U.S. patent application number 13/641480 was filed with the patent office on 2013-08-01 for methods for measuring enzyme activity useful in determining cell viability in non-purified samples.
The applicant listed for this patent is Shawn Mark O'Hara, Daniel Zweitzig. Invention is credited to Shawn Mark O'Hara, Daniel Zweitzig.
Application Number | 20130196318 13/641480 |
Document ID | / |
Family ID | 44799329 |
Filed Date | 2013-08-01 |
United States Patent
Application |
20130196318 |
Kind Code |
A1 |
O'Hara; Shawn Mark ; et
al. |
August 1, 2013 |
METHODS FOR MEASURING ENZYME ACTIVITY USEFUL IN DETERMINING CELL
VIABILITY IN NON-PURIFIED SAMPLES
Abstract
The present invention relates generally to the field of
detection of microorganisms, in particular detection of bacteria,
to methods for measuring enzyme activity, such as DNA polymerase
activity, and particularly relates to such methods performed on
microbial crude lysates, useful for determining microbial enzyme
activities which can be linked to amplification signal generators
such as real-time Polymerase Chain Reaction (PCR) techniques,
thereby enabling determination of microbial pathogens in samples
such as unpurified blood and other body fluids. This invention also
relates to reagents for use in such methods, and to test kits
comprising such reagents for carrying out the methods.
Inventors: |
O'Hara; Shawn Mark;
(Richboro, PA) ; Zweitzig; Daniel; (Feastervile,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
O'Hara; Shawn Mark
Zweitzig; Daniel |
Richboro
Feastervile |
PA
PA |
US
US |
|
|
Family ID: |
44799329 |
Appl. No.: |
13/641480 |
Filed: |
April 15, 2011 |
PCT Filed: |
April 15, 2011 |
PCT NO: |
PCT/US2011/032600 |
371 Date: |
March 21, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61324939 |
Apr 16, 2010 |
|
|
|
61325413 |
Apr 19, 2010 |
|
|
|
61324949 |
Apr 16, 2010 |
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Current U.S.
Class: |
435/6.11 ;
435/6.12 |
Current CPC
Class: |
C12Q 1/42 20130101; C12Q
1/04 20130101; C12Q 1/6888 20130101; C12Q 1/25 20130101; C12Q 1/48
20130101 |
Class at
Publication: |
435/6.11 ;
435/6.12 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for detecting polymerase activity as an indicator of
the presence of a micro-organism in a sample, which method
comprises: (a) contacting the sample with a nucleic acid molecule
which acts as a substrate for polymerase activity in the sample;
(b) incubating the thus contacted sample under conditions suitable
for polymerase activity; and (c) specifically determining the
presence (and/or the amount) of a nucleic acid molecule resulting
from the action of the microorganism polymerase on the substrate
nucleic acid molecule, thereby to indicate the presence of the
microorganism.
2. The method of claim 1, wherein the polymerase is either a DNA or
RNA polymerase.
3. The method of claim 1, wherein the microorganism detected is a
viable microorganism in the sample.
4. The method of claim 1, wherein the microorganism detected is an
intact microorganism in the sample.
5. The method of claim 4, wherein the intact microorganism detected
is one in which the nucleic acid polymerase gene and its translated
active protein polymerase is essential for viability of the
microorganism.
6. The method of claim 1, wherein the polymerase substrate is
immobilized.
7. The method of claim 1, wherein the sample in which the
microorganism is detected is a normally sterile body fluid.
8. The method of claim 1, wherein the sample is prepared using a
differential cell lysis sample preparation method, thereby allowing
only viable microorganism derived polymerase activity to modify the
polymerase specific substrate.
9. The method of claim 1, wherein the sample in which the
microorganism is detected is prepared from crude cell lysates or
purified cell fractions.
10. The method of claim 9, wherein the method further comprises
conducting a complete or partial microorganism genome and or
transcriptome sequence analysis.
11. The method of claim 10, wherein the complete or partial
microorganism genome and or transcriptome sequence analysis can be
performed simultaneously, in concert, or in parallel using a single
sample preparation.
12. The method of claim 10, wherein the complete or partial
microorganism genome and or transcriptome sequence analysis of the
microorganisms further comprises a method for the diagnostic
measure and detection of agents with anti-microbial and or
anti-polymerase activity useful in the management of patients.
13. An assay kit comprised of reagents useful in the method of any
of claims 1 through 12, for screening normally sterile body fluids
for the presence of absence of microorganisms therein and to
provide diagnostic, prognostic patient management information.
14. A method of detecting an enzyme selected from the group
consisting of NAD-dependent ligase or phosphatase or a mixture
thereof as an indicator of the presence of a microorganism in a
sample, which method comprises: (a) contacting the sample with a
nucleic acid molecule which acts as a substrate for enzyme activity
of the selected enzyme or mixture in the sample, while not allowing
interfering signals from DNA polymerase; (b) incubating the thus
contacted sample under conditions suitable for enzyme activity; and
(c) specifically determining the presence (and/or the amount) of an
enzyme modified nucleic acid molecule resulting from the action of
the selected enzyme or mixture on the substrate nucleic acid
molecule, thereby to indicate the presence of the
microorganism.
15. The method of claim 14, wherein the microorganism is one in
which the selected enzyme or mixture is (or has been)
expressed.
16. The method of claim 14 or claim 15, wherein the sample is
prepared using a differential cell lysis sample preparation method,
thereby allowing only viable microorganism derived ligase and/or
phosphatase activity to modify the ligase specific substrate.
17. An assay kit capable of performing an assay for detecting an
enzyme selected from the group consisting of NAD-dependent ligase
or phosphatase or a mixture thereof as an indicator of the presence
of a microorganism in a sample, said kit comprising: (a) a
substrate containing a nucleic acid molecule for activity of the
selected enzyme or mixture in the sample, while not allowing
interfering signals from DNA polymerase; (b) Incubation means for
incubating the sample and substrate under conditions suitable for
enzyme activity; and (c) Means for specifically determining the
presence (and/or the amount) of a nucleic acid molecule resulting
from the action of the selected enzyme or mixture on the substrate
nucleic acid molecule, as an indication of the presence of the
microorganism.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a non-provisional application, which is
incorporated by reference herein and claims priority, in part, of
U.S. Provisional Application No. 61/324,939, filed Apr. 16, 2010,
U.S. Provisional Application No. 61/324,949, filed Apr. 16, 2010
and U.S. Provisional Application No. 61/325,413, filed Apr. 19,
2010.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
detecting microorganisms, and more particularly to the detection of
bacteria. Also provided by the invention are improved methods of
detecting microorganisms that are highly sensitive, are applicable
to non-purified samples, and have numerous applications, together
with assay kits, which rely upon the presence of ligase and/or
phosphatase as an indicator of bacterial viability.
BACKGROUND OF THE INVENTION
[0003] Measuring the presence and levels of certain molecules which
are associated with cell viability is important in a number of
contexts. For example, measuring levels of ATP is useful in
mammalian cells for growth analysis and toxicology purposes.
[0004] Culture approaches can be used to detect small numbers of
bacteria but such techniques require several days to complete,
especially when attempting to detect small numbers of bacteria and
also when detecting slower growing microorganisms.
[0005] Alternatively, tests may be carried out based upon measuring
the presence of a molecule which can be linked to the presence in
the sample of a contaminant cell or organism. The most commonly
detected molecule is Adenosine Triphosphate (ATP). Detection of DNA
and RNA has also been proposed, although the correlation between
the presence of DNA and RNA and viability is not clear-cut due to
the variable persistence of nucleic acids in cells post death (Keer
& Birch, Journal of Microbiological Methods 53 (2003) 175-183).
Detection of adenylate kinase as an indicator of viability has also
been proposed (Squirrell D J, Murphy M J, Leslie R L, Green J C D:
A comparison of ATP and adenylate kinase as bacterial cell markers:
correlation with agar plate counts, in Bioluminescence and
Chemiluminescence Progress and Current Applications. Edited by:
Stanley R A, Kricka L J. John Wiley and Sons; 2002 and WO
96/02665). A routinely employed method for determining ATP levels
involves the use of bioluminescence. The method uses the ATP
dependency of the reaction in which light emitting luciferase
catalyzes oxidation of luciferin. The method may be used to measure
relatively low concentrations of ATP. Kits useful for detecting ATP
using bioluminescence are commercially available from Roche, New
Horizons Diagnostics Corp, Celsis etc. However, a number of
problems exist with respect to bioluminescence detection. For
example, detection of microbial ATP only, in the presence of ATP
from non-microbial sources can be a problem. This problem has been
solved to a certain degree by use of filters which can separate
bacteria from nonbacterial sources of ATP, thus providing a more
accurate signal.
[0006] Accordingly, it can be seen that a number of problems exist
with respect to the conventional art of microbe detection. In order
to further address such problems, detection of ligases has been
proposed, such as described in published patent application
WO/1996/002665, published Feb. 1, 1996, there is disclosed a method
for determining the presence and/or amount of microorganisms and/or
their intracellular material present in a sample characterized in
that the amount of adenylate kinase in the sample is estimated by
mixing it with adenosine diphosphate (ADP), determining the amount
of adenosine triphosphate (ATP) produced by the sample from this
ADP, and relating the amount of ATP so produced to the presence/or
amount of adenylate kinase and to microorganisms and/or their
intracellular material, wherein the conversion of ADP to ATP is
carried out in the presence of magnesium ions at a molar
concentration sufficient to allow maximal conversion of ADP to ATP.
The amount of magnesium present is preferably such that there is
sufficient to provide one mole of magnesium for one mole of ADP
such that all of the ADP molecules may be associated with at least
one magnesium ion.
[0007] In published patent application WO/2009/007719, published
Jan. 15, 2009, entitled DETECTION OF MICRO-ORGANISMS BASED ON THEIR
NAD-DEPENDENT DNA LIGASE ACTIVITY ligases, in particular
NAD-dependent ligases, are disclosed as a useful indicator of the
presence of a (viable) microorganism in a sample. Ligases are
enzymes which catalyze ligation of nucleic acid molecules. The
ligation reaction requires either ATP or NAD+ as co-factor
depending upon the ligase concerned. In this disclosure, the use of
NAD-dependent ligase activity is utilized as an indicator of the
presence of a (viable) microorganism in a sample. The link between
NAD-dependent ligase activity and viability is central to the
invention disclosed in this application, (Korycka-Machala et al.,
Antimicrobial Agents and Chemotherapy, August 2007, p 2888-2897),
since it allows the activity of this enzyme to be used as an
indicator of viable microbial cells, in particular of bacterial
origin, in the sample. However, in the experiments leading to the
development of the present invention, it was found that the
techniques and teachings described in this published patent
application WO/2009/007719 could not be applied to the
determination of viable microorganisms in unpurified samples, such
as crude microbial lysates, blood or blood cultures, thereby
constituting a major drawback of the technology as described in
this reference. However, it has been discovered that these
methodologies, too, have problems. For example, it has been found
that in general the conventional ligase substrate assay design and
resultant detection signal thereof, as disclosed in the
above-reference patent application, is not ligase specific when
applied to its intended sample type (blood derived microbe crude
cell lysates). It is these problems which the present invention
seeks to address and to overcome.
SUMMARY OF THE INVENTION
[0008] In contrast to the conventional methods described above, in
one aspect the present invention is directed to the detection of
enzymes such as polymerases, in preferred embodiments DNA or RNA
polymerases, as a useful indicator of the presence of a (viable)
microorganism or microbe in a sample, in particular a sample that
is, for example, a crude microbial lysate or unpurified blood or
blood culture. The association discovered in accordance with the
present invention between enzyme, e.g., polymerase, activity and
viability of microorganisms or microbes enables the detection of
activity of these enzymes to be used as an indicator of viable
microbial cells, in particular of bacterial origin, in the
sample.
[0009] Similarly, the invention provides, in a preferred
embodiment, methods for detecting a DNA or RNA polymerase as an
indicator of the presence of a microorganism in a sample. Such a
method can comprise:
(a) contacting the sample with a nucleic acid molecule which acts
as a substrate for polymerase activity in the sample, (b)
incubating the thus contacted sample under conditions suitable for
polymerase activity; and (c) determining the presence (and/or the
amount) of a nucleic acid molecule resulting from the action of the
microorganism polymerase on the substrate nucleic acid molecule to
indicate the presence of the microorganism.
[0010] In addition, the present invention provides reagents useful
in the foregoing described methods, and assay kits comprising such
reagents useful for performing the methods.
[0011] In another aspect, the present invention provides
improvements to the methods, compositions and kits described in
published patent application WO/2009/007719, published Jan. 15,
2009, entitled DETECTION OF MICRO-ORGANISMS BASED ON THEIR
NAD-DEPENDENT DNA LIGASE ACTIVITY, which published application
identifies ligases, in particular NAD-dependent ligases, as a
useful indicator of the presence of a (viable) microorganism or
microbe.
[0012] The entire disclosure contained in said WO/2009/007719 is
hereby incorporated by reference and made a part of this
application.
[0013] The present invention accordingly provides improvements to
the methods, and compositions and kits based thereon as disclosed
in WO/2009/007719, of detecting an enzyme selected from the group
consisting of NAD-dependent ligase, or a phosphatase, or a mixture
of the foregoing, as an indicator of the presence of a
microorganism in a sample, which improved methods comprise:
(a) contacting the sample with a nucleic acid molecule which acts
as a substrate for enzyme activity in the sample, while not
allowing interfering signals from DNA polymerase, (b) incubating
the thus contacted sample under conditions suitable for enzyme
activity; and (c) determining the presence (and/or the amount) of
an enzyme modified nucleic acid molecule resulting from the action
of the selected enzyme or mixture on the substrate nucleic acid
molecule to indicate the presence of the microorganism.
[0014] Thus, it will be appreciated that the improved methods of
the invention are useful for identifying all microorganisms in
which such enzymes or mixtures thereof are (or have been)
expressed.
[0015] As stated herein, the first step in the method comprises
contacting the sample with a nucleic acid molecule which acts as a
substrate for enzyme activity in the sample, while not allowing
interfering signals from DNA polymerase. It is thus to be
appreciated that any suitable ligatable molecule which can be
specifically detected once ligated may be utilized in the methods
of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1, drawings A. through D. show template diagrams and
graphical representations of results produced by experiments
conducted in accordance with the present invention as described
herein.
[0017] FIG. 2, drawings A. through D. are graphical representations
showing that Non-Ligate-able, Polymerase favorable substrates are
sensitive and specific in microbe derived crude cell lysates.
[0018] FIG. 3 is a graphical representations showing
Non-Ligate-able, Polymerase favorable substrates are sensitive and
specific in Microbe Spiked Blood Culture derived crude cell
lysates.
DETAILED DESCRIPTION OF THE INVENTION
[0019] Thus, from the foregoing description it can be appreciated
that the methods of the present invention are useful for
identifying all microorganisms in which an enzyme, such as a
suitable polymerase, is (or has been) expressed. In certain
embodiments, the methods of the invention are applied to the
detection of viable microorganisms and thus may be considered as a
method for detecting a viable microorganism in a sample. In
particular, in a preferred embodiment the methods of the invention
may be useful for identifying bacteria or microorganisms in which
the nucleic acid polymerase gene and its translated active protein
polymerase is essential for viability. However, microorganisms,
such as bacteria, recently rendered non-viable (for example through
treatment with an anti-bacterial) may retain detectable polymerase
activity until the enzyme is degraded.
[0020] In the development of the present invention, a paradigm
shift in sample preparation of functional cellular biochemical
components was discovered in that the present invention enables
assays to be performed directly on samples from gently lysed cells,
without the expensive complications added by traditional, and often
extreme, denaturation based isolation protocols. Thus, it has been
found that the present invention enables the detection of viable
organisms in samples such as crude clinical lysates, including
without limitation cell fractions from whole blood and or blood
cultures, and from large volumes of the same, which are typically
10-20 ml, preferable in the range of 0.1-100 ml. The invention is
particularly useful for the detection of all organisms associated
with septicemia, and for those associated with conditions including
but not limited to bacteremia, fungemia, and virus and parasitic
conditions. It has unexpectedly been found that in accordance with
the present invention detection of such organisms can be
accomplished in such non-purified samples as described above, in
contrast to the teachings of the conventional art in which sample
derived polymerase inhibition, as well as the presence of
interfering proteinases and nucleases, has been a barrier to such
assay methods when performed on non-purified samples.
[0021] As described above, ligases, in particular NAD-dependent
ligases, have been disclosed as a putatative useful indicator of
the presence of a (viable) microorganism in a sample. However, in
contrast, the present invention provides other viable microbe cell
derived enzymes, useful rather than ligases, that can, similarly,
be used to link their activity from viable cells to a high
sensitivity signal generator such as amplification by techniques
such as PCR and the like. This feature of the invention also
potentially enables differentiating bacteria from fungi. In an
example of an embodiment of the invention, the following may be
used in this regard: [0022] a. Kinases add PO4 which could be used
to enable a ligase or stop a polymerase [0023] b. Phosphates can be
used to remove a PO4 and enable Polymerases [0024] c. DNA & RNA
Polymerases can be used to extend substrates to enable downstream
traditional PCR or isothermal amplifications [0025] d. Isothermal
amplifications can be run off of endonuclease enzyme activities
[0026] e. Ribosomes [0027] f. miRNA mechanisms [0028] g. Gyrase
[0029] h. Helicase [0030] i. Exonucleases, 5'-3', 3'-5' i.e.
removing a blocking groups such as PO4, TaqMan etc [0031] j.
Endonucleases [0032] k. Proteases [0033] l. DNases [0034] m. RNases
[0035] n. UDGlycosolases [0036] o. Repair enzymes
[0037] In a preferred embodiment of the present invention, it has
been discovered that measurement of DNA polymerase activity, in
accordance with the invention, enables the determination of cell
viability from microbe crude lysates. This may be verified using
more selective modified oligo substrates in combination with very
selective "hot starts" (as well known in the art) and controlling
for RT, 37, 60C activities.
[0038] In one embodiment of the invention, the invention has
application to blood product screening, especially of platelets as
in this application any microbe growth is cause for discarding of
product, and differentiation of bacteria from fungus is not
necessary. In a further embodiment of the invention, phosphatases
may be employed and are likely another excellent candidate enzyme
to enable polymerase activity, as they remove either a 5' or 3'
phosphate leaving an --OH-- and thus could enable any polymerase by
the removal of a designed 5' Taq included, or ligase (remove 3').
In addition, phosphatases are robust and may help to differentiate
yeast and bacteria via optimization of pH. It will therefore be
appreciated that any suitable enzyme that will enable polymerase as
contemplated by the teachings herein may be useful in the practice
of the present invention.
[0039] In the practice of the present invention, detection of
microorganisms may include recently viable microorganisms, up until
the point where DNA polymerase has been degraded, as appropriate.
If a distinction between viable and recently viable microorganisms
is required, a simple time course or comparison of polymerase
activity between two or more time points, under appropriate
conditions, should be sufficient to determine whether polymerase
activity increases, persists or diminishes over time. In a
preferred embodiment, if the polymerase activity is found to
persist for, or increases over, an extended period or at (a) later
time point (s) (compared to the initial measurement), this may
indicate that the microorganisms are viable. If polymerase activity
diminishes at (a) later time point (s), this may indicate that the
detected activity was from recently viable microorganisms. This
time course measurement approach may be especially useful when
applied toward antibiotic susceptibility testing (AST) and as well
as determination of other appropriate therapies. Detection methods
are discussed in detail herein. In specific preferred embodiments
of the invention, the microorganism is a bacterium, as herein
described, and the methods of the invention may be more generally
applicable (Wilkinson et al., Molecular Microbiology (2001) 40(6),
1241-1248). The bacteria may, as well, be mesophillic and/or
thermophillic bacteria, for example.
[0040] A "sample" in the context of the present invention is
defined to include any sample in which it is desirable to test for
the presence of a microorganism, in particular a bacterium. Thus
the sample can consist of a clinically provided crude microbe
lysate, or may comprise a clinical sample of blood or blood
culture, or comprise a sample suitable for an in vitro assay
system, for example. Samples may also comprise beverage or food
samples or preparations thereof, or pharmaceutical or cosmetic
products such as personal care products including shampoos,
conditioners, moisturizers etc., all of which are tested for
microbial contamination as a matter of routine. The sample may
comprise tissue or cells and may comprise sputum or a platelet
sample. In addition, the methods and kits of the invention may be
used to monitor contamination of surfaces, such as for example in
locations where food is being prepared. In a preferred embodiment,
contamination is indicated by the presence of polymerase activity.
The contamination may be from any microbial source, in particular
bacterial contamination. Furthermore, the invention is also useful
in monitoring environmental conditions such as water supplies,
wastewater, marine environments etc. The invention is also useful
in monitoring bacterial growth in fermentation procedures and in
air sampling where bacteria or spore content can be assessed in
hospital, industrial facilities or in biodefense applications.
[0041] The methods of the invention rely on the fact that if there
are one or more (viable) micro-organisms, in particular bacteria,
present in the sample, enzyme activity, preferably DNA polymerase
activity, will be present. The enzyme can thus, under appropriate
conditions, catalyse a reaction to generate a novel detectable
nucleic acid molecule (in a subsequent process). The novel nucleic
acid molecule may be detected by any suitable means such as
hereinafter described, thereby allowing a determination of the
presence of the microorganisms in the sample under test.
[0042] Thus, if the microorganism is not present in the sample,
there will be no enzyme (e.g., polymerase) activity in the sample
and thus the novel detectable nucleic acid molecule will not be
generated.
[0043] The methods of the present invention provide significant
technical advantages, due in large part to the fact that a novel
nucleic acid molecule is generated as part of the method. In the
methods of the present invention, unreacted nucleic acid molecule
will not contribute to the signal, and as a result no false
positive signals should be produced when the methods are carried
out.
[0044] Furthermore, the methods provided by the invention are
highly sensitive, and may provide detection of the enzyme (e.g.,
polymerase) present in the sample down to femtogram and possibly
even attogram levels. The sensitivity is derived from the fact that
every bacterial cell contains thousands of enzyme molecules, and
thus each can catalyse multiple events under suitable conditions.
Unlike direct PCR approaches, which must target one or a few copies
of a gene per cell or use additional steps or reagents to detect
ribosomal or messenger RNA, the approach described herein targets
the detection of multiple copies of the enzyme per cell in a simple
assay format.
[0045] As stated herein, the first step in a method according to
the invention comprises contacting the sample with a nucleic acid
molecule which acts as a substrate for the enzyme, for example
polymerase, activity in the sample.
[0046] Suitable substrate nucleic acid molecules for use in the
invention are described in more detail below. The nucleic acid
molecules may incorporate synthetic nucleotide analogues as
appropriate or may be RNA or DNA based for example, or mixtures
thereof. They may be labelled, such as using a fluorescent label,
or FRET pair, in certain embodiments to facilitate detection.
Suitable detection methods are described herein.
[0047] "Nucleic acid" is defined herein to include any natural
nucleic acid and natural or synthetic analogues that are capable of
generating a detectable nucleic acid molecule by the action of
polymerase. Suitable nucleic acid molecules may be composed of, for
example, double or single-stranded DNA and double or
single-stranded RNA.
[0048] Though the nucleic acid substrate may comprise a blunt-ended
double-stranded DNA molecule, in an embodiment of the invention the
nucleic acid substrate for the polymerase comprises two double
stranded DNA molecules with a complementary overhang and 5'
phosphate groups at the ends to be joined. In one specific
embodiment, the complementary overhang is between 2 and 10, such as
3 or 5 base pairs. In an alternative embodiment, the nucleic acid
substrate comprises a DNA molecule with a nick containing a 5'
phosphate. Synthesized nucleic acid molecules are commercially
available and can be made to order with a terminal 5.sup.1
phosphate group attached. This has the technical advantage that
100% of the nucleic acid molecules used in the methods of the
invention will be labeled with a 5' phosphate group.
[0049] In especially preferred embodiments of the invention, if
polymerase is present in the sample, it will catalyse and a novel
nucleic acid molecule (incorporating an overall novel sequence)
will be formed which can be detected by a subsequent process, as
detailed herein (such as PCR for example).
[0050] Thus, the substrate nucleic acid molecule may, in fact,
comprise two or more nucleic acid molecules as appropriate. This
applies generally to the methods and kits of the invention.
[0051] In certain embodiments, the nucleic acid substrate comprises
two double stranded nucleic acid molecules with single-stranded
complementary overhangs.
[0052] It is to be appreciated that the novel methods of the
present invention can be used to differentiate ligase from
polymerase by taking a sample suspected of containing both and
testing for both polymerase and ligase in parallel in separate
reaction vessels, then subtracting the signals, thus in fact
determining the true ligase levels found in the sample. This can be
represented by the following equation:
[polymerase signal-ligase signal (polymerase+ligase)=true ligase
signal]
[0053] It also is to be appreciated that in any embodiment of the
present invention, the action of polymerases on nucleic acids is
well known and thus it can be seen that many different types of
nucleic acid substrates can be selected for use and will have the
advantages of utilization in the novel methods of the invention, as
herein described. Preferably, the nucleic acid substrate is present
in excess, and in particular in large molar excess, over the
polymerase in the sample. This is an important technical
distinction over prior art methods. Because a novel polymerized
nucleic acid molecule is detected, only the presence of this
molecule in the sample is essential for the detection methods to
work effectively. Thus, it is not detrimental to the methods of the
invention if other nucleic acid molecules are present in the sample
such as from the bacteria to be detected or from mammalian or
fungal sources which may be found in the sample to be tested for
example.
[0054] The present invention can be more fully described by
reference to the following examples of experimental work conducted
in accordance with the invention. Also, while certain of the
preferred embodiments of the present invention have been described
and specifically exemplified above, it is not intended that the
invention be limited to such embodiments.
Example 1
Discovery of a Ligase Independent Mechanism
[0055] Three different DNA substrates (A) were incubated with E.
coli ligase or no ligase and subjected to PCR containing full
length DNA ligase substrate specific PCR primers in the
presence/absence of UNG. PCR was monitored via SYBR green (qPCR)
and the resultant reactions were subjected to gel analysis (B).
Three different DNA substrates (A) were incubated with E. coli
ligase or no ligase and subjected to PCR containing S1-Extension
detection primers in the presence/absence of UNG. PCR was monitored
via the commercially--available Zeus-Probe (qPCR) methodology (Zeus
Scientific, Inc., Raritan, N.J.) and the resultant reactions were
subjected to gel analysis (C). Decreasing amounts of a
non-ligatable DNA substrate (S1/AS only) was incubated with three
different commercially available DNA polymerases and subjected to
Zeus-Probe qPCR analysis. The results of these experiments are
illustrated graphically in FIG. 1.
Example 2
Non-Ligate-Able, Polymerase Favorable Substrates were Found to be
Sensitive and Specific in Microbe Derived Crude Cell Lysates
[0056] Decreasing amounts microbes were beadmill-lysed and
incubated with a DNA substrate (S1/AS only) in the presence of DNA
polymerase buffer and dNTP's at 37.degree. C. for 30 min. (A). The
lysates were then subjected to Zeus-Probe qPCR containing
S1-extension specific primers. The results are displayed
graphically in FIG. 2.
Example 3
Non-Ligate-Able, Polymerase Favorable Substrates were Found to be
Sensitive and Specific in Microbe Spiked Blood Culture Derived
Crude Cell Lysates
[0057] Decreasing amounts microbes were spiked into 10 ml of blood
broth. The microbes were subsequently recovered, subjected to
beadmill-lysis and incubated with a DNA substrate (S1/AS only) in
the presence of DNA polymerase buffer and dNTP's at 37.degree. C.
for 30 min. (A). The lysates were then subjected to Zeus-Probe qPCR
containing S1-extension specific primers. The results are displayed
graphically in FIG. 3.
[0058] Accordingly, in yet another aspect the present invention
improves upon the invention described and claimed in
WO/2009/007719. In accordance with the present invention, it has
been discovered that the putative DNA ligase specific substrate in
accordance with the disclosure of said WO/2009/007719 yields robust
signals from either purified DNA polymerase or purified DNA ligase,
such that the methods set forth therein are not rendered DNA ligase
specific when applied to the intended sample type, such as
septicemia samples. For example, in the development of the present
invention, septicemia samples using the sample preparation methods
taught by WO/2009/007719 were input into the assay protocols as
taught therein as crude microbe cell lysates containing a high
abundance of DNA polymerases. DNA polymerase(s) are abundant in all
living cells. It was found that the assays as disclosed in
WO/2009/007719 are incapable of discriminating between any DNA
polymerase and DNA ligase derived signals, when inputting
non-ligase-purified samples, which from a practical standpoint
include all clinical sample inputs, because isolating ligase is
neither a practical nor routine procedure as disclosed in this
reference, when attempting to obtain results from clinical samples.
Rather, experiments conducted in accordance with what is taught by
this reference were found to produce an assay signal contaminated
by DNA polymerase, not a DNA ligase specific signal, which is
clearly the desired result in accordance with this reference.
[0059] These findings described above are contrary to the ability
of a system, produced in accordance with the teachings of
WO/2009/007719, to specifically detect DNA ligase from viable
cells. It further precludes the intended ability of the disclosed
assay of this reference to differentiate viable cell-derived NAD
dependent bacterial ligase from ATP dependent fungal ligase, as
active polymerases are common to all viable cells and cannot be
differentiated from any ligases in such an assay system. Having
thus identified this critical problem, which is clearly
unanticipated by this reference or any other known art, the present
invention provides improvements which enable specific ligase
signals to be detected from non-purified ligase samples, such as
crude microbe lysates, by providing alternative, substitute DNA
substrates, as hereinafter described, that do not allow interfering
signals from DNA polymerases to be detected.
[0060] The present invention therefore also provides improved
methods, and compositions and kits based thereon, of detecting an
enzyme selected from the group consisting of NAD-dependent ligase,
or phosphatase, or a mixture thereof as an indicator of the
presence of a microorganism in a sample, the methods
comprising:
(a) contacting the sample with a nucleic acid molecule which acts
as a substrate for enzyme activity in the sample, while not
allowing interfering signals from DNA polymerase, (b) incubating
the thus contacted sample under conditions suitable for enzyme
activity; and (c) determining the presence (and/or the amount) of
an enzyme modified nucleic acid molecule resulting from the action
of the selected enzyme or mixture on the substrate nucleic acid
molecule to indicate the presence of the microorganism.
[0061] Thus, the improved methods of the invention are useful for
identifying all microorganisms in which an NAD-dependent ligase, or
a phosphatase, or mixtures thereof, are (or have been)
expressed.
[0062] In a preferred embodiment of the invention the first step in
the improved method disclosed herein comprises contacting the
sample with a nucleic acid molecule which acts as a substrate for
NAD-dependent ligase activity in the sample, while not allowing
interfering signals from DNA polymerase. Any suitable enzyme
modified, or ligatable, molecule which can be specifically
detected, once ligated, may be utilized in the methods of the
invention.
[0063] The substrate nucleic acid molecules for use in the methods,
and inclusion in the kits, of the present invention, must be of
sequence and structure such that the NAD-dependent ligase can act
on the molecule to produce a detectable enzyme modified or ligated
(novel) nucleic acid molecule, and such that it does not allowing
interfering signals from DNA polymerase.
[0064] It is to be appreciated that in the development of the
present invention, it was noted that the elimination of the
polymerase chain reaction (PCR) Taq-DNA polymerase derived
background was not a viable solution to the lack of specificity
that has been found in the current substrate design as disclosed in
WO/2009/007719, as it was determined to be a separate detection
system issue that would have to be addressed separately and is
therefore outside the scope of the present disclosure.
[0065] Accordingly, in the present case for experiments leading up
to the present invention it was specifically set as a goal to block
all DNA polymerase activity with a inhibitor additive that does not
interfere with ligase. In order to accomplish this, it was noted
that DNA polymerases have well-documented enzyme functions that
need to be neutralized/controlled:
(a) 5'-3' DNA polymerase activity (b) 3'-5' exonuclease activity
(c) 5'-3' exonuclease activity (d) inherent esterase activity
[0066] It has been determined in accordance with the present
invention that suitable substrate nucleic acid molecule strategies
for use in the novel methods of the present invention, which are
suitable in substitution for those substrate molecules disclosed as
being used in the methods of WO/2009/007719, may include, but are
not limited to, the following: [0067] 1. Modified nucleotides that
inhibit the polymerase from any activity [0068] 2.
Dideoxynucleotides ddCTP, ddGTP that stop the polymerase upon first
base addition and sequester-neutralize its activity while ligase
enjoys productive reactions using dATP [0069] 3.
Dideoxyoligonucleotides that prevent AS oligo from being extended
[0070] 4. S1 oligos with polymerase inhibition modified bases
incorporated to block their activity on this DNA substrate [0071]
5. DNA polymerase specific antibodies that inhibit this
activity--these are well known in the art of PCR [0072] 6. Aptamer
oligo inhibitory complexes. [0073] 7. DNA substrate hybridization
strategies that eliminate polymerase extended substrates from being
detected in downstream amplifications such as PCR-by shortening AS
on the 5' side combined with a true "Hot Start," as such term is
known in the art [0074] 8. DNA substrate hybridization strategies
that eliminate polymerase from binding and extending by shortening
AS on 3' side but do not effect ligase [0075] 9. Relative rate
kinetics combined with polymerase extension length balanced in
favor of ligase. [0076] 10. Per-PCR S1 3'-dideoxy competition (full
length, or a 13mer that is the complementary to the 3' of the AS)
[0077] 11. Pre-PCR S1 3' Phosphate competition [0078] 12. Complete
removal of AS using optimal UNG (standard UNG enzyme) conditions
[0079] 13. Complete removal of AS using Thermostable UNG (NEB).
Will enable heat treatment of UNG to eliminate contaminating
ligase/polymerase, PCR mm must have dTTP [0080] 14. Make an AS that
has deoxyuridine (UNG removal) and the rest RNA bases to allow
UNG/Rnase co-treatment prior to PCR [0081] 15. Need to get dideoxy
3' AS [0082] 16. Shorten the 3' end of the AS to reduce Taq docking
at higher temps (i.e. 65 deg) [0083] 17. AS covalently attached to
a solid support during ligation/extension step [0084] 18.
3'-dideoxy S2 reverse complement (full length, or maybe just a
13mer that is the complementary to the S1 Pol extension) [0085] 19.
For background reduction--"Hot Start" strategies, as well known in
the art--100% elimination of unwanted oligo or extended oligo
hybridizations. [0086] a. True Physical--not easy to do as all
contact materials must be at the hot temperature of about 90
degrees C., and must never drop below a threshold temperature of
about 65 degrees C., the problem being that the transfer process
creates a temperature drop, which should be avoided. [0087] b. Non
enzyme Hot Starts, e.g., drop in 2 mM MgCl (0.1 mM EDTA protected),
primers, dNTPs or other essential components. [0088] c. Chem-primer
Hot Start.
[0089] Although it has been shown that the improvements of the
present invention can be realized by the substitution of the
suitable substrate nucleic acid molecules described herein for
those described in WO/2009/007719, it is to be appreciated that the
present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the
invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing
description. All such modifications are intended to fall within the
scope of the present invention. Moreover, all embodiments described
herein are considered to be broadly applicable and combinable with
any and all other consistent embodiments, as appropriate.
[0090] It will be appreciated by those of ordinary skill in the art
that the broad fundamental principles and teachings of the present
invention are capable of being applied to optimize all variations
of denaturant-enabled-crude lysate (bead mills &
ultrasonics)-direct-probe/SYBR-PCR analysis of various biological
tissue samples (including, but not limited to, blood, body fluid,
and soft tissues) for not only microorganisms or microbes as
specifically described above, but also for various pathogens, such
as any bacteria, fungi, virus, parasites, etc.
[0091] Although specific references are made herein to PCR, It is
further to be appreciated that the improvements of the present
invention are not limited to PCR or similar methodologies.
Amplification assays contemplated for use in the present invention
include, but are not limited to, other well-known nucleic-acid
based techniques such as DNA amplification assays, PCR assays
incorporating thermostable polymerases, and isothermal
amplifications methods. It is to be appreciated that one skilled in
the art may conceive of various suitable amplification methods that
will be useful in the practice of the present invention, and that
therefore the invention is not intended to be limited thereby.
[0092] It is also to be appreciated that the present invention has
applications in any and all methods, procedures and processes
involving DNA diagnostics. Examples of such applications include
but are not limited to those involving food, water safety,
bioterrorism, medical/medicines and/or anything involving pathogen
detection. In the food industry, the present invention can be used
to monitor the efficacy of preservatives. The method of the
invention has the potential to be applied to all cells. Although
bacterial cells are exemplified in the example, one of ordinary
skill in the art can easily see that the methods of the invention
can be applied to many other cell types. The invention can also be
used for the identification of substances that can disrupt
membranes and/or kill cells, e.g. bacterial cells. The
identification of new disinfectants and/or antibiotics are now a
priority since multidrug resistance organisms have flourished and
spread in health institutions and patients.
[0093] It will further be appreciated that the methods of the
invention, in combination with quantitative PCR as a tool, can
quickly and successfully identify the impact of a disinfectant
and/or antibiotic without having to spend time culturing the cells
and waiting for growth. In some instances, organisms can take days
to weeks to culture, and thus it can take significant time to see
if the candidate substance has been able to kill cells, like
microorganisms. In other instances, certain organisms will not grow
in cell culture, therefore making it difficult to determine if a
substance was effective. Thus, applying the novel methods of the
invention can save time and resources for identification of novel
disinfectants and/or antibiotics.
[0094] A further advantage of the novel methods according to the
invention is their ease of use. For example, using these methods,
large amounts of samples can easily be tested for the presence of
viable cells, e.g. bacteria. For example, samples may be tested for
the presence of potentially live bacteria with intact cell
membranes. In another embodiment, environmental samples may be
tested for the presence of viable cells, e.g. bacteria. These
samples may be, for example, collected from soil or be parts of
plants. The methods according to the invention can further be used
for testing of treated waste water both before and after
release.
[0095] The methods according to the invention may further be used
for testing medicinal samples, e.g., stool samples, blood cultures,
sputum, tissue samples (also cuts), wound material, urine, and
samples from the respiratory tract, implants and catheter
surfaces.
[0096] Another field of application of the methods according to the
invention can be the control of foodstuffs. In other embodiments,
the food samples are obtained from milk or milk products (yogurt,
cheese, sweet cheese, butter, and buttermilk), drinking water,
beverages (lemonades, beer, and juices), bakery products or meat
products. The method of the invention can determine if
preservatives in the food or antimicrobial treatment of food (such
as pasteurization) has prevented cell growth. A further field of
application of the method according to the invention is the
analysis of pharmaceutical and cosmetic products, e.g. ointments,
creams, tinctures, juices, solutions, drops, etc.
[0097] In addition, the methods of the invention can identify
potentially viable members of a microbial community for ecological
studies, health of specific soils for agricultural and/or
ecological systems. Traditionally identifying a bacterial community
has been performed using cultivation-based approaches or plate
counts. The more colonies that are counted, the more bacteria are
estimated to be in the original sample. Problems, however, arise
from sometimes long incubation times (in the range of days) making
this method unsuitable for timely and accurate results. These
drawbacks are utilizing the methods of the invention.
[0098] Non-limiting examples of bacteria that can be subjected to
analysis using the methods of the invention or to detect potential
viability in a sample using the method of the invention comprise,
for example: B. pertussis, Leptospira pomona, S. paratyphi A and B,
C. diphtheriae, C. tetani, C. botidinum, C. perfringens, C. feseri
and other gas gangrene bacteria, B. anthracis, P. pestis, P.
multocida, Neisseria meningitidis, N. gonorrheae, Hemophilus
influenzae, Actinomyces {e.g., Norcardia), Acinetobacter,
Bacillaceae {e.g., Bacillus anthrasis), Bacteroides {e.g.,
Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia {e.g.,
Borrelia burgdorferi), Brucella, Campylobacter, Chlamydia,
Coccidioides, Corynebacterium {e.g., Corynebacterium diptheriae),
E. coli {e.g., Enterotoxigenic E. coli and Enterohemorrhagic E.
coli), Enterobacter (e.g. Enter obacter aerogenes),
Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi,
Salmonella enteritidis, Serratia, Yersinia, Shigella),
Erysipelothrix, Haemophilus (e.g., Haemophilus influenza type B),
Helicobacter, Legionella (e.g., Legionella pneumophila),
Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma,
Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium
tuberculosis), Vibrio (e.g., Vibrio cholerae), Pasteurellacea,
Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa),
Rickettsiaceae, Spirochetes (e.g., Treponema spp., Leptospira spp.,
Borrelia spp.), Shigella spp., Meningiococcus, Pneumococcus and all
Streptococcus (e.g., Streptococcus pneumoniae and Groups A.sub.3 B,
and C Streptococci), Ureaplasmas. Treponema pollidum,
Staphylococcus aureus, Pasteurella haemolytica, Corynebacterium
diptheriae toxoid, Meningococcal polysaccharide, Bordetella
pertusis, Streptococcus pneumoniae, Clostridium tetani toxoid, and
Mycobacterium bovis. The above list is intended to be merely
illustrative and by no means is meant to limit the invention to
detection to those particular bacterial organisms.
[0099] A particularly preferred embodiment of the present invention
utilizes PCR. General procedures for PCR are taught in U.S. Pat.
No. 4,683,195 (Mullis, et al.) and U.S. Pat. No. 4,683,202 (Mullis,
et al.). However, optimal PCR conditions used for each
amplification reaction are generally empirically determined or
estimated with computer software commonly employed by artisans in
the field. A number of parameters influence the success of a
reaction. Among them are annealing temperature and time, extension
time, Mg.sup.2+, pH, and the relative concentration of primers,
templates, and deoxyribonucleotides. Generally, the template
nucleic acid is denatured by heating to at least about 95.degree.
C. for 1 to 10 minutes prior to the polymerase reaction.
Approximately 20-99 cycles of amplification are executed using
denaturation at a range of 90.degree. C. to 96.degree. C. for 0.05
to 1 minute, annealing at a temperature ranging from 48.degree. C.
to 72.degree. C. for 0.05 to 2 minutes, and extension at 68.degree.
C. to 75.degree. C. for at least 0.1 minute with an optimal final
cycle. In one embodiment, a PCR reaction may contain about 100 ng
template nucleic acid, 20 uM of upstream and downstream primers,
and 0.05 to 0.5 mm dNTP of each kind, and 0.5 to 5 units of
commercially available thermal stable DNA polymerases.
[0100] A variation of the conventional PCR is reverse transcription
PCR reaction (RT-PCR), in which a reverse transcriptase first
coverts RNA molecules to single stranded cDNA molecules, which are
then employed as the template for subsequent amplification in the
polymerase chain reaction. Isolation of RNA is well known in the
art. In carrying out RT-PCR, the reverse transcriptase is generally
added to the reaction sample after the target nucleic acid is heat
denatured. The reaction is then maintained at a suitable
temperature (e.g. 30-45.degree. C.) for a sufficient amount of time
(10-60 minutes) to generate the cDNA template before the scheduled
cycles of amplification take place. One of ordinary skill in the
art will appreciate that if a quantitative result is desired,
caution must be taken to use a method that maintains or controls
for the relative copies of the amplified nucleic acid. Methods of
"quantitative" amplification are well known to those of skill in
the art. For example, quantitative PCR can involve simultaneously
co-amplifying a known quantity of a control sequence using the same
primers. This provides an internal standard that may be used to
calibrate the PCR reaction.
[0101] Another alternative of PCR is quantitative PCR (qPCR). qPCR
can be run by competitive techniques employing an internal
homologous control that differs in size from the target by a small
insertion or deletion. However, non-competitive and kinetic
quantitative PCR may also be used. Combination of real-time,
kinetic PCR detection together with an internal homologous control
that can be simultaneously detected alongside the target sequences
can be advantageous.
[0102] Primers for PCR, RT-PCR and/or qPCR are selected within
regions or specific bacteria which will only amplify a DNA region
which is selected for that specific organism. Alternatively,
primers are selected which will hybridize and amplify a section of
DNA which is common for all organisms. Primer selection and
construction is generally known in the art. In general, one primer
is located at each end of the sequence to be amplified. Such
primers will normally be between 10 to 35 nucleotides in length and
have a preferred length from between 18 to 22 nucleotides. The
smallest sequence that can be amplified is approximately 50
nucleotides in length (e.g., a forward and reverse primer, both of
20 nucleotides in length, whose location in the sequences is
separated by at least 10 nucleotides). Much longer sequences can be
amplified. One primer is called the "forward primer" and is located
at the left end of the region to be amplified. The forward primer
is identical in sequence to a region in the top strand of the DNA
(when a double-stranded DNA is pictured using the convention where
the top strand is shown with polarity in the 5' to 3' direction).
The sequence of the forward primer is such that it hybridizes to
the strand of the DNA which is complementary to the top strand of
DNA. The other primer is called the "reverse primer" and is located
at the right end of the region to be amplified. The sequence of the
reverse primer is such that it is complementary in sequence to,
i.e., it is the reverse complement of a sequence in, a region in
the top strand of the DNA. The reverse primer hybridizes to the top
end of the DNA. PCR primers should also be chosen subject to a
number of other conditions. PCR primers should be long enough
(preferably 10 to 30 nucleotides in length) to minimize
hybridization to greater than one region in the template. Primers
with long runs of a single base should be avoided, if possible.
Primers should preferably have a percent G+C content of between 40
and 60%. If possible, the percent G+C content of the 3' end of the
primer should be higher than the percent G+C content of the 5' end
of the primer. Primers should not contain sequences that can
hybridize to another sequence within the primer (i.e.,
palindromes). Two primers used in the same PCR reaction should not
be able to hybridize to one another. Although PCR primers are
preferably chosen subject to the recommendations above, it is not
necessary that the primers conform to these conditions. Other
primers may work, but have a lower chance of yielding good
results.
[0103] PCR primers that can be used to amplify DNA within a given
sequence can be chosen using one of a number of computer programs
that are available. Such programs choose primers that are optimum
for amplification of a given sequence (i.e., such programs choose
primers subject to the conditions stated above, plus other
conditions that may maximize the functionality of PCR primers). One
computer program is the Genetics Computer Group (GCG recently
became Accelrys) analysis package which has a routine for selection
of PCR primers.
[0104] The oligonucleotide primers and probes disclosed below can
be made in a number of ways. One way to make these oligonucleotides
is to synthesize them using a commercially-available nucleic acid
synthesizer. A variety of such synthesizers exists and is well
known to those skilled in the art.
[0105] Nucleic acid may also be detected by hybridization methods.
In these methods, labeled nucleic acid may be added to a substrate
containing labeled or unlabeled nucleic acid probes. Alternatively,
unlabeled or unlabeled nucleic acid may be added to a substrate
containing labeled nucleic acid probes. Hybridization methods are
disclosed in, for example, Micro Array Analysis, Marc Schena, John
Wiley and Sons, Hoboken N.J. 2003.
[0106] Methods of detecting nucleic acids can include the use of a
label. For example, radiolabels may be detected using photographic
film or a phosphoimager (for detecting and quantifying radioactive
phosphate incorporation). Fluorescent markers may be detected and
quantified using a photodetector to detect emitted light (see U.S.
Pat. No. 5,143,854 for an exemplary apparatus). Enzymatic labels
are typically detected by providing the enzyme with a substrate and
measuring the reaction product produced by the action of the enzyme
on the substrate. Colorimetric labels are detected by simply
visualizing the colored label. In one embodiment, the amplified
nucleic acid molecules are visualized by directly staining the
amplified products with a nucleic acid-intercalating dye. As is
apparent to one skilled in the art, exemplary dyes include but not
limited to SYBR green, SYBR blue, DAPI, propidium iodine, and
ethidium bromide. The amount of luminescent dyes intercalated into
the amplified DNA molecules is directly proportional to the amount
of the amplified products, which can be conveniently quantified
using a conventional detection devices according to manufacturers'
instructions. A variation of such an approach is gel
electrophoresis of amplified products followed by staining and
visualization of the selected intercalating dye. Alternatively,
labeled oligonucleotide hybridization probes (e.g. fluorescent
probes such as fluorescent resonance energy transfer (FRET) probes
and colorimetric probes) may be used to detect amplification. Where
desired, a specific amplification of the genome sequences
representative of the biological entity being tested, may be
verified by sequencing or demonstrating that the amplified products
have the predicted size, exhibit the predicted restriction
digestion pattern, or hybridize to the correct cloned nucleotide
sequences.
[0107] The present invention also comprises kits. For example, the
kit can comprise a substrate containing a nucleic acid molecule for
activity of the selected enzyme or mixture in the sample (while not
allowing interfering signals from DNA polymerase), incubation means
for incubating the sample and substrate under conditions suitable
for enzyme activity, and means for specifically determining the
presence (and/or the amount) of a nucleic acid molecule resulting
from the action of the selected enzyme or mixture on the substrate
nucleic acid molecule (as an indication of the presence of the
microorganism). Such a kit can also comprise other reagents
suitable for conducting the novel methods of the invention, for
screening normally sterile body fluids for the presence of absence
of microorganisms therein and to provide diagnostic, prognostic
patient management information, as well as primers useful for
amplifying nucleic acid molecule corresponding to organisms
specifically or generally, buffers and reagents for isolating DNA,
and reagents for PCR. The kit can further include detectably
labeled oligonucleotide, which hybridizes to a nucleic acid
sequence encoding a polypeptide corresponding to organisms of
interest. The kit can also contain a control sample or a series of
control samples which can be assayed and compared to a test sample
contained. Each component of the kit can be enclosed within an
individual container and all of the various containers can be
within a single package, along with instructions for interpreting
the results of the assays performed using the kit.
[0108] It is also to be appreciated that the methods provided by
the invention further comprise conducting a complete or partial
microorganism genome and or transcriptome sequence analysis
utilizing the principles and teachings provided herein, and wherein
the complete or partial microorganism genome and or transcriptome
sequence analysis can be performed simultaneously, in concert, or
in parallel using a single sample preparation as herein described.
It is also to be appreciated that the novel methods herein of the
invention can provide for the diagnostic measure and detection of
agents with anti-microbial and or anti-polymerase activity, useful
in the management of patients.
[0109] The contents of all references, patents and published patent
applications cited throughout this application, are incorporated
herein by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
[0110] The foregoing detailed description has been given for
clearness of understanding only and no unnecessary limitations
should be inferred therefrom as modifications will be obvious to
those skilled in the art. It is not an admission that any of the
information provided herein is prior art or relevant to the
presently claimed inventions, or that any publication specifically
or implicitly referenced is prior art.
[0111] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0112] Although the present invention has been described in detail,
the specific examples herein are provided by way of specific
illustration of embodiments of the invention and for purposes of
clarity of understanding. It will be readily apparent to those of
ordinary skill in the art, in light of the teachings of this
invention as set forth herein, that many changes and modifications
may be made to these embodiments thus described without departing
from the spirit or scope of the invention.
[0113] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth and as follows in the scope of the appended
claims.
* * * * *