Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism

Definitions

• the present invention relates to methods for selectively excluding, from molecular detection, DNA of dead cells from a mixture containing live and dead cells, and in particular relates to improved methods for performing direct Polymerase Chain Reaction (PCR) techniques in blood and other body fluids for correlation with viable microbe cells from Bacteremia, Fungemia, Viremia and other types of parasite containing samples.
• PCR Polymerase Chain Reaction
• the improved methods provided by the invention are particularly advantageous for the diagnosis of septicemia.
• TTR time to result
• Encouraging issues for improvement of quality and reproducibility of molecular diagnostic applications in bloodstream infections include selective enrichment procedures for bacterial nucleic acids, blocking or elimination methods of excess human DNA, and use of viability markers to discriminate clinically relevant findings, as shown in experience from microbial safety analysis.
• disease-oriented multiplex PCR, pathogen microarrays and proteomic profiling have the potential to evolve as important rapid and high-throughput diagnostic means for infectious disease diagnosis.
• Metabolic and reproductive activity, and, in the case of pathogenic microorganisms, the potential health risk are limited to the live portion of a mixed microbial population.
• Four physiological states are used in the conventional art to distinguish, in flow cytometry using fluorescent stains: reproductively viable, metabolically active, intact and permeabilized cells. Depending on the conditions, all stages except the permeabilized cells can have the potential of recovery upon resuscitation and thus have to be considered potentially live. Due to the relatively long persistence of DNA after cell death in the range between days to 3 weeks, DNA -based diagnostics tend to overestimate the number of live cells. DNA extracted from a sample can originate from cells in any of the four mentioned physiological states including the dead permeabilized cells. Detection of the latter, however, is not desired.
• the most important criterion for distinguishing between viable and irreversibly damaged cells is membrane integrity. Sorting out noise derived from membrane-compromised cells helps to assign metabolic activities and health risks to the intact and viable portion of bacterial communities. Live cells with intact membranes have been distinguished by their ability to exclude DNA -binding dyes that easily penetrate dead or membrane -compromised cells.
• EMA-PCR was reported to be an easy-to-use alternative to microscopic or flow-cytometric analyses to distinguish between live and dead cells.
• This diagnostic DNA-based method combines the use of a live-dead discriminating dye with the speed and sensitivity of realtime PCR.
• Ethidium monoazide (EMA) a DNA -intercalating dye with the azide group allowing covalent binding of the chemical to DNA upon exposure to bright visible light (maximum absorbance at 460 nm), has been used in this regard.
• Cells are exposed to EMA for 5 minutes allowing the dye to penetrate dead cells with compromised cell walls/membranes and to bind to their DNA. Photolysis of EMA using bright visible light produces a nitrene that can form a covalent link to DNA and other molecules.
• PMA is identical to propidium iodide (PI), except that the additional presence of an azide group allows crosslinkage to DNA upon light- exposure.
• PI has been extensively used to identify dead cells in mixed populations.
• PMA concentration and incubation time were optimized with one gram-negative and one gram-positive organism before applying these parameters to the study of a broad-spectrum of different bacterial species.
• the disclosed method purportedly limits molecular diagnostics to the portion of a microbial community with intact cell membranes. This is achieved by exposing a mixture of intact and membrane- compromised cells to a phenanthridium derivative.
• PCR is performed using genomic DNA from the mixture as a template.
• WO/2001/077379 discloses methods of detecting cells in a sample and for obtaining quantitative information about cell populations within a sample.
• a method is disclosed for distinguishing between living and dead cells in a sample. The method comprises contacting the sample with a viability probe which modifies the nucleic acid of dead cells within the sample, and detecting nucleic acid from the cells in the sample.
• Also described is a method of detecting cells in a sample comprising: (a) contacting the sample with a viability probe which labels the nucleic acid of dead cells within the sample; (b) separating the nucleic acid from the cells into labeled and non-labelled fractions; and (c) detecting the nucleic acid in one or both of the fractions.
• PCR correlates with viable microbe cells derived from blood, employing a combination of selective blood cell lysis, washing (and or) DNase along with subsequent microbe cell lysis and PCR.
• the present invention seeks to realize the potential TTR advantage of molecular nucleic-acid based techniques, including PCR, by dramatically simplifying costly DNA isolations and sample preparation, and by not isolating DNA, but rather by performing a rapid and simple direct-analysis on crude microbe lysates after a rapid separation of the dead microbe DNA and cells, resulting in the selective enrichment of viable microbe cells.
• This is particularly and unexpectedly advantageous in the diagnosis of septicemia, and is accomplished according to a preferred embodiment of the present invention by:
• PCR inhibitors from blood can be eliminated via a simple combination of chemical denaturants (chaotropes: detergents, pH, salts, organic chemical based differential salvation via dipole moment such as alcohols and amine containing compounds & enzymes such as nucleases, proteinases etc.) and washing, thereby circumventing DNA isolation and enabling microbe lysate-Direct-PCR.
• chemical denaturants chaotropes: detergents, pH, salts, organic chemical based differential salvation via dipole moment such as alcohols and amine containing compounds & enzymes such as nucleases, proteinases etc.
• the ratio of live/dead microbes present in blood and blood culture can then be used as a measure of the effectiveness of a therapy and of testing the efficacy of treatment.
• Figure 1 shows, in table form, the results of experiments conducted to compare filter-bead mill- in situ microbe lysis and analyte analysis via DNA Polymerase (PolMA), and genomic DNA via quantitative gene specific PCR.
• Figure 2 shows an illustration in diagram form of a strategy for detection of microbes in lysates according to the invention.
• Figure 3 shows flow diagrams illustrating that the addition of trypsin and DNase enables significant reduction of clogging observed during the processing of two "difficult" clinical samples in accordance with the present invention.
• a chaotropic agent also known as chaotropic reagent and chaotrope, is a substance which disrupts the three dimensional structure in macromolecules such as proteins, DNA, or R A, and denatures them. Chaotropic agents interfere with stabilizing inter -molecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects. Often structural features, as detected by means such as circular dichroism can be titrated in a chaotrope concentration-dependent fashion. Chaotropic reagents include, for example: Urea 6 - 8 mol/1
• high generic salts can have chaotropic properties, by shielding charges and preventing the stabilization of salt bridges. Hydrogen bonding is stronger in nonpolar media, so salts, which increase the dipole moment of the solvent, can also destabilize hydrogen bonding.
• chaotrope concentration-dependent fashion Often structural features, as detected by means such as circular dichroism can be titrated in a chaotrope concentration-dependent fashion.
• Some examples of historically useful chaotropic reagents in biochemistry and molecular biology include: Urea 6 - 8 mol/1 , guanidinium chloride 6 mol/1, lithium perchlorate 4.5 mol/1, alchohols, amines (especially quaternary amines), detergents (especially nonionic), pH change, betaine, proline, carnitine, trehalose, NP-40 and the like , as well as BSA.
• DoE design of experiment
• the design of experiment (DoE) process has been used for optimization of effective formulation ranges and combinations of ranges of various chaotropes (mixtures or reagents, or "cocktails”) to: a) denature dead cell structures such that they are easily separated from live cells based on their size (filtration) and density (centrifugation); and b)
• Preferential blood cell lysis conditions yield preferential homogenization of blood cells from blood-microbe mixtures such as found in septicemia blood culture samples. Homogenization needs to occur at a sufficient level (creating a fluid) which enables passage of unwanted blood cells fluid through a filter from the Feed side (retaining desired microbe cells) through to the filtrate side effectively separating these two populations. These lysis conditions would enable the microbial cells to remain intact and thus enable rapid/sensitive filter-based separation of homogenized blood cells by retaining microbe cells.
• differential blood cell Lysis and sufficient homogenization of their resulting cell debris are employed to reduced blood cells down to a fluid level enabling differential filterability where the filter retains microbes on the Feed side, thus separating the intact microbes, for subsequent sterile fluids analyses.
• Filter pore sizes known to those in the art as pore sizes measuring between 0.45um, 0.22um, O. lum in diameter should be sufficient. However these effective pore sizes could be both smaller than 0.1 and larger than 0.45 depending on the microbe and differential cell debris size filterability.
• Conditions include but are not limited to optimized combinations of detergent, proteinases, chaotrops, denaturants, and nucleases to achieve the desired effects.
• Microbe specific filter-in situ is defined herein as employing physical and biochemical cell wall lysis methods while microbes are captured on the Feed side of the filter and /or subsequent microbe specific analyte assays applied in situ.
• in situ means lysis and or subsequent analysis occurs after differential separation of undesired interfering cells (i.e. Blood cells) while desired microbe cells are still retained on the Feed side of the filter.
• undesired interfering cells i.e. Blood cells
• desired microbe cells are still retained on the Feed side of the filter.
• the physical forces employed to lyse these now separated, intact and filter-contained microbes are those common to those skilled in this art including but not limited to enzymatic cell wall digestion.
• filter-in situ sonication of all microbes by direct probe contacting the residual liquid retained by surface tension on the filter side containing the separated microbes, alternatively by sonic probe contacting the opposite side of the filter from the microbes and transferring its lytic energy via through the pores not through the solid filter material.
• efficiency of filter-bead-mill in situ for microbe lysis of bacteria and yeast occurs as well in a closed microfuge tube as it does directly on the filter Feed surface after capturing microbes spiked in blood where the blood cells were differentially lysed and filter separated.
• filter-in situ as defined herein is an elegant simplification of septicemia sample preparation enabling more efficient processing with less manipulations, less potential for contamination, more flexible formats both manually and for automated device designs.
• filtration is employed as the term is commonly used in the art, that is, a mechanical or physical operation which is used for the separation of solids from fluids (liquids or gases) by interposing a medium through which only the fluid can pass.
• fluids liquids or gases
• Example 1 Experiments were conducted to compare filter-bead mill-in situ microbe lysis and analyte analysis via DNA Polymerase (PolMA), and genomic DNA via quantitative gene specific PCR. The results are presented in the tables illustrated in Figure 1 of the drawings. Interpretation of delta Ct values must be greater than two to be considered a significant
• Example 2 This example of an embodiment of the invention demonstrates the suitability of the present invention for circumventing the necessity for conventional DNA isolation techniques, and for enabling microbe lysate-direct-probe-based-PCR techniques to be performed a.
• Staphylococcus Aureus (SA) was spiked into standard blood cultures, (Candida consensus assay, E.Coli, E faecium) followed by WBC detergent + base lysis, pelletizing, and washing.
• any PCR measurement of at least two separate time points using separate but equal aliquots from a single blood culture that shows a significant increase in a microbe target signal must be due to microbe growth, thereby indicating the presence of viable microbes (disregarding contamination effects). It is to be appreciated that non-growth based single point positive PCR analysis of blood will indicate the presence of a viable microbe when all dead cell DNA has been eliminated, prior to viable microbe lysis and PCR setup - baring any PCR process induced contamination. This can be demonstrated by by DNasing and Washing away dead cell DNA.
• 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. It is to be appreciated that the present invention has applications in any and all methods, procedures and processes involving DNA diagnostics.
• 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.
• 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.
• 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.
• applying the novel methods of the invention can save time and resources for identification of novel disinfectants and/or antibiotics.
• a further advantage of the novel methods according to the invention is ease of use.
• samples can easily be tested for the presence of viable cells, e.g. bacteria.
• samples may be tested for the presence of potentially live bacteria with intact cell membranes.
• environmental samples may be tested for the presence of viable cells, e.g. bacteria.
• 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.
• 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.
• Another field of application of the methods according to the invention can be the control of foodstuffs.
• 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.
• the methods of the invention solve the problem of long incubation times (in the range of days) making the older methods unsuitable for timely warning and preventive action.
• modern PCR based methods can give false positive results (testing positive for an organism although the organism is not viable).
• research has recently discovered that some organisms can, under certain circumstances, lose the ability to replicate although they are still viable.
• These 'viable but not culturable' (VBNC) bacteria cannot be detected using traditional cultivation but might regain their ability to grow if transferred to a more appropriate environment.
• contaminated water, sewage, food, pharmaceuticals and/or cosmetics can prevent contaminated products from being released to the public.
• the methods of the invention can save resources, by minimizing false positives (testing positive for a pathogen although the pathogen is not viable) and rapid testing of samples, as compared to the current time consuming methods.
• 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.
• 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 roblems, 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.
• 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, in addition to SA as previously described: 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.
• Enterobacter aerogenes Enterobacter aerogenes
• Enterobacteriaceae Enterobacteriaceae
• 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 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 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.
• 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.).
• optimal PCR conditions used for each amplification reaction are generally empirically determined or estimated with computer software commonly employed by artisans in the field.
• annealing temperature and time e.g., annealing temperature and time, extension time, Mg 2+ , pH, and the relative concentration of primers, templates, and deoxyribonucleotides.
• extension time e.g., extension time
• Mg 2+ e.g., adenosine triphosphate
• pH e.g., adenosine
• 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.
• RT-PCR reverse transcription PCR reaction
• 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.
• 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°C) for a sufficient amount of time (10-60 minutes) to generate the cDNA template before the scheduled cycles of amplification take place.
• qPCR quantitative PCR
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• PCR Another alternative of PCR useful in connection with the invention is isothermal nucleic acid amplification assay for the detection of specific DNA or RNA targets.
• isothermal amplification of nucleic acids are homogeneous real-time strand displacement amplification, Phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing, rolling-circle amplification of duplex DNA sequences assisted by PNA openers or loop-mediated isothermal amplification of DNA analytes.
• 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.
• Methods of detecting nucleic acids can include the use of a label.
• 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.
• the amplified nucleic acid molecules are visualized by directly staining the amplified products with a nucleic acid-intercalating dye.
• exemplary dyes include but not limited to SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold 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 Flurolmager (Molecular Dynamics) or other equivalent devices according to manufacturers' instructions.
• Flurolmager Molecular Dynamics
• a variation of such an approach is gel electrophoresis of amplified products followed by staining and visualization of the selected intercalating dye.
• labeled oligonucleotide hybridization probes e.g.
• fluorescent probes such as fluorescent resonance energy transfer (FRET) probes and colorimetric probes
• FRET fluorescent resonance energy transfer
• 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.
• kits can comprise 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 also 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

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