May 20, 2011. The
entire contents of each of the above-referenced applications are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention pertains to the field of detection,
identification and characterization of microorganisms in complex,
cell-containing biological fluids and tissues.
BACKGROUND
[0003] The ability to detect and characterize low levels of
microorganisms in biological samples is valuable for many
applications including diagnosing and treating infections in both
humans and animals, infectious disease research, detecting food
contamination and identifying the causative organisms, monitoring
product quality during food processing, monitoring environmental
quality and so on.
[0004] Culture is often used to facilitate the detection and
characterization of microorganisms in biological samples. The
samples are incubated in an atmosphere and at a temperature that is
conducive to the growth of microorganisms, possibly with the
addition of nutrient media to sample. Under these conditions, the
microorganisms will multiply and can reach high concentrations.
After growth to a sufficient concentration is achieved, a variety
of methods can be used for the detection and characterization of
the microorganisms. These methods include staining,
fluorescence-in-situ-hybridization (FISH),
polymerase-chain-reaction (PCR) and
matrix-assisted-laser-desorption-ionization (MALDI) mass
spectrometry. The drawback to culture is that it is slow, typically
proceeding over many hours. Direct, i.e. non-culture, methods would
therefore be preferred in those cases where rapid detection and
characterization is important.
[0005] A range of bioanalytical methods rely on the lysis of cells
for the release of intracellular components. Such components
include organelles such as mitochondria, lysosomes, and endoplasmic
reticulum, molecular assemblies such as microtubules and ribosomes
and molecules such as proteins, carbohydrates and nucleic acids.
Following lysis, the intracellular components can be subjected to
analysis by for example electrophoresis, chromatography, mass
spectrometry or optical spectroscopy. Likewise, molecular methods
such as PCR, microarray analysis and sequencing rely on cell lysis
for the release of intracellular DNA and RNA for amplification and
other kinds of processing. To meet these needs, various cell lysis
methods have been developed. Such methods include osmotic,
chemical, mechanical (e.g. grinding with beads), hydrodynamic (e.g.
pressure cell) and acoustic (i.e. sonication with ultrasound).
[0006] Ultrasound (acoustic waves beyond the audible range) has
been used to lyse cells to release contents for molecular analysis
often in conjunction with beads. See Seiter, J. A. and Jay, J. M.
1980.sup.5. U.S. Pat. No. 5,374,522 (Murphy et al.) describes the
use of an ultrasonic bath to disrupt cells such as Mycobacterium
tuberculosis in a sample to which beads of glass or other materials
in the range of 50 microns to 1 mm have been added. Such disruption
released RNA and DNA into solution for hybridization with genetic
probes. In U.S. Pat. No. 6,431,476, Taylor et al. teach a method
for disrupting cells or viruses in a chamber with an ultrasonic
transducer. Chandler et al. (U.S. Pat. No. 6,506,584) teach
treating liquid with ultrasound in a flow-through device. The
treatment can include cell lysis. U.S. Pat. No. 6,686,195 (Colin et
al.) teaches lysing cells in a tube brought into direct contact
with a shaped sonotrode. In U.S. Pat. No. 6,881,541 Petersen et al.
teach a method for extracting nucleic acid from a sample using
ultrasound. In U.S. Pat. No. 6,887,693 McMillan et al. teach a
method for lysing components of a fluid sample that have been
captured on a solid support. In U.S. Pat. No. 6,893,879, Petersen
et al. teach a method for extracting an analyte from a fluid
sample. U.S. Pat. No. 6,939,696 (Llorin et al.) teaches disrupting
microorganisms in a sonicator at high pH in a lube without beads.
In these references, the goal is to disrupt or lyse cells, whether
mammalian or bacterial, to release the cell content for analysis.
Belgrader et al. (U.S. Pat. No. 7,541,166) describe an apparatus
that allows a sample or parts of a sample to be moved into a
sonication chamber multiple times, allowing differing sonication
levels to be applied to more and less sensitive cells such as
epithelial and sperm cells releasing their DNA for analysis at
different times.
[0007] In analyzing cell-containing biological samples, it is
sometimes advantageous to lyse a subpopulation of the cells present
in the sample. For example, when it is desired to perform a
differential analysis of the white blood cells in blood using a
Coulter counter, it is convenient to lyse the red blood cells while
leaving the white blood cells intact. Various lysis solutions have
been developed to achieve this result. See for example, U.S. Pat.
No. 3,874,852 (Hamill), U.S. Pat. No. 4,185,964 (Lancaster), U.S.
Pat. No. 4,521,518 (Carter et al.), U.S. Pat. No. 5,284,940 (Lin et
al.), and U.S. Pat. No. 5,958,781 (Wong et al.). It is worth noting
that red blood cells lyse fairly readily compared to the white
blood cells and selective red blood cell lysis can be accomplished
simply with osmotic shock. Agents that selectively lyse bacteria
but not mammalian cells have potential utility in combating
infections. Oren and Shay studied melittin diastereomers that lyse
bacteria but not mammalian cells.sup.3. Selective lysis can be
useful for biological research. Grifantini and coworkers were able
to isolate adherent bacteria co-cultured with epithelial cells for
gene expression studies by selectively lysing the epithelial cells
with saponin..sup.4
[0008] Direct assays for the detection of microorganisms in
biological fluids are often hampered by the presence of endogenous
cells in high numbers. In general, such assays can be simplified if
a method for selectively removing the endogenous cells were
available. Zierdt and his colleagues published a lysis method in
1977.sup.1. This method uses a mild detergent solution containing
an enzyme mixture (Rhozyme prepared from Aspergillus oryzae
cultures). In a subsequent paper.sup.2, Zierdt refined the solution
by substituting the less toxic detergent Tween 20 for the Triton
X-100 used in the original protocol. The Zierdt method is able to
process a suitable volume of blood, 1 mL for example, in 1 hour,
yielding a clear, red solution that can be filtered through a 0.6
micron track-etch filter 8 mm in diameter in approximately 3
minutes using a pressure differential of 2.5 psi. A key advantage
of the Zierdt method is that the product is filterable through
filters with pores small enough to retain microorganisms. Following
filtration, the filter can be placed on a nutrient plate under
suitable conditions, allowing colonies to grow from individual
cells. The colonies can then be counted and further analyzed for
the identity and antibiotic susceptibility of the organisms.
Alternatively, FISH or other fluorescent labeling methods can be
applied to the cells and fluorescence microscopy used to directly
visualize the cells on the filter. This offers the possibility of
rapid detection and identification of microorganisms in a range of
complex samples including blood and other clinical specimens.
Hence, a method that is able to selectively lyse mammalian cells
faster than the Zierdt method would be advantageous.
[0009] In addition to the presence of cells, other constituents of
biological samples can also hamper the detection of microorganisms.
For example, bronchial samples are often highly viscous due to the
presence of phlegm and other lung exudates. Urine specimens may
contain significant amounts of protein as well as cells and mucus.
These materials impede the detection of microorganisms by
microscopic methods. Various reagents are used to overcome the
obstacles to detection posed by these sample constituents. For
example, N-acetyl-L-cysteine (NALC), combined with sodium citrate
is a digestant that breaks up mucus in sputum and other bronchial
samples. The sodium citrate stabilizes the NALC by binding heavy
metal ions that may be present. Such reagents have proven to be
useful, but their action is often slow and their effectiveness
limited.
[0010] It is therefore an object of the present invention to
provide a method for the rapid and efficient lysis of mammalian
cells in biological samples while leaving microorganisms (bacteria
and fungi) in the sample substantially intact.
[0011] It as a further object of the invention to provide a method
for treating viscous, cell and protein containing biological
samples to render them liquid and freely flowing without disrupting
microorganisms that may be present.
[0012] It is a further object of the invention to provide a method
for making highly cellular and/or viscous biological samples
filterable through small pore size filters in order to retain and
concentrate microorganisms on the filter for further analysis.
SUMMARY OF THE INVENTION
[0013] The present invention is directed to achieving the
objectives above by means of the surprising discovery that
endogenous mammalian cells in a biological sample can be rapidly
and effectively lysed while leaving the cells of any microorganisms
that may be present substantially intact by mixing the sample with
a lysis solution as described herein and subjecting the mixed
sample to high-frequency ultrasound of prescribed frequency, power,
duty cycle and duration.
[0014] The invention is also directed to liquefying highly viscous
biological samples in a rapid manner while preserving substantially
all the microorganisms present in viable form, by mixing the sample
with an appropriate lysis solution as described herein and
subjecting the sample to high-frequency ultrasound of prescribed
frequency, power, duty cycle and duration.
[0015] This invention also provides a method for rapidly and
effectively capturing microorganisms in intact and viable form from
highly cellular and/or viscous biological samples by mixing the
sample with an appropriate lysis solution as described herein and
subjecting the sample to high-frequency ultrasound of prescribed
frequency, power, duty cycle and duration and filtering the treated
sample through a filter.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 is a graph that shows the filterability of whole
blood: lysis solution with and without ultrasonic treatment.
[0017] FIG. 2 shows the results of Fluorescence in situ
Hybridization (FISH) experiments for Coagulase Negative
Staphylococcus (CNS). The slide-bound membranes were examined using
a fluorescent microscope, a 60.times. oil objective, and the
AdvanDx PNA FISH filter cube (XF 53) for fluorescent organisms. CNS
was detected in all 4 samples.
[0018] FIG. 3 is a graph that shows peptide nucleic acid (PNA) FISH
detection of bacteria in platelet concentrates by CFU/mL.
[0019] FIG. 4 shows the results of FISH experiments for the
detection of Bacillus cereus in concentrated platelets.
[0020] FIG. 5 are two panels that show the results of FISH
experiments for the detection of Staphylococcus aureus (top) and
Serratia inarcescens (bottom) in clinical bronchoalveolar
lavage.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Described by the present invention are methods for selective
lysis of endogenous cells in a biological sample and method for
detecting, identifying, characterizing or quantifying
microorganisms in a biological sample, where the sample comprises a
mixture of endogenous cells and microorganisms. The present
inventors have found that endogenous cells in a biological sample
can be rapidly and effectively lysed while leaving the cells of any
microorganism that may be present in the sample substantially
intact.
[0022] The term "endogenous cells" is meant to refer to those cells
that are produced by or originate from or are growing within an
organism, tissue or biological sample. For example, in certain
preferred embodiments, an endogenous cell may be a mammalian
cell.
[0023] The term "biological sample" is meant to refer to cell
containing samples. In certain embodiments, a biological sample may
be a body fluid, for example, but not limited to, blood or blood
fractions, blood culture fluid, respiratory secretions,
cerebrospinal fluid, urine, stool, wound exudates and
naso-pharyngeal fluid or mucus. In other embodiments, the
biological sample may be platelets, platelet concentrate or a
mammalian cell culture. In still other embodiments, the biological
sample may be food or edible products.
[0024] In preferred embodiments, the phrase "substantially intact"
is meant to mean that the microorganisms are viable (i.e. they are
capable of growing) or that their cells appear to be intact when
imaged under a microscope in either stained or unstained form. In
related preferred embodiments, the phrase "substantially intact" is
meant to refer to at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, or more of the microorganisms in the sample are
recovered.
[0025] In certain exemplary embodiments, the method comprises
mixing the biological sample with a lysis solution, and subjecting
the mixture to ultrasound at a controlled temperature, thereby
selectively lysing the endogenous cells in the biological
sample.
[0026] Selective Lysis
[0027] As mentioned above, a method for selectively lysing
mammalian cells while leaving microorganisms intact was developed
by Zierdt and his colleagues. The method, published in 1977, uses a
mild detergent solution containing an enzyme mixture (Rhozyme
prepared from Aspergillus oryzae cultures). 30 mL of blood mixed
with conventional blood culture medium (brain heart infusion broth,
sodium polyanethol sulfate, p-aminobenzoic acid, 3% CO.sub.2) is
mixed with 20 mL lysis solution (0.1% Triton X-100 in 0.01M
NaHCO.sub.3--Na.sub.2CO.sub.3 buffer with 3% of stock Rhozyme 41
solution) and incubated for 30 minutes at 37.degree. C. Samples
lysed in this manner are capable of being filtered through 0.45
micron pore size filters. Zierdt subsequently refined the solution
by substituting the less toxic detergent Tween 20 for the Triton
X-100 used in the original protocol. Later, Zierdt studied a
variety of detergents useful in blood lysis for their efficacy in
lysing blood and their toxicity to bacteria as components of blood
culture media..sup.6 In addition to Triton X-100 and Tween 20, Brij
96 and digitonin performed well.
[0028] However, the Zierdt method has the drawbacks of a lengthy
incubation, a large (10:1) dilution of the sample, and the presence
of residual blood cell nuclei in the lysed sample.
[0029] Lysis Solutions
[0030] Lysis solutions can be useful in assays for microorganisms
for the purpose of lysing endogenous cells as well as liquefying
and clarifying mucus and phlegm containing samples. In addition to
the Rhozyme-based lysis solution described above, various
compositions of lysis solutions have been developed often
containing detergents, enzymes, salts and buffering agents.
[0031] Saponins, produced by certain plants, are ambipathic
glycoside detergent compounds that bind cholesterol. Saponins have
been found to be particularly effective for the selective lysis of
mammalian cells in microbial cell assays.
[0032] Gordon Dorn in U.S. Pat. No. 4,164,449 teaches a method of
concentrating microbial cells from blood by lysing the blood with
saponin, centrifuging the lysed blood and removing the residual
blood components from the microorganisms that are now in the
pellet. The saponin is preferably treated to remove toxic
components according the method taught in U.S. Pat. No. 3,883,425
also by Dorn which uses ultrafiltration to remove low-molecular
weight components considered to be toxic to microorganisms.
[0033] In U.S. Pat. No. 5,501,960, Dorn teaches the use of sodium
polyanethol sulfonate in combination with purified saponin to
improve the recovery of microorganisms from specimens containing
blood components.
[0034] The Dorn method requires mixing the blood with the
saponin-containing lysis solution followed by 30 minutes of
centrifugation. After centrifugation, the majority of the
supernatant is removed and discarded. The microorganism-containing
pellet is resuspended and distributed onto growth media for
culture. After culture, colonies can be counted and analyzed. While
quantitative, this method requires overnight culture and is
somewhat labor intensive.
[0035] In U.S. Pat. No. 5,043,267, Richards teaches the use of
saponin to lyse blood containing phagocytosed pathogens to release
degraded pathogen while leaving unphagocytosed pathogens intact.
Antigens from the degraded pathogens are detected with an
immunoassay while the intact pathogens arc cultured for
confirmation of the assay result. Richards extends the Dorn method
and allows detection of certain microbial antigens in one hour. The
antigens are cell membrane constituents (lipoteichoic acid and
peptidoglycan) which are not very specific. The ability of this
method to identify microorganisms is therefore limited.
[0036] According to the invention, lysis solutions can comprise
detergents or detergents combined with proteinase. In particularly
preferred embodiments, the lysis solution comprises a detergent and
a proteinase. Detergents useful in the invention include, but are
not limited to, saponin, nonionic surfactants such as Triton X-100
and polysorbate surfactants such as Tween 20. In preferred
embodiments, detergent concentrations can range from 0.1 to 10%.
Proteinases useful in the invention include proteinases derived
from Aspergillus (e.g. Aspergillus melleus) which have broad enzyme
activity and those with more specific activity like Streptokinase
which speeds the dissolution of fibrin clots. Commercially
available Proteinase from Aspergillus melleus in the range of 8
Units/mL to 160 Units/mL has been shown to work. Other enzymes can
be combined with proteinase to promote the breakdown of certain
biomolecules. Cholesterol esterase, lipase and DNase are examples
of enzymes that can be used in combination with proteinase.
Reducing agents such as TCEP can be helpful for liquefying mucoid
samples by reducing the disulfide bonds in mucin strands.
Chaotropic agents such as guanidinium chloride can also aid in the
dissolution of mucin gels by disrupting non-covalent bonds.
Hypotonic salt solutions can also promote lysis.
[0037] According to certain preferred exemplary embodiments, the
present inventors have found that a combination of saponin with
proteinase from Aspergillus melleus in a phosphate buffer is
particularly effective.
[0038] Accordingly, the present invention features a lysis solution
comprising a detergent and a proteinase, preferably a lysis
solution comprising Saponin and Proteinase. In certain embodiments,
the lysis solution further comprises a Sodium Phosphate buffer, pH
8.
[0039] The lysis solution preferably comprises 0.1%, 0.2%, 0.3%,
0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.11%, 1.12%,
1.13%, 1.14%, 1.15%, 1.16%, 1.17%, 1.18%, 1.19%, 1.2%, 1.21%,
1.22%, 1.23%, 1.24%, 1.25%, 1.26%, 1.27%, 1.28%, 1.29%, 1.30%,
1.31,%, 1.32%, 1.33%, 1.34%, 1.35%, 1.36%, 1.37%, 1.38%, 1.39%,
1.40%, 1.41%, 1.42%, 1.43%,1.44%, 1.45%, 1.46%, 1.47%, 1.48%,
1.49%, 1.50%, 1.55%, 1.60%, 1.65%, 1.70%, 1.72%, 1.75%, 1.80%,
1.82%, 1.85%, 1.90%, 1.92%, 1.95%, 2.0%, 2.2%, 2.5%, 3.0%, 3.1%,
3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%,
4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%,
5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%,
6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%,
7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%,
8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%,
9.8%, 9.9%, or 10.0% Saponin. Preferably, the lysis solution
comprises 1.15% Saponin.
[0040] The lysis solution preferably comprises 5.0, 5.25, 5.5, 6.0,
6.25, 6.5, 7.0, 7.25, 7.5, 8.0, 8.25, 8.5, 9.0, 9.25, 9.5, 10.0,
10.25, 10.5, 11.0, 11.25, 11.5, 12.0, 12.25, 12.5, 13.0, 13.25,
13.5, 14.0, 14.25, 14.5, 15.0, 15.25, 15.5, 16.0, 16.25, 16.5,
17.0, 17.25, 17.5, 18.0, 18.25, 18.5, 19.0, 19.25, 19.5, 20.0,
20.25, 20.5, 21.0, 21.25, 21.5, 22.0, 22.25, 22.5, 23.0, 23.25,
23.5, 24.0, 24.25, 24.5, 25.0, 25.25, 25.5, 26.0, 26.25, 26.5,
27.0, 27.25, 27.5, 28.0, 28.25, 28.5, 29.0, 29.25, 29.5, 30.0,
31.25, 31.5, 32.0, 32.25, 32.5, 33.0, 33.25, 33.5, 34.0, 34.25,
34.5, 35.0, 35.25, 35.5, 36.0, 36.25, 36.5, 37.0, 37.25 or 37.5
Units Proteinase. In preferred embodiments, the lysis solution
comprises 11.25 Units Proteinase.
[0041] Buffer concentrations from 0.01M to 0.1M have been tested.
Tris and DNase running buffer have been used in place of sodium
phosphate buffer. Accordingly, in further related embodiments,
other buffers known to one skilled in the art can be used. It is
therefore likely that other buffers can be substituted.
[0042] In certain preferred embodiments, the lysis solution
comprises [0043] 0.1M Sodium Phosphate buffer, pH 8; [0044] 1.15%
Saponin; and [0045] 11.25 Units/mL Proteinase.
[0046] In further preferred embodiments, the Saponin is from
Quillaja bark. Preferably, the Proteinase is from Aspergillus
melleus.
[0047] In other exemplary embodiments, the lysis solution comprises
[0048] 0.1M Sodium Phosphate buffer, pH 8 [0049] 1.15% Saponin from
Quillaja bark (Sigma 54521-25G) [0050] 11.25 Units/mL Proteinase
from Aspergillus melleus, Type XXIII (Sigma P4032-25G)
[0051] Sonics
[0052] As mentioned above, ultrasound has been used to lyse cells
to release the intracellular contents for molecular analysis. Beads
of glass or other materials are often added to the sample to
increase the mechanical action of the ultrasound. Transducers of
various shapes have been used. In the prior work the outcome has
been the comprehensive lysis of all the cells in the sample. By
contrast, in the methods of the present invention the selective
lysis of particular cell populations is achieved.
[0053] Two rather different types of ultrasound generating
equipment are available. They are distinguished by the operating
frequency. One type operates in the frequency range of 20 kHz (just
above the range of human hearing) to about 80 kHz. The other type
operates in the frequency range of 500 kHz to 1.5 MHz or higher and
is often called megasonics. In the former, the wavelength of the
sound waves ranges from about 80 mm to about 20 mm; while in the
latter, the wavelength ranges from about 3 mm to about 1 mm. The
shorter wavelengths produced in the megasonic range allow better
localization of the sonic energy to the biological sample that may
have a volume of approximately 1 cm.sup.3. Moreover, bubbles
produced by cavitation will generally be smaller at higher
frequencies since the shorter cycles give them less time to grow
before they collapse in the compressive phase of the cycle. As used
herein, the term "high frequency ultrasound" is meant to refer to
ultrasound in the megasonic range.
[0054] Some embodiments described below employ the Covaris S2 high
frequency ultrasound system. In that system, a concave transducer
is used to focus the acoustic energy on the sample. The transducer
operates in a water bath in which the sample tube is immersed. The
acoustic energy is coupled to the sample by the water. The system
operates at approximately 500 KHz in a pulsed mode. The number of
cycles per burst, the duty cycle, time duration and the intensity
are settable on the instrument.
[0055] Covaris Settings:
[0056] The following settings have been found to provide optimum
results with the Covaris S2.
[0057] Heat water bath to 37.degree. C.
[0058] Degas water bath for 30 min prior to use per manufacturer's
instruction.
[0059] Mix 1 mL of blood with 1 mL of lysis solution in a 3 mL
round-bottom glass tube.
[0060] Treat sample for 100 seconds at 10% duty cycle, 1 intensity,
1000 cycles/burst.
[0061] Treat sample for 60 seconds at 10% duty cycle, 2 intensity,
1000 cycles/burst.
[0062] Treat sample for 60 seconds at 10% duty cycle, 2 intensity,
200 cycles/burst.
[0063] Lower intensity (intensity setting 1) can be used in the
last two steps if the duration is increased.
[0064] Other embodiments use a non-focused, high-frequency
ultrasound system manufactured by ProSys, Inc. In this system, a
planar transducer emits a directed beam of ultrasound. The diameter
of the beam is governed by the size of transducer. The system
operates at approximately 1 MHz. The duty cycle, time duration and
the intensity are settable on the instrument. Sonic energy from the
transducer can be coupled into the biological sample by water, a
gel, or an elastomer.
[0065] ProSys Settings:
[0066] The following settings have been found to provide optimum
results with the ProSys.
[0067] 100 ms Pulse
[0068] 50% Duty Cycle
[0069] 45 Watts
[0070] 5 minute duration
[0071] The present inventors have found that when used as described
above, these systems are effective in achieving the selective lysis
of mammalian cells while leaving microorganisms intact and
viable.
[0072] Systems operating at lower frequencies (20 to 40 kHz) are
available from other suppliers such as Branson Ultrasonics. These
do not focus the acoustic energy with a focusing transducer but can
concentrate the energy with a transducer probe having a narrow tip.
Our experience with these systems has shown them to be much less
effective in lysing blood cells than the high frequency
systems.
[0073] Filtration
[0074] It can be advantageous to filter lysed samples through
filters having pores sufficiently small to retain microorganisms in
the sample. As previously mentioned, the retained microorganisms
can be supplied with nutrients and grown into colonies on the
membrane for counting and further analysis or they can be
visualized on the filter by fluorescence microscopy following
treatment with fluorescent probes or antibodies. 0.6 micron and
0.45 micron filters are commonly used to capture bacteria and fungi
from liquid samples. 0.45 micron and 0.2 micron filters are
commonly used for filter sterilization of water and media. Blood
and other cell-containing biological samples are not directly
filterable with these filters because the filters are rapidly
clogged by these highly cellular liquids. Filterability is a good
measure of the effectiveness of a lysis procedure.
[0075] Various types of filters can be used. Membrane filters made
of nylon, polycarbonate, polyester and aluminum oxide have been
used. Track-etch membranes of polycarbonate or polyester are useful
if it is desired to image microorganisms in a sample after the
lysis procedure. These membranes feature smooth, flat surfaces with
well-controlled cylindrical pores. Anopore (aluminum oxide) filters
are also flat with well-controlled pores and thus useful for
imaging. Pore sizes from 0.2 to 1 micron are effective for
retention of bacteria and yeast with 0.45 to 0.8 micron being most
useful. Larger pore sizes (up to 4 microns) can be used if only
yeast (fungi) are of interest.
[0076] Detection and Identification of Microorganisms
[0077] Various assays for the detection and identification of
microorganisms have been developed. Three general classes of assays
are in common use. The first class encompasses culture-based
methods whereby any microorganisms present in the biological sample
are allowed to grow perhaps with the admixture of nutrient media
into the sample. The growth of the microorganism(s) can be detected
in various ways, such as by changes in the turbidity or the pH of
the sample or by the evolution of CO.sub.2 driven by the metabolic
activity of the microorganisms during their growth. Microorganisms
can be identified on the basis of which of a range of biochemical
nutrient sources they are able to utilize for growth. Likewise,
their resistance to various antibiotics can be assessed by
characterizing their ability to grow in the presence of different
concentrations of the antibiotics of interest. Culture-based
methods are widely used for both identification and
characterization of microorganisms. The primary disadvantage of
these methods is the length of time (8 to 24 hours) required to get
results.
[0078] The second class of assays involve the use of stains,
binding agents or probes to confer a detectable color or label to
the cells of any microorganisms that may be present. Microscopic
examination is generally used to visualize the colored or labeled
cells.
[0079] The Gram stain is an example of a stain that is commonly
used in microbiology. It involves the use of crystal violet and
iodine to stain fixed bacterial cells. Gram positive bacteria can
be distinguished from Gram negative bacteria by their ability to
retain the purple color of the crystal violet stain after washing
with alcohol or acetone. Gram negative bacteria lose the purple
color during the wash and are stained pink by the counter-stain,
usually safranin or basic fuchsin, applied after the wash.
[0080] Antibodies are examples of binding agents. Antibodies that
recognize and bind to bacterial cell-surface molecules have been
developed. Such antibodies can be chemically modified to
incorporate fluorescent tags. They can be utilized in assays such
as direct fluorescence assays (DFA) in which one or more
fluorescently tagged antibodies are mixed with the biological
sample to be tested and incubated to allow antibody binding.
Following a wash step, the sample is examined with a fluorescent
microscope to detect cells to which the fluorescent antibodies have
bound. Other examples of binding agents include aptamers, peptides,
lectins and phages.
[0081] Probes are molecules that incorporate nucleobases. They can
bind to DNA or RNA by hydrogen bonding of the nucleobases in the
probe to complementary nucleobases in the DNA or RNA in a process
called base pairing. Probes can be made up of DNA, PNA (peptide
nucleic acid), LNA (locked nucleic acid), and related molecules and
combinations thereof. The number and sequence of the nucleobases in
a probe determine what target sequence the probe will bind to
according to the rules of base pairing, as well as the strength and
specificity of the binding. The strength of the binding under
various conditions of salt concentration and pH depends on type(s)
of the component molecules (DNA, PNA, LNA and others) that make up
a probe. Probes can incorporate fluorescent labels that make them
detectable by fluorescence imaging.
[0082] Many different fluorescent labels (fluorophores) have been
developed for use in biological assays. There are labels with
excitation and emission wavelengths ranging from the ultraviolet to
the near-infrared regions of the electromagnetic spectrum. Labels
further differ in the width of the excitation and emission bands,
the Stokes shift, and the fluorescence lifetime. Representative
fluorescent labels include fluorescein, tetramethyl rhodamine,
Texas Red, and Cy5.
[0083] Fluorescence-in-situ-hybridization (FISH) is an assay
utilizing fluorescently labeled probes. In one type of FISH assay,
probes directed at ribosomal RNA (rRNA) are used. The sequence of
rRNA varies from species to species. This allows FISH assays to be
made species-specific through the proper design of the probe
sequence. Agents that help to preserve RNA directly such as TCEP
and the cationic, quaternary ammonium salts, tetra- and
hexa-decyltrimethylammonium bromide, like those found in RNAprotect
Bacteria Reagent (Qiagen 76506) or indirectly by maintaining the
bacterial cell wall (e.g. MgSO4) can be beneficial to these assays.
PNA FISH assays are commercially available for diagnostic use in
hospital clinical microbiology laboratories for the identification
of microorganisms in suspected blood stream infections. In these
assays, the probes comprise fluorescently labeled PNA
molecules.
[0084] The advantage of this second class of assays is that they
result in intact cells that can be microscopically examined This
allows the size, shape and clustering characteristics of the cells
to be assessed along with the staining behavior. PNA FISH is
particularly advantageous because of its generality, high
specificity and easily visualized fluorescence.
[0085] The third class of assays for the detection and
identification of microorganisms encompasses those that involve the
use of molecular methods, including amplification techniques such
as PCR. In contrast to the first two classes of assays in which the
cells to be detected remain intact, in these assays the target is
microbial DNA, RNA or proteins that have been released by the lysis
or rupture of the cells. In the case of PCR, the product(s) of the
amplification can be detected by sequencing, through the use of
microarrays, by the use of intercalating dyes or with probes
carrying a detectable label such as one or more fluorophores or
nanoparticles. Amplification-based methods suffer from certain
drawbacks including false negatives due to inhibitors present in
many biological samples, and false positives caused by remnant DNA
from microorganisms killed by host defense mechanisms.
PREFERRED EMBODIMENTS OF THE INVENTION
[0086] Selective ultrasonic lysis of whole blood, concentrated
platelets, respiratory secretions, urine, or blood culture for the
purpose of microbial detection is carried out by treating the
sample with a buffered solution comprised of saponin or Tween-20
and proteinase in a ratio ranging from 1:1 to 1:4; lysing the
mixture with focused or planar high-frequency ultrasonic waves; and
concentrating the lysed sample via filtration or centrifugation.
Detection can then be accomplished by the following additional
steps: rinsing the concentrate; probing the rinsed concentrate with
fluorescently labeled PNA probes and hybridizing the probes to
specific rRNA targets; stringent washing to remove unbound and
non-specifically hybridized probe; and analyzing the sample to
detect fluorescent microorganisms. These are the preferred
protocols.
[0087] Reagent Preparation
[0088] The lysis solutions were optimized individually for each
sample type. Saponin was chosen for the lysis of whole blood and
platelets for its superior ability to lyse blood cell membranes
while leaving bacterial cells intact. Tween-20 was used in place of
saponin for respiratory secretions because the sample was easier to
filter after treatment with Tween-20 while still preserving the
microorganisms.
[0089] Whole Blood and Blood Culture Formulation [0090] 1) Add 115
mg saponin from Quillaja bark purified to remove low molecular
weight contaminants (Sigma S4521) to 10 mL 0.1M sodium phosphate
buffer, pH 8. [0091] 2) Vortex briefly to dissolve. [0092] 3) Add
112.5 Units of Proteinase from Aspergillus melleus Type XXIII
(Sigma S4032) to the solution. [0093] 4) Vortex briefly to
dissolve. [0094] 5) Filter solution with a 32 mm, polyethersulfone
(PES) 0.2 um syringe filter.
[0095] Concentrated Platelet Formulation [0096] 1) Add 58 mg
saponin from Quillaja bark purified to remove low molecular weight
contaminants (Sigma S4521) to 10 mL 0.1M sodium phosphate buffer,
pH 8. [0097] 2) Vortex briefly to dissolve. [0098] 3) Add 56.25
Units of proteinase from Aspergillus melleus Type XXIII (Sigma
S4032) to the solution. [0099] 4) Vortex briefly to dissolve.
[0100] 5) Filter solution with a 32 mm PES 0.2 um syringe
filter.
[0101] Possible perturbations: The concentration of saponin and/or
proteinase may be increased to promote filterability or decreased
to improve recovery. Additional agents to promote degradation of
fibrin clots and filterability such as Streptokinase may be
added.
[0102] Respiratory Secretions Formulation [0103] 1) Add 115 .mu.L
of Tween-20 to 10 mL 0.1M sodium phosphate buffer, pH 8. [0104] 2)
Vortex well to mix completely. [0105] 3) Add 112.5 Units of
proteinase from Aspergillus melleus Type XXIII (Sigma S4032).
[0106] 4) Vortex briefly to dissolve. [0107] 5) Filter solution
with a 32 mm PES 0.2 um syringe filter.
[0108] Urine Formulation [0109] 1) Add 25 .mu.L of Tween-20 to 50
mL of 1.times. Phosphate Buffered Saline (Sigma P7059). [0110] 2)
Filter solution with a 32 mm PES 0.2 um syringe filter.
[0111] Possible perturbations: Proteinase may be added to this
formulation to increase filterability.
[0112] Sample Preparation [0113] 1) Add 1 mL of lysis solution to a
3 mL round bottom glass tube (Covaris 520067). [0114] 2) Add 1 mL
of sample: whole blood anti-coagulated with sodium heparin,
concentrated platelets anti-coagulated with acid-citrate-dextrose
(ACD), or respiratory secretions without preservative. [0115] 3)
Cap tube (SUN-SRi 200596) and invert to mix several times.
[0116] Possible perturbations: 1) 200 .mu.L of Bond Breaker TCEP
Solution (Thermo Scientific 77720) may be added to the preparation
to protect RNA and/or to improve filterability of respiratory
secretions. 2) For very thick, mucoid respiratory secretions or for
blood culture with 10.sup.5 CFU/mL or more less sample may be added
to the preparation and the difference in volume may be replaced
with additional lysis solution, water, or buffer. 3)
Anti-coagulants other than those listed above may be used for whole
blood and concentrated platelets.
[0117] Ultrasonic Lysis
[0118] The sample was treated with acoustic energy to promote
mixing, selectively lyse human cells over bacteria and yeast, and
break apart sample matrix for improved filtration. This has been
done with focused acoustic energy in the Covaris S2 and non-focused
acoustic energy with the ProSys.
[0119] Covaris Method [0120] 1) Heat water bath to 37.degree. C. by
setting chiller to 37.7.degree. C. [0121] 2) Degas water bath for
30 min prior to use per manufacturer's instruction. [0122] 3) Place
3 mL glass tube into tube holder (custom-built tube holder with
fixed positioning of the tube in the vertical and horizontal axes).
[0123] 4) Treat sample for 100 seconds at 10% duty cycle, 1
intensity, 1000 cycles/burst [0124] 5) Treat sample for 60 seconds
at 10% duty cycle, 2 intensity, 1000 cycles/burst [0125] 6) Treat
sample for 60 seconds at 10% duty cycle, 2 intensity, 200
cycles/burst
[0126] Possible perturbations: 1) Reduce treatment time in step 5
and 6 to 30 seconds and cycles/burst to 500 in step 5 and to 100 in
step 6 to improve recovery in platelets. 2) Treat urine with 3
cycles of step 6 only. 3) Treat blood culture with 1 cycle of step
6 only.
[0127] Concentration, Hybridization, and Detection
[0128] The lysate was concentrated on an aluminum and SiO.sub.2
coated polycarbonate track etched membrane (PCTE) filter bonded to
a plastic slide with a ring press and supported by a stainless
steel fit. The slide was held in a custom-built, heated slide
holder with a vacuum manifold. The filtration area was 52
mm.sup.2.
[0129] Concentration Method [0130] 1) Filter entire lysate using a
vacuum equivalent 5 to 15 inches of Hg. [0131] 2) Rinse filter and
holder 3 times with 830 .mu.L each of 1.times. PBS while vacuuming.
[0132] 3) Turn off and purge vacuum.
[0133] Possible perturbations: 1) The lysate may be rinsed with a
1% solution of dextran sulfate, RNAprotect, and/or 400 mM
MgCl.sub.2 in 1.times. PBS to improve hybridization and detection.
2) The lysate may be concentrated using centrifugation rather than
filtration. 3) Less than the entire volume of lysate may be
filtered for samples from which only high colony count organisms
are relevant like urine.
[0134] Hybridization and Wash Method [0135] 1) Filter PNA FISH Flow
Hybridization Buffer immediately prior to use with a 13 mm, 0.2
.mu.m. polytetrafluorethylene (PTFE) syringe filter. [0136] 2) Add
400 .mu.L of filtered or PNA FISH Flow Hybridization Buffer
containing 100 nM to 500 nM or 50 nM probe for bacteria or yeast
respectively to the holder. [0137] 3) Cover the holder to prevent
evaporation. [0138] 4) Heat the retentate and hybridization buffer
in the holder for 30 minutes at 55.degree. C. [0139] 5) Vacuum away
hybridization buffer. [0140] 6) Turn off and purge vacuum. [0141]
7) Add 500 .mu.L of PNA FISH Flow Wash Buffer to the holder. [0142]
8) Cover holder to prevent evaporation. [0143] 9) Heat the
retentate and wash buffer in the holder for 10 minutes at
55.degree. C. [0144] 10) Vacuum away wash buffer. [0145] 11) Turn
off and purge vacuum. [0146] 12) Repeat steps 6-10.
[0147] Possible perturbations: 1) Add 40 ul of Bond Breaker TCEP
Solution to the holder with the hybridization buffer to protect RNA
and improve hybridization and detection. 2) Add 10% methanol to the
wash buffer to preserve Gram negative cells during wash step. 3)
Use Tween-20 in the wash buffer rather than Triton-X to preserve
Streptococcus pneumoniae. 4) Add 1% solution of dextran sulfate,
RNAprotect, and/or 400 mM MgCl.sub.2 to the wash buffer improve
hybridization and detection.
[0148] Detection [0149] 1) Remove slide from the holder and allow
to air dry. [0150] 2) Add 1 drop of mounting media (20% (v/v) 1M
Tris-HCl pH 7.6, 80% (v/v) glycerol and 2% (w/v) DABCO) and a 15
mm, round, glass coverslip (Ted Pella, Inc. 26024). [0151] 3) View
and image filter immediately after adding mounting media on a
fluorescent microscope or automated scanner.
[0152] Possible perturbations: The concentrated, hybridized lysate
may be fixed to and viewed, imaged, and/or scanned on a solid
surface rather than a filter.
[0153] Mass Spectrometry Analysis
[0154] Following lysis, mass spectrometry (MS) can be used for
detection, identification, characterization or quantification of
microorganisms in a sample.
[0155] In an embodiment, the detection, identification,
characterization or quantification is done by a mass spectrometer,
which may be one of the following:
matrix-assisted-laser-desorption-ionization (MALDI) mass
spectrometry (e.g. MALDI-TOF MS), Tandem MS, ESI-TOF, ESI-iontrap,
LC-MS, GC-MS, ion mobility MS, laser desorption ionization mass
spectrometry (LDI-MS) and quadrupole-MS. Other mass spectrometry
devices and methods now existing or which may be developed are also
within the scope of the present invention.
[0156] Mass spectrometry is a sensitive and accurate technique for
separating and identifying molecules. Generally, mass spectrometers
have two main components, an ion source for the production of ions
and a mass-selective analyzer for measuring the mass-to-charge
ratio of ions, which is and converted into a measurement of mass
for these ions. Several ionization methods are known in the art and
described herein.
[0157] Different mass spectrometry methods, for example, quadrupole
mass spectrometry, ion trap mass spectrometry, time-of-flight mass
spectrometry, gas chromatography mass spectrometry and tandem mass
spectrometry, can utilize various combinations of ion sources and
mass analyzers which allows for flexibility in designing customized
detection protocols. In addition, mass spectrometers can be
programmed to transmit all ions from the ion source into the mass
spectrometer either sequentially or at the same time. Furthermore,
a mass spectrometer can be programmed to select ions of a
particular mass for transmission into the mass spectrometer while
blocking other ions.
[0158] Mass spectrometers can resolve ions with small mass
differences and measure the mass of ions with a high degree of
accuracy. The high degree of resolution and mass accuracy achieved
using mass spectrometry methods allows the use of large sets of
tagged probes because the resulting reporter tags can be
distinguished from each other. The ability to use large sets of
tagged probes is an advantage when designing multiplex
experiments.
[0159] Another advantage of using mass spectrometry is based on the
high sensitivity of this type of mass analysis. Mass spectrometers
achieve high sensitivity by utilizing a large portion of the ions
that are formed by the ion source and efficiently transmitting
these ions through the mass analyzer to the detector. Because of
this high level of sensitivity, even limited amounts of sample can
be measured using mass spectrometry.
[0160] Mass spectrometry methods are well known in the art (see
Burlingame et al. Anal. Chem. 70:647R-716R (1998); Kinter and
Sherman, Protein Sequencing and Identification Using Tandem Mass
Spectrometry Wiley-Interscience, New York (2000)).
[0161] In recent years, MALDI-TOF mass spectrometry has emerged as
a powerful tool for the identification of bacteria and other
microorganisms. The advantages of this approach include relatively
straightforward sample preparation and rapid analysis. Intact
bacterial cells from, for example, a colony can be mixed with MALDI
matrix and applied directly to the MALDI sample plate. Pattern
recognition applied to the complex spectra that are obtained allows
identification of bacteria, often to the strain level (see Lay.
Mass Spectrometry Reviews 20: 172-194 (2001)).
[0162] Mass spectrometry and MALDI-TOF in particular is well suited
for the analysis of microorganisms obtained using the methods of
the invention.
EXAMPLES
[0163] The examples below demonstrate the methods for clinical
samples such as blood, platelet concentrates and bronchoalveolar
lavage. The method will have utility for many other types of
samples in which the detection of microorganisms at low levels is
of value. These include biological samples such as tissue, stool,
lavage fluids, needle aspirates and saliva. Another category
includes foods such as milk, meats, cheese and vegetables.
Example 1
Filterability Comparison of the Selective Ultrasonic Lysis Approach
with the Zierdt Method
[0164] Whole, sodium heparin anti-coagulated blood was mixed 1:1 or
1:10 with a lysis solution based on Zierdt's refined lysis solution
(Zierdt, J. Clin. Microbiol., 1982) which contained Rhozyme 41
(Rohm and Hass, Philadelphia, Pa.), a crude proteinase mixture
extracted from Aspergillus oryzae and Tween-20 in sodium phosphate
buffer. Rhozyme 41 was no longer available; proteinase from
Aspergillus melleus was substituted. The mixtures of blood and
lysis solution were subjected to one hour incubation at 37.degree.
C., focused ultrasonic waves in the Covaris, or no treatment. Then
they were tested for filterability and examined microscopically to
assess the number of residual cells.
[0165] Two lysis solutions were made--one with and one without
detergent. The lysis solutions were made by adding 350 .mu.L of
Tween-20 to 49.65 mL of 0.01 M sodium phosphate buffer and mixing
thoroughly. Then, if required, 250 mg of proteinase were added and
briefly vortexed to dissolve. Lysis solutions were filtered with a
0.2 .mu.m, 32 mm, PES syringe filter.
[0166] Samples were prepared by adding 0.5 mL of whole blood and
0.5 mL of lysis solution (1:1) to a 2 mL, round-bottom, snap-cap,
plastic microcentrifuge tube (Eppendorf 022363352) or 0.1 mL of
blood and 0.9 mL of lysis solution (1:10). The samples were capped
and inverted to mix. The samples remained at these concentrations
during the incubation at 37.degree. C. or treatment with the
Covaris. The 1:1 sample was diluted 1:5 before being examined
microscopically or tested for filterability in order to obtain the
same blood to fluid ratio as the 1:10 samples.
[0167] Samples were heated in a 37.degree. C. water bath for one
hour in accordance with Zierdt's lysis procedure (Zierdt, J. Clin.
Microbiol., 1982). Other samples were treated with the Covaris S2
instead of heat. The Covaris bath was filled with deionized water,
degassed for 30 minutes, and chilled to 7.degree. C. to promote
better sound transmission according to the manufacturer's
recommendations. The Covaris bath was not chilled for all of the
testing in order to promote proteinase activity. When the bath was
not chilled it reached 25.degree. C. The Covaris samples were
treated for 60 consecutive seconds at an intensity of 3, 10% duty
cycle, and 200 cycles per burst. Control samples were mixed with
lysis solution and tested immediately after mixing.
[0168] All samples were examined for residual cells using bright
field microscopy and a 20.times. objective. Slides were made by
pipetting 15 .mu.L of thoroughly mixed sample (1:10) onto a glass
slide and adding a 22.times.22 mm coverslip. An average number of
cells was taken over multiple fields of view.
[0169] All samples were tested for filterability as a measure of
how well the cells had been lysed. The barrel from a 3 mL syringe
was fitted with a 13 mm, 0.45 .mu.m, nylon syringe filter. The
outlet of the syringe filter was attached to a vacuum pulling at
5'' Hg. The filter was primed with 0.5 mL of 1.times. PBS. Another
1 mL of 1.times. PBS was filtered and timed to obtain a
normalization value for each filter. Finally 1 mL of sample mixed
with lysis solution and either treated with heat or sonic energy or
untreated was filtered and timed.
TABLE-US-00001 TABLE 1 Filterability and Microscopic Appearance of
Whole Blood Following Various Lytic Treatments Blood:Lysis
Microscopy Filtration Solution Lysis Solution and Treatment
(cells/20X field) (sec) 1:10 Detergent 25 .infin. Detergent &
proteinase 75 27 Detergent & proteinase for 1 hr @ 1 23
37.degree. C. (Zierdt method) Detergent with Covaris @ 7.degree. C.
0 15 Detergent & proteinase with Covaris 0 14 @ 25.degree. C.
1:1 Detergent & proteinase for 1 hr @ Not tested in this
experiment; this 37.degree. C. does not filter Detergent with
Covaris @ 7.degree. C. 12-18 61 Detergent & proteinase with
Covaris 12-18 15 @ 25.degree. C.
[0170] Results showed that treatment with ultrasonic energy
produced a lysate that was more filterable and contained less
cellular debris than the Zierdt method (Table 1). Although not
tested during these experiments, other work showed the Zierdt
method produced a product that was not filterable if used in a 1:1
mixture with whole blood. They also demonstrated that the
composition of the lysis fluid has secondary contributions to
filterability. However, it was unclear whether the significant gain
in filterability for the 1:1 sample with detergent and proteinase
over the sample with detergent only was due to the proteinase or
the warmer Covaris bath. Further experiments were conducted at the
same temperature. The results showed adding proteinase to the
ultrasonic lysis increases filterability and may also reduce the
number of residual blood cells even though the sonic treatment only
lasted 60 seconds and it was at room temperature (Table 2). This
indicates that the acoustic energy may have been speeding up
enzymatic reactions as well as shearing cells.
TABLE-US-00002 TABLE 2 Filterability and Microscopic Appearance of
Whole Blood Treated with Proteinase and Ultrasonic Energy
Blood:Lysis Lysis Microscopy Filtration Solution Solution and
Treatment (cells/20X field) (sec) 1:1 Detergent with Covaris 10 38
@ 20.degree. C. Detergent & proteinase 2-3 20 with Covaris @
20.degree. C.
[0171] International patent application WO 2009/015484 A1 (Peytavi
et al.) demonstrates the concentration of microbial cells from
whole blood using high concentrations of heat-treated saponin and
centrifugation. 10% heat-treated saponin in the lysis solution with
proteinase and Covaris treatment did not improve filterability
(Table 3).
TABLE-US-00003 TABLE 3 Filterability of Whole Blood Treated with
High Concentration Saponin and Ultrasonic Energy Saponin
Concentration in Lysis Solution Filtration (sec) 10%, heat-treated
69 1.4%, untreated 51 0.7%, untreated 56
Example 2
Recovery of Microorganisms from Whole Blood After Selective Lysis
with Focused Acoustic Energy
[0172] Nine different microorganisms were inoculated into whole,
sodium heparin treated blood. The blood was mixed with lysis
solution and plated before and after ultrasonic lysis. The plates
were incubated overnight and colonies were counted in the morning
to determine percent yield after Covaris treatment in blood and
lysis solution.
[0173] Candida albicans, Candida krusei, Enterococcus faecium,
Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus
epidermidis were subcultured to non-selective agar media. The
plates were incubated at 37.degree. C. overnight. The following
morning they were inoculated to broth media from the freshly
subcultured agar media and allowed to incubate at 37.degree. C. for
2.5 hours. Bacteria and yeast were diluted serially with 1.times.
PBS with 0.05% Tween-20 to 1:1,000 or 1:10,000.
[0174] Lysis solution was made by adding 350 .mu.L of Tween-20 to
49.65 mL of 0.01M sodium phosphate buffer and mixing thoroughly.
Then 250 mg of proteinase were added and briefly vortexed to
dissolve. The lysis solution was filtered with a 0.2 .mu.m, 32 mm,
PES syringe filter.
[0175] Samples were prepared by adding 0.5 mL of whole blood, 0.5
mL of lysis solution, and 10 to 40 .mu.L. of the diluted
microorganisms to a 2 mL, round-bottom, snap-cap, plastic
microcentrifuge tube. The samples were capped and inverted to mix.
100 .mu.L of the blood mixture were plated using a plate spinner
and a disposable, sterile T-spreader before and after treatment
with the Covaris. The plates were incubated overnight at 37.degree.
C. overnight. Colonies were counted and recorded the following
morning to determine percent yield (Table 4). This experiment was
performed three times.
[0176] Samples were treated with the Covaris S2. The Covaris bath
was filled with deionized water and degassed for 30 minutes. The
water bath was maintained at 20.degree. C. The Covaris samples were
treated for 60 consecutive seconds at an intensity of 2, 10% duty
cycle, and 200 cycles per burst.
TABLE-US-00004 TABLE 4 Recovery from Whole Blood after Ultrasonic
Lysis Yield Average Standard Organism Experiment 1 Experiment 2
Experiment 3 Yield Deviation C. albicans 300% 263% 243% 268% 29 C.
krusei 63% 217% 129% 136% 77 E. faecium 92% 134% 267% 164% 91 E.
faecalis 150% 123% 165% 146% 21 E. coli 88% 61% 63% 70% 15 K.
pneumoniae 93% 41% 50% 61% 28 P. aeruginosa 41% 56% 48% 48% 8 S.
aureus 171% 164% 143% 159% 15 S. epidermidis 93% 70% 192% 118%
65
[0177] The results showed some yields were greater than 100%. In
this experiment, the microorganisms were quantitated by the number
of colonies formed after overnight growth. Cells in a cluster or
chain form single colonies and thus represent single colony forming
units (cfu). If disrupted, such clusters can form multiple
colonies. We have observed that immediately following treatment
with the Covaris, organisms that normally occur in clusters are
generally seen to be present in single cell form.
[0178] The results also indicated some loss of viability. The
impact was greatest on Gram negative rods.
Example 3
Detection of Staphylococcus aureus from Selective Lysis of Whole
Blood Versus Routine Blood Culture
[0179] Staphylococcus aureus (SA) that had been diluted serially
was inoculated into sterile, fresh, whole, sodium heparin treated
blood and incubated to allow for phagocytosis. The blood was then
split into two samples and subjected to either selective lysis or
turned into a mock blood culture. The portion that underwent
selective lysis was filtered. The filter was placed on an agar
plate and incubated overnight. Colonies were counted in the
morning. The portion that was made into a mock blood culture was
incubated two days and checked for growth using Gram stain and
subculture to an agar plate. The results of both methods were
compared to the number of CFU initially inoculated into the blood
and to each other.
[0180] SA (ATCC 29213) was subcultured to trypticase soy agar with
5% sheep blood and incubated at 37.degree. C. overnight. The
following morning it was inoculated into trypticase soy broth and
incubated at 37.degree. C. at 180 rpm for 2 hours. The broth was
then diluted serially 1:10 with 1.times. phosphate buffered saline
with 0.05% Tween-20 to 1.times.10.sup.-9. 100 .mu.l of the
10.sup.-5 and 10.sup.-6 dilutions and 1 mL of the 10.sup.-7,
10.sup.-8, and 10.sup.-9 dilutions were filtered with the Microfil
V Filtration Device (Millipore MVHAWG124) that had been pre-wetted
with 1.times. phosphate buffered saline. The device contains a 47
mm, mixed cellulose ester filter with 0.45 .mu.m pores and a
printed grid for counting colonies. The filter was removed from the
device, placed on trypticase soy agar, and incubated overnight at
30.degree. C. The filters were examined for growth the following
morning, and colonies were counted. These counts were used to
estimate how many CFU were added to the aliquots of blood.
[0181] The lysis solution was prepared by adding 140 mg of saponin
to 10 mL of 0.1M sodium phosphate buffer, pH 8 and vortexing to
dissolve. Then 51 mg of proteinase were added and vortexed briefly
to dissolve. The solution was filtered with a 0.2 .mu.L, 32 mm, PES
syringe filter.
[0182] Five 2.5 mL aliquots of blood were inoculated with 50 .mu.L
each of the last five SA dilutions, 10.sup.-5 to 10.sup.-9, and
incubated for 1 hour at 37.degree. C. to allow for phagocytosis.
The samples were then mixed and split into separate 1 mL aliquots.
The excess 0.55 mL from each sample was discarded. Each 1 mL
aliquot was either mixed with 1 mL of lysis solution or 3 mL of
BacT/ALERT SA blood culture media (Biomerieux 259789).
[0183] The mock blood cultures were incubated for 2 days at
37.degree. C. and 180 rpm. They were Gram stained and subcultured
semi-quantitatively to trypticase soy agar each morning to monitor
for growth. The agar subcultures were incubated overnight at
37.degree. C. and examined for growth the following morning.
[0184] The bath on the Covaris was filled with deionized water,
heated to 37.degree. C., and degassed for 30 minutes. The aliquots
that were mixed with lysis solution were loaded into the custom
tube holder designed to fix the X and Y axis. The samples were
warmed and mixed for 100 seconds at an intensity of 1, 10% duty
cycle, and 1000 cycles per burst.
[0185] Then the intensity was increased to 2 for 60 seconds.
Finally, the cycles per burst were decreased to 200 for 60
seconds.
[0186] The lysed samples were filtered on Microfil V filtration
devices that had been pre-wetted with 1.times. phosphate buffered
saline. The filters were rinsed with more 1.times. phosphate
buffered saline. The filters were removed from the device, placed
on trypticase soy agar, and incubated overnight at 30.degree. C.
The filters were examined for growth the following morning, and
colonies were counted.
TABLE-US-00005 TABLE 5 Comparison of Selective Lysis of Whole Blood
and Blood Culture Number of Approx. CFU Colonies from Added to
Selectively Lysed Growth or No Each Split Whole Blood on Growth of
Blood Sample Day 1 Culture 64.6 159 Growth Day 1 6.8 37 Growth Day
1 0.56 0 No Growth Day 2 0.04 0 No Growth Day 2 0 0 No Growth Day
2
[0187] The data demonstrate that selective lysis of whole blood for
the detection of SA was as sensitive as blood culture. Selective
lysis, however, has the advantage that isolated colonies were
available for analysis after overnight incubation; whereas, the
blood culture would require another overnight incubation before
overnight colonies were available (Table 5).
Example 4
Detection of Coagulase-Negative Staphylococcus from Clinical, Whole
Blood Samples
[0188] Four leftover clinical samples of ethylenediaminetetraacetic
acid (EDTA) anti-coagulated whole blood from suspected
catheter-related blood stream infections (CR-BSI), reported
clinically as 10.sup.6 cfu/mL Coagulase Negative Staphylococcus
(CNS), were received frozen. They were defrosted, mixed with lysis
solution, treated with focused ultrasonic energy, and filter
concentrated. The retentate was probed using the PNA Flow FISH
method on the membrane and examined using fluorescent
microscopy.
[0189] Lysis solution was prepared by adding 115 mg of saponin to
10 mL of 0.1M sodium phosphate buffer, pH 8 and vortcxing to
dissolve. 11.25 Units/mL of proteinase were added and vortexed
briefly to dissolve. The solution was filtered using a 0.2 .mu.m,
32 mm, PES syringe filter.
[0190] Samples were prepared by adding 1 mL of lysis solution and 1
mL of defrosted blood to a 3 mL, round bottom, glass Covaris tube.
The samples were mixed by inversion.
[0191] The bath on the Covaris was filled with deionized water,
heated to 37.degree. C., and degassed for 30 minutes. The tubes
were loaded into the custom tube holder designed to fix the X and Y
axis. The samples were warmed and mixed for 100 seconds at an
intensity of 1, 10% duty cycle, and 1000 cycles per burst. Then the
intensity was increased to 2 for 60 seconds. Finally, the cycles
per burst were decreased to 200 for 60 seconds.
[0192] The lysed samples were filter-concentrated, hybridized,
washed, and mounted as described in the Preferred Embodiments
section. The retentate was probed with a three probe mixture
containing a S. aureus specific, fluorescein-labeled probe, a CNS
specific, TAMRA-labeled probe, and universal bacteria, Cy5-labeled
probe.
[0193] The slide-bound membranes were examined using a fluorescent
microscope, a 60.times. oil objective, and the Advanllx PNA FISH
filter cube (XF 53) for fluorescent organisms. CNS was detected in
all 4 samples (FIG. 2).
Example 5
Whole Blood Lysis with Non-Focused Acoustic Energy
[0194] Whole blood anti-coagulated with sodium heparin was
inoculated with E. coli, mixed with lysis solution and subjected to
non-focused ultrasound from the ProSys megasonic bowl instrument.
Samples were plated before and after treatment with the ProSys to
determine recovery and tested for filterability.
[0195] An adjustable tube holder for the ProSys megasonic bowl was
devised by attaching a clip to a manual positioning stage of the
kind used for optics prototyping. The tool clip was kept level to
keep the sample parallel to the transducer surface. The positioning
stage allowed the height of the tube to be precisely adjusted in
order to maximize the amount of activity within the sample while it
was being treated with the ProSys. The ProSys bowl was filled with
deionized water. The transducer was used to heat the water in the
bowl until it reached 35.degree. C.
[0196] Lysis solution was made by adding 115 mg of saponin to 10 mL
of 0.1 M sodium phosphate buffer and vortexing to dissolve. Then
112.5 units of proteinase were added and briefly vortexed to
dissolve. The lysis solution was filtered with a 0.2 .mu.m, 32 mm,
PES syringe filter.
[0197] Samples were prepared by adding 1 mL of whole blood and 1 mL
of lysis solution to a 15 mL Falcon tube. The tubes were sealed
with a cyclic-olefin polymer (COP), pressure-sensitive adhesive
tape (Adhesives Research, ARseal 90404) and mixed by inversion.
[0198] The tubes were clipped topside down to the tube holder and
lowered into the bath until fully submerged. They were treated with
the ProSys for 5 consecutive minutes with 45 watts, 100 ms pulse,
and 50% duty cycle. The temperature was maintained in the bowl
between 36.degree. C. and 38.degree. C. by removing warm water and
replacing it with icy, deionized water. The lysate was removed from
the tube holder and tested for filterability as described in
Example 1. Identical samples were subjected to focused sonic lysis
by the Covaris in parallel with the ProSys samples for comparison
(Table 6). This experiment was performed four times.
TABLE-US-00006 TABLE 6 Comparison of ProSys and Covaris Whole Blood
Lysate Filterability Experiment Ultrasonic System Filtration (sec)
1 ProSys 55 1 ProSys 60 1 ProSys 46 1 ProSys 62 1 Covaris 57 2
ProSys 63 2 ProSys 63 2 ProSys 70 2 Covaris 56 3 ProSys 40 3 ProSys
44 3 ProSys 43 3 ProSys 50 3 Covaris 52 4 ProSys 46 4 ProSys 47 4
ProSys 46 4 ProSys 60 4 Covaris 55
[0199] The results showed that the lysis produced by the
non-focused acoustic energy from the ProSys compared favorably to
the focused ultrasonic energy from the Covaris. The results also
indicate that the lysis can be done reliably and reproducibly.
Recovery assays were done with E. coli to test whether lysis caused
by the ProSys was selective for blood cells.
[0200] Escherichia coli was subcultured to a trypticase soy agar
(TSA) plate and incubated at 37.degree. C. overnight. The following
morning it was inoculated to trypticase soy broth (TSB) media from
the freshly subcultured plate and allowed to incubate at 37.degree.
C. for 2 hours. 350 .mu.L of sterile broth were added to 600 .mu.L
of broth culture. The E. coli were further diluted serially with
1.times. PBS with 0.05% Tween-20 to 1:10,000. 20 .mu.L of diluted
culture were added to samples prepared as described above.
[0201] 100 .mu.L of the samples were plated before and after
treatment with the ProSys (as described above) using a plate
spinner and a disposable, sterile T-spreader. The plates were
incubated overnight at 37.degree. C. Colonies were counted and
recorded the following morning to determine percent yield.
Identical samples were subjected to focused sonic lysis by the
Covaris in parallel with the ProSys samples for comparison (Table
7).
TABLE-US-00007 TABLE 7 E. coli Recovery from Whole Blood after
Lysis with the ProSys Ultrasonic CFU CFU System Pre-Lysis
Post-Lysis Yield ProSys 29 11 38% ProSys 25 17 68% ProSys 32 12 38%
ProSys 20 13 65% Covaris 28 15 54%
[0202] The recovery results for lysis with the ProSys compared
favorably to the Covaris.
Example 6
Detection of Bacteria in Concentrated Platelets
[0203] Platelets from one unit of blood (450 mL) were separated and
concentrated in approximately 30 mL of plasma by centrifugation. 1
mL aliquots of platelet concentrate were inoculated with bacteria,
mixed 1:1 with lysis solution, and selectively lysed using the
Covaris S2. The lysates were filtered, and the retentates were
probed with fluorescently labeled universal bacteria PNA probe. The
filters were examined on a fluorescent microscope for the presence
of bacteria. The platelets were plated pre- and post-lysis to
determine the detection limits of this invention.
[0204] The platelets were stored on a rotational shaker
(Manufacturer: VWR, Model: S-500 Orbital Shaker) at ambient
temperature. The speed of oscillation was set between 3 and 4 such
that an overall speed of 70 rotations per minute was achieved.
Platelets were extracted from the bag in a laminar flow hood using
aseptic technique.
[0205] Lysis solution was prepared daily. 115 mg of saponin were
added to 10 mL of 0.1 M sodium phosphate buffer, pH 8.0 and mixed
to homogeneity via gentle shaking and inversion. 37 mg of
proteinase were added and mixed gently to avoid foaming (foaming
indicates possible denaturation of protein). The lysis solution was
filtered with a 32 mm, 0.2 .mu.m, PES syringe filter. The tube was
protected from light to prevent degradation of the enzyme.
[0206] Serratia marcescens (ATCC 14756), Enterobacter cloacae (ATCC
13047), Salmonella choleraesuis (ATCC 10708), Salmonella
enteritidis (NCTC 4444), Escherichia coli (ATCC 35218), Klebsiella
pneumoniae (ATCC 13882), Pseudomonas aeruginosa (10145),
Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis
(14990), Streptococcus agalactiae (ATCC 13813), Propionibacterium
acnes (ATCC 11827), Bacillus cereus (ATCC 10876) were subcultured
to non-selective agar media. The plates were incubated at
37.degree. C. overnight or at room temperature for 3 days. The
following morning they were inoculated to TSB from the freshly
subcultured agar media and allowed to incubate at 37.degree. C. for
2 to 4 hours. P. acnes was grown for 1 to 2 days. Broth cultures
were diluted 1:10 serially with sterile broth to 1:1,000,000.
10.sup.-3 to 10.sup.-6 were the four dilutions used for
testing.
[0207] Samples were prepared by mixing 1.1 mL, of concentrated
platelets and 100 .mu.L of diluted bacteria in a 3 mL, glass,
round-bottom Covaris tube. Two 100 uL aliquots were removed for
plating. 1 mL of lysis solution was added to the inoculated
platelets. The Covaris tubes were capped and inverted to mix. 200
uL of TCEP were added to platelet solution; and the contents of the
Covaris tubes were pipetted vigorously to ensure complete mixing of
the sample and to dissociate the gel-like residue that is generated
upon addition of TCEP to platelets. The two 100 .mu.L aliquots
(replicates) from the inoculated platelets were plated using a
plate spinner and disposable T-spreaders. After incubation the
colonies were counted, and the number of CFU/mL added to the
platelets was determined.
[0208] The bath on the Covaris was filled with deionized water,
heated to 37.degree. C., and degassed for 30 minutes. The tubes
were loaded into the custom tube holder designed to precisely
position the tube. Each sample was processed via the Covaris to
accelerate the lysis of platelets. The samples were warmed and
mixed for 100 seconds at an intensity of 1, 10% duty cycle, and
1000 cycles per burst. For the next 30 seconds, the intensity was
set to 2 and the cycles per burst was set to 500. For the final 30
seconds, the cycles per burst was set to 100.
[0209] The lysate was filter-concentrated, hybridized, washed, and
mounted as described in the Preferred Embodiments section. It was
probed with a TAMRA-labeled universal bacteria PNA probe. Gram
positive specimens were prepared using 1.times. lysis solution.
Gram negative samples were prepared using 1/2 X lysis solution with
200 uL TCEP and 10% methanol (v/v) flow wash buffer. The gram
negative protocol also worked on gram positives with the same
efficacy.
[0210] The slide-bound membranes were examined on the fluorescent
microscope using the 60.times. oil objective and the AdvanDx PNA
FISH filter cube (XF 53). The goal was to determine which was the
most dilute sample with detectable, fluorescent organisms on the
membrane in order to establish the lower limit of detection (FIGS.
3 & 4). The results showed that all of the isolates were
detectable between 1000 CFU/mL and 10,000 CFU/mL, and S.
epidermidis was detectable between 100 CFU/mL and 1,000 CFU/mL by
the method of this invention.
Example 7
Filterability of Bronchoalveolar Lavage
[0211] Large volume or pooled leftover clinical samples of
bronchoalveolar lavage (BAL) were obtained from a hospital
laboratory. They were mixed with different processing solutions,
treated with the Covaris to break apart mucous, debris, and cells,
and tested for filterability.
[0212] Samples were prepared by homogenizing large volume or pooled
BAL with forceful pipetting and vortexing. 0.5 mL to 1 mL of sample
was mixed by inversion and vortexing with 1 mL to 1.5 mL of
processing solution in a 3 mL round-bottom, glass Covaris tube.
[0213] The bath on the Covaris was filled with deionized water,
heated to 37.degree. C., and degassed for 30 minutes. The tubes
were loaded into the custom tube holder designed to precisely
position the tube. The samples were warmed and mixed for 100
seconds at an intensity of 1, 10% duty cycle, and 1000 cycles per
burst. For the next 60 seconds, the intensity was set to 2. For the
final 60 seconds, the cycles per burst was set to 200.
[0214] All samples were tested for filterability as a measure of
how well the BAL had been processed. The barrel from a 3 mL syringe
was fitted with a 13 mm, 0.45 .mu.m, nylon syringe filter. The
outlet of the syringe filter was attached to a vacuum pulling at
5'' Hg. The filter was primed with 0.5 mL of 1.times. PBS. Another
1 mL of 1.times. PBS was filtered and timed to obtain a
normalization value for each filter. Finally 2 mL of sample mixed
with processing solution either treated with sonic energy or
untreated was filtered and timed (Table 8).
TABLE-US-00008 TABLE 8 Development of BAL Processing Solution %
Filtered Filtra Before tion- Processing Solution Clogging (sec) 0.5
mL BAL + 1.5 mL solution (pooled BAL #8-11) Sodium Phosphate Buffer
10 n/a Sodium Phosphate Buffer + Covaris 33 n/a Saponin + Covaris
70 n/a Saponin + DNase + Covaris 100 161 DNase + Covaris 20 n/a
Triton X-100 + Covaris 100 36 Guanidinium Cl + Covaris 50 n/a
Sputolysin + Covaris 25 n/a NaLC + Sodium Citrate + Covaris 40 n/a
TCEP + Covaris 40 n/a 1 mL BAL + 1 mL solution (pooled BAL #8-11)
Triton X-100 + Covaris 45 n/a 1 mL BAL + 1 mL solution (large
volume BAL # 12) Sodium Phosphate Buffer 15 n/a Sodium Phosphate
Buffer + Covaris 30 n/a Triton + Protease + Covaris 100 26 Triton
X-100 + Covaris 50 n/a Triton + Protease + DNase + Covaris 100 40
Triton + DNase + Covaris 100 63 Guanidinium + Triton + Covaris 100
33 NaLC + Sodium Citrate + Triton + Covaris 100 76 Triton +
Protease + DNase + Guanidinium + Covaris 100 29 Triton + Protease +
DNase + NaLC + Covaris 100 35 Repeat Triton + Protease + Covaris
100 28 Repeat Triton + Protease + DNase + Covaris 100 39 1 mL BAL +
1 mL solution (large volume BAL # 14) Sodium Phosphate Buffer 5 n/a
Sodium Phosphate Buffer + Covaris 10 n/a Mucolexx + Covaris 10 n/a
Triton + Protease + NaLC + Covaris 55 n/a Triton + Protease + NALC
+ NaCitrate + Covaris 45 n/a Triton + Protease + Guanidinium +
Covaris 100 140 Triton + Protease + Covaris 30 n/a Triton +
Protease + TCEP + Covaris 100 35 Triton + Protease + NALC + DNase +
Covaris 30 n/a Triton + Protease + NALC + DNase in Running 55 n/a
Buffer + Covaris Triton + Protease + TCEP + DNase in Running 100 69
Buffer + Covaris 1 mL BAL + 1 mL solution (large volume BAL # 17)
Sodium Phosphate Buffer 10 n/a Sodium Phosphate Buffer + Covaris 18
n/a Triton + Protease + Covaris 45 n/a Triton + Protease + TCEP +
DNase in Running 100 43 Buffer + Covaris Triton + Protease + TCEP +
Covaris 100 26
[0215] Results showed that a processing solution with Triton X 100,
proteinase, and TCEP followed by treatment with the Covaris
produced the most reliably filterable sample. Others additives such
as DNase may or may not improve the efficacy of the processing
solution. However, there was some concern about the harmful effects
that Triton X 100 may have on bacteria so some alternatives to
Triton X 100 were also tested (Table 9).
TABLE-US-00009 TABLE 9 Triton X 100 Alternatives for BAL Processing
Solution % Filtered Filtration Processing Solution Before Clogging
(sec) 0.5 mL BAL + 1.5 mL solution (pooled BAL #64-66) NaCl +
Protease + TCEP + Covaris 100 32 DexS04 + Protease + TCEP + Covaris
100 42 SDS + Protease + TCEP + Covaris 100 24 Triton + Protease +
TCEP + Covaris 100 30 Saponin + Protease + TCEP + Covaris 100 28
Tween-20 + Protease + TCEP + Covaris 100 29 1 mL BAL + 1 mL
solution (pooled BAL #67-68) Saponin + Protease + TCEP + Covaris 18
n/a Saponin + Protease + TCEP + Covaris 26 n/a Saponin + Protease +
TCEP + High [DNase] + Covaris 14 n/a Saponin + Protease + TCEP +
Low [DNase] + Covaris 16 n/a Saponin + Protease + TCEP + High
[Gelsolin] + Covaris 22 n/a Saponin + Protease + TCEP + Low
[Gelsolin] + Covaris 26 n/a 0.5 mL BAL + 1.5 mL solution (pooled
BAL #67-68) Saponin + Protease + TCEP + Covaris 82 n/a Tween-20 +
Protease + TCEP + Covaris 100 35 Saponin + Tween-20 + Protease +
TCEP + Covaris 100 73
[0216] Results showed that Tween-20 along with proteinase, and TCEP
followed by ultrasonic treatment was a promising alternative to
Triton X 100 in the processing solution for BAL.
Example 8
Detection of Bacteria from Clinical Bronchoalveolar Lavage
Samples
[0217] Leftover positive clinical BAL samples (BAL #18 and #43
clinical reports: "few Staphylococcus aureus and Usual throat
organism" and ".gtoreq.10K cfu/mL Serratia marcescens"
respectively) were mixed with processing solution, treated with
focused ultrasonic energy, and filter concentrated. The retentate
was probed using the PNA Flow FISH method on the membrane and
examined using fluorescent microscopy.
[0218] Processing solution was prepared by adding 115 .mu.L of
Triton X 100 to 10 mL of 0.1M sodium phosphate buffer, pH 8 and
vortexing to mix thoroughly. 11.25 Units/mL of proteinase were
added and vortexed briefly to dissolve. The solution was filtered
using a 0.2 .mu.m, 32 mm, PES syringe filter.
[0219] Samples were prepared by homogenizing with forceful
pipetting and vortexing. 1 mL of processing solution, 1 mL of
homogenized BAL, and 0.2 mL of TCEP were added to a 3 mL, round
bottom, glass Covaris tube. The samples were mixed by inversion and
vortexing until uniformly liquid throughout.
[0220] The bath on the Covaris was filled with deionized water,
heated to 37.degree. C., and degassed for 30 minutes. The tubes
were loaded into the custom tube holder designed to fix the X and Y
axis. The samples were warmed and mixed for 100 seconds at an
intensity of 1, 10% duty cycle, and 1000 cycles per burst. Then the
intensity was increased to 2 for 60 seconds. Finally, the cycles
per burst were decreased to 200 for 60 seconds.
[0221] The processed BAL was filter-concentrated, hybridized,
washed, and mounted as described in the Preferred Embodiments
section. It was probed with species specific S. aureus
TAMRA-labeled or universal bacteria fluorescein-labeled PNA
probe.
[0222] The slide-bound membranes were examined using a fluorescent
microscope, a 60.times. oil objective, and the AdvanDx PNA FISH
filter cube (XF 53) for fluorescent organisms. S. aureus was
detected in the BAL with reported S. aureus, and bacilli were
detected in the BAL with reported S. marcescens (FIG. 5).
INCORPORATION BY REFERENCE
[0223] All patents, patent applications, and published references
cited herein are hereby incorporated by reference in their
entirety. While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
REFERENCES
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Solution Nontoxic to Pathogenic Bacteria. J. Clin. Microbiol.
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of Bacteria but Not Mammalian Cells by Diastereomers of Melittin:
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J, Ratti G, Petracca R, Galli G, Agnusdei M, Giuliani M M, Santini
L, Brunelli B, Tettelin H, Rappuoli R, Randazzo F, Grandi G. 2002.
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Polyacrylamide Gel Electrophoresis to the Characterization and
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