Methods for rapidly detecting heat resistant molds and yeast

Abstract

An improved, faster method of selectively enriching a liquid sample, which may be a beverage, for heat resistant yeast or mold and detecting the live organisms. The method includes the step of exposing dead cells and free DNA to a reagent that intercalates within DNA and prevents the DNA from participating in a PCR reaction. The DNA from the remaining live cells may then be detected using a PCR technique. Kits and systems for the use of the method are included.

Description

[0001] METHODS FOR RAPIDLY DETECTING HEAT RESISTANT MOLDS AND YEAST FIELD OF THE INVENTION The present embodiments relate to systems, kits, and methods for detecting important microbial spoilage organisms that can survive heat fill and pasteurization methods. BACKGROUND The true purpose of food and beverage spoiler testing is to detect contamination of live organisms that can be transmitted via food and beverage, either directly or indirectly through the food contact surfaces, and make people sick or spoil food and beverage products. To this end, many food and beverage processors have a kill-step or other mitigation process to eliminate most if not all living microorganisms while leaving components of killed organisms in the product. Consequently, food and beverage samples with existing organisms, although killed and thus not a threat to human health or product quality, can be carried over to the finished good. The same phenomenon can be true for environmental surface samples. Polymerase chain reaction (PCR) technology or any molecular (nucleic acid amplification) technology amplifies and detects the target DNA, an indirect indicator of the pathogen presence. The carried over DNA from the killed organisms can contribute to the final PCR signal independent of the DNA that is coming from the live organisms. More specifically, these techniques do not distinguish between DNA from live and dead organisms. This deters from the true purpose of testing, which is to detect the live organisms only. This can cause a significant loss of time, effort, product, and money due to essentially false positives in testing. In particular, the beverage industry struggles with detection of mold and yeast spoilage organisms that can survive hot fill and pasteurization. The currently available methods for detecting these organisms are often laborious and lengthy, delivering results in weeks (˃ 20 days). From a quality management standpoint, these lengthy methods are not compatible with rapid production and shipping schedules in the beverage industry. There is a need for a rapid method for the detection of heat resistant mold and yeast as a preventive QA / QC tool. SUMMARY OF THE INVENTION Embodiments herein are directed to novel methods, kits, and systems which may be used for a rapid enrichment and detection of viable heat resistant mold and yeast species. In one embodiment, the disclosure is a method which includes the steps of heat shocking a liquid sample to kill heat-sensitive organisms. Then a growth medium is added to the liquid sample to create a culture mixture. Next, the culture mixture is incubated at a temperature in which heat resistant contaminant cells can divide, thus enriching the remaining heat resistant organisms. The organisms are then exposed to a viability PCR reagent which intercalates within DNA that is not within a live cell. The contaminant cells are then separated from the viability PCR reagent for analysis. In some embodiments, the liquid sample is mixed with a growth medium prior to the step of heat shocking. The sample and medium mixture may then proceed to the step of incubating the culture mixture at a temperature in which heat resistant contaminant cells can divide, thus selectively enriching the remaining heat resistant organisms. The organisms are then exposed to a viability PCR reagent which intercalates within DNA that is not within a live cell. The contaminant cells are then separated from the viability PCR reagent for analysis. In some embodiments, the method is a method of selectively enriching a liquid sample for and detecting live heat resistant yeast and mold. In other embodiments, the method can be used to detect non-heat resistant (or heat sensitive) organisms that have survived the heat shock step and / or pasteurization. In some embodiments, the step of heat shocking includes placing the liquid sample in an environment comprising a temperature of at least about 60°C. In some embodiments, the step of heat shocking includes placing the liquid sample in an environment comprising a temperature of at least about 75°C. In some embodiments the step of heat shocking includes the step of maintaining the liquid sample in the heated environment for at least about 15 minutes. In some embodiments the step of heat shocking includes the step of maintaining the liquid sample in the heated environment for at least about 30 minutes. Some embodiments further include the step of cooling the liquid sample after heat shocking and prior to incubating the culture mixture. In some embodiments, step of cooling includes exposing the liquid sample to a temperature of between about 45°C and about 55°C. In some embodiments, the step of cooling is performed for about 10 minutes. In some embodiments of the enrichment step, the temperature at which heat resistant contaminant cells can divide is between about 10°C and about 35°C. In some embodiments, the temperature at which the heat resistant contaminant cells can divide is between about 30°C to about 35°C. In some embodiments, the temperature at which heat resistant contaminant cells can divide is 30 ±2°C. In some embodiments, the heat resistant contaminant cells are incubated for at least 48 hours, for at least 72 hours, or for 72±2 hours. In some embodiments the viability PCR reagent with which the enriched organisms are mixed is a fluorogenic nucleic acid stain. In some embodiments, the fluorogenic nucleic acid stain is PMAxx™, propidium monoazide, or ethidium monoazide. In some embodiments, the fluorogenic nucleic acid stain is photoactivated after mixing with the enriched organisms. In some embodiments, the step of photoactivating the fluorogenic nucleic acid stain includes exposing the viability PCR reagent to a light source. In some embodiments, the light source has a wavelength of between about between 465nm and about 475 nm. In some embodiments the step of separating the heat resistant contaminant cells from the viability PCR reagent and the culture mixture includes centrifuging the viability PCR reagent and the culture mixture and removing a supernatant. Some embodiments, further include the step of digesting the heat resistant contaminant cells after the step of separating the heat resistant contaminant cells from the viability PCR reagent and the culture mixture. This serves to release the DNA from inside live cells. This DNA does not have viability PCR reagent intercalated within it and thus, can be amplified by a PCR reaction. In some embodiments, the digestion step includes reacting the heat resistant contaminant cells with an enzyme capable of digesting cell walls. In some embodiments, this enzyme is lyticase. In some embodiments, the digestion step is conducted at a temperature of about 30°C-about 37°C. In some embodiments, the enzyme is deactivated after the digestion step by incubating the contaminant cells and enzyme mixture at about 95°C for about 10 minutes. Some embodiments, further include the step of comprising the step of detecting DNA released from the digested heat resistant contaminant cells. In some embodiments, the detection step may include performing a polymerase chain reaction (PCR), isothermal amplification, or ligase chain reaction (LCR) reaction. While the disclosed method may be applied to any liquid which is thought to have heat resistant yeast or mold, in some embodiments, the liquid is a beverage. In some embodiments, the liquid is a juice. In some embodiments, the juice has a pH of below 7.0. The disclosure also includes a kit. In some embodiments the kit includes any combination of an enzyme capable of digesting cell walls, a viability PCR reagent, and at least one pair of PCR primers. The pair of primers includes a first primer that specifically hybridizes to a target nucleic acid molecule derived from a heat resistant yeast or mold and a second primer that specifically hybridizes to the complement of the target nucleic acid molecule. The primers hybridize under amplification conditions in which they may produce an amplicon. In some embodiments, the kit may further include one or more of a DNA polymerase, deoxynucleotide triphosphates, a buffer, and a probe. In some embodiments, the sequence of the probe is substantially homologous to or substantially complementary to a sequence of the target nucleic acid. In some embodiments, the kit may include at least one PCR tube. Each of the PCR tubes may include one or more of the at least one pair of PCR primers, a DNA polymerase, deoxynucleotide triphosphates, a buffer, and a probe. The disclosure also includes a system. In some embodiments, the system includes a kit as described herein and an instrument configured to perform a DNA amplification assay on the DNA released from the digested heat resistant contaminant cells. In some embodiments, kit included in the system further includes at least one PCR tube which includes one or more of the following: a DNA polymerase, deoxynucleotide triphosphates, a buffer, and a probe. In some embodiments, the PCR tubes further include at least one primer pair. Each of the at least one primer pair includes a first primer that specifically hybridizes to a target nucleic acid molecule derived from a heat resistant yeast or mold and a second primer that specifically hybridizes to the complement of the target nucleic acid molecule. In some embodiments of the system, the instrument comprises a sample temperature controlling device. What is described is: A.1. A for detecting heat resistant yeast or mold cells in a liquid sample, the method comprising: a. obtaining a liquid sample suspected of being contaminated with heat resistant yeast or mold cells and / or spores; b. heat shocking the liquid sample to kill heat sensitive organisms; c. adding a growth medium to the liquid sample to create a culture mixture; d. incubating the culture mixture at a temperature that allows the heat resistant yeast or mold cells to divide and / or grow; e. adding a viability PCR reagent to the culture mixture; f. separating the heat resistant yeast or mold cells from the viability PCR reagent and the culture mixture; and g. detecting DNA from heat resistant yeast or mold cells, and thereby detecting the presence of heat resistant yeast or mold cells in the liquid sample. A.2. The method of clause A.1, wherein the step of adding a growth medium to the liquid sample to create a culture mixture is performed before the step of heat shocking the liquid sample. A.3. The method of clause A.1, wherein the step of adding a growth medium to the liquid sample to create a culture mixture is performed after the step of heat shocking the liquid sample. A.4. The method of clause A.1, wherein the step of heat shocking comprises placing the liquid sample in an environment comprising a temperature of at least about 60°C. A.5. The method of clause A.1, wherein the step of heat shocking comprises placing the liquid sample in an environment comprising a temperature of at least about 75°C. A.6. The method of clauses A.4 or A.5, wherein the liquid sample is maintained in the environment for at least about 15 minutes. A.7. The method of clauses A.4 or A.5, wherein the liquid sample is maintained in the environment for at least about 30 minutes. A.8. The method of clause A.1, further comprising the step of cooling the liquid sample after heat shocking the liquid sample and prior to incubating the culture mixture. A.9. The method of clause A.8, wherein the step of cooling comprises exposing the liquid sample to a temperature of between about 45°C and about 55°C. A.10. The method of clause A.8, wherein the step of cooling is performed for about 10 minutes. A.11. The method of clause A.1, wherein the culture mixture is incubated in step (d) at a temperature between about 10°C and about 35°C. A.12. The method of clause A.11, wherein the culture mixture is incubated in step (d) at a temperature between about 30°C to about 35°C. A.13. The method of clause A.11, wherein the culture mixture is incubated in step (d) at a temperature of about 30 ±2°C. A.14. The method of clause A.1, wherein incubating the culture mixture in step (d) is performed for at least 48 hours. A.15. The method of clauseA.1, wherein incubating the culture mixture in step (d) is performed for at least 72 hours. A.16. The method of clause A.1, wherein incubating the culture mixture in step (d) is performed for about 72±2 hours. A.17. The method of clause A.1, wherein the viability PCR reagent comprises a fluorogenic nucleic acid stain. A.18. The method of any of clauses A.17-A.19, wherein the viability PCR reagent comprises PMAxx™, propidium monoazide, or ethidium monoazide. A.19. The method of clause A.17 or A.18. further comprising the step of photoactivating the fluorogenic nucleic acid stain after mixing the heat resistant yeast or mold cells with the fluorogenic nucleic acid stain. A.20. The method of clause A.18, wherein the step of photoactivating the fluorogenic nucleic acid stain comprises exposing the viability PCR reagent to a light source. A.21. The method of clause A.1, wherein the step of separating the heat resistant yeast or mold cells from the viability PCR reagent and the culture mixture comprises centrifuging the viability PCR reagent and the culture mixture to pellet the heat resistant yeast or mold cells, removing a supernatant and optionally resuspending the pellet. A.22. The method of clause A.1, further comprising a step of digesting the heat resistant yeast or mold cells after the step of separating the contaminant cells from the viability PCR reagent and the culture mixture. A.23. The method of clause A.22, wherein the step of digesting comprises reacting the heat resistant yeast or mold cells with an enzyme capable of digesting cell walls. A.24. The method of clause A.22, wherein the step of digesting comprises reacting the heat resistant yeast or mold cells with lyticase or zymolase. A.25. The method of clauses A.23 or A.24, further comprising the step of incubating the heat resistant yeast or mold cells at about 95°C for about 10 minutes. A.26. The method of clause A.1, further comprising the step of detecting DNA in the heat resistant yeast or mold cells. A.27. The method of clause A.26, wherein the step of detecting DNA comprises performing a polymerase chain reaction (PCR), isothermal amplification, or ligase chain reaction (LCR) reaction. A.28. The method of clause A.27, wherein DNA is detected using a fluorescent DNA probe. A.29. The method of A.28, wherein the DNA probes comprise FRET hybridization probes for detection of heat resistant yeast and / or mold DNA molecules. A.30. The method of clause A.1, wherein the liquid is a beverage. A.31. The method of clause A.1, wherein the liquid is a juice. A.32. The method of clause A.31, wherein the juice has a pH of below 7.0. A.33. The method of any one of clauses A.1-A.32, wherein the method further comprises detecting non-heat resistant (or heat sensitive) organisms that survived the heat shock step and / or pasteurization. B.1. A kit, comprising: an enzyme capable of digesting cell walls; at least one pair of primers, each pair of primers comprising: a first primer that specifically hybridizes to a target nucleic acid molecule derived from a heat resistant yeast or mold and a second primer that specifically hybridizes to a complement of the target nucleic acid molecule under amplification conditions capable of producing an amplicon, and a viability PCR reagent. B.2. The kit of clause B.1, further comprising at least one of the following: a DNA polymerase, deoxynucleotide triphosphates, a buffer, and at least one probe. B.3. The kit of clause B.2, wherein the at least one probe comprises a sequence that is substantially homologous to or substantially complementary to a sequence of the target nucleic acid. B.4. The kit of clause B.1, wherein the at least one pair of primers and at least one of a DNA polymerase, deoxynucleotide triphosphates, a buffer, and at least one probe are provided in each of at least one PCR tube. C.1. A system, comprising: a kit of any one of clauses B.1-B.4; and an instrument configured to perform a DNA amplification assay. C.2. The system of clause C.1, further comprising at least one PCR tube, wherein each of the at least one PCR tube comprises at least one of the following: a DNA polymerase, deoxynucleotide triphosphates, a buffer, and a probe. C.3. The system of clause C.1, wherein the kit comprises the kit of clause B.2. C.4. The system of clause C.1, wherein the instrument comprises a sample temperature controlling device. BRIEF DESCRIPTION OF THE DRAWINGS FIGs.1A-1B. Illustrate workflows for a method of detecting heat resistant yeast or mold in accordance with the present invention. FIG.1A illustrates a workflow for detecting heat resistant yeast or mold in a liquid sample. FIG. 1B illustrates a workflow for detecting heat resistant yeast or mold in a liquid sample. FIG. 2. illustrates a block diagram of an exemplary embodiment of a thermal cycling system in accordance with aspects of the disclosure. FIGs.3A-3B. illustrate melt curves results obtained using the GENE-UP®system from grape juice concentrate samples inoculated with B. fulva and tested in accordance with the present invention. FIGs.4A-4B. illustrate melt curves results obtained using the GENE-UP®system from cranberry juice concentrate samples inoculated with B. fulva and tested in accordance with the present invention. FIGs.5A-5B. illustrate melt curves results obtained using the GENE-UP®system from red grapefruit juice concentrate samples inoculated with A. chevalieri and tested in accordance with the present invention. DETAILED DESCRIPTION OF THE INVENTION The present disclosure provides for a novel methods, kits, and systems which may be used for the enrichment and detection of viable heat resistant mold and yeast species. The methods and systems exclude detection of heat sensitive organisms. In other embodiments, the methods, kits and systems described herein can be used for the detection of non-heat resistant (or heat sensitive) organisms that have survived a heat treatment or pasteurization step that is used to kill non-heat resistant cells. Kits for use in the methods and systems are also disclosed. Detection of such heat resistant species may be used in the food and beverage industry to predict spoilage potential of products which are or may be contaminated with heat resistant species even after undergoing heat fill or pasteurization methods. The methods and systems disclosed herein are faster and less laborious than those currently available. Various methods are described in the embodiments herein. The embodiments can be combined with one another. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. All references cited herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the subject matter is not entitled to antedate such disclosure by virtue of prior invention. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the disclosure or any exemplified term. Likewise, the disclosure is not limited to its preferred embodiments. That the disclosure may be more readily understood, select terms are defined. Unless defined otherwise, all technical and scientific terms have the same meaning as is commonly understood by one of ordinary skill in the art to which the embodiments disclosed belongs. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the mixtures and methods of the disclosure and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether a term is elaborated or discussed herein. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The articles “a,” “an,” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a sample” means one sample or more than one sample. As used herein, the adverbs “about” or “approximately” means that the numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical limitation is used, unless indicated otherwise by the context, “about” means the numerical value can vary by ±5% and remain within the scope of the disclosed embodiments. Thus, about 100 means 95 to 105. An “amplicon,” as described herein, is an amplification product. An amplicon can be produced by amplifying a nucleic acid sequence from a test sample. An amplicon can include, but is not limited to, a PCR product. A “PCR product,” as described herein, refers to any product produced as a result of a PCR reaction. Without wishing to be bound by theory, an amplicon can be produced by amplifying a nucleic acid molecule through many means, and the amplicon can be detected through many means. Non-limiting examples of DNA amplification reactions include polymerase chain reaction (PCR), isothermal amplification, ligase chain reaction (LCR), and rolling circle replication (RCR). Non-limiting examples of PCR reactions include emulsion PCR, real time PCR (RT-PCR), multiplex PCR, long-range PCR, single-cell PCR, fast-cycling PCR, methylation-specific PCR (MSP), hot start PCR, high-fidelity PCR, rapid amplified polymorphic DNA analysis (RAPD), rapid amplification of cDNA ends (RACE), in situ PCR, differential display PCR, and bridge PCR (bPCR) amplification. Non-limiting examples of isothermal reactions include isothermal amplification is LOOP-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), thermophilic helicase- dependent amplification (tHDA), rolling-circle amplification (RCA),multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), nucleic acid sequence- based amplification (NASBA), self-sustained sequence reaction (3SR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), and bridge amplification. An amplicon can be detected following amplification, e.g., with a DNA gel, a lateral flow detection device, or a vertical flow detection device. Alternatively, the predictive region, or a fragment thereof, or a complement thereof, may be amplified and the resulting amplicon may be detected in real-time or post-amplification. In one aspect, positive amplification of DNA may be monitored in real-time using dsDNA binding dyes. As used herein, the term “beverage” means a liquid for drinking. As used herein, the term “juice” refers to juice at any stage in the process of manufacturing juice prior to packaging into any container that can hold juice. As used herein, the term “sample” means any fluid medium or liquid that may contain a particular item or that is suspected of containing a particular item. In some embodiments, samples may be used which are high in dissolved solids without further processing. In some embodiments, samples containing high solids (non-dissolved) may be analyzed through the use of a filter or used in conjunction with additional manual steps. In some embodiments, samples are non-filtered. In some embodiments, samples are filtered. In some embodiments, samples are purified. In some embodiments, samples are non-purified. Samples may be a liquid, a suspension, extracted or dissolved sample, or a supercritical fluid. If a sample is going to be used in a flow device (vertical or lateral) some flow properties should exist in the sample or sample extract or be added to the sample or sample extract to allow flow through the devices and systems described herein. Examples of samples include, but are not limited to, food swabs, food extracts, food suspensions, food cultures, yeast cultures, amplification reactions, PCR reactions, and the like which are liquids or are suspended or dissolved in a liquid. The sample may also be derived from another sample. For example, a PCR reaction may be performed on a nucleic acid mixture that has been extracted, isolated, and / or purified from another sample (e.g., juice). The PCR reaction would be considered to be a sample derived from another sample. As used herein, the term “detecting” or “detection” is used in the broadest sense to include qualitative and / or quantitative measurements of an analyte. As used herein, the term “probe” is defined as a detectable, illustratively partially single-stranded and often entirely single-stranded, polynucleotide, for example, an oligonucleotide, capable of specifically hybridizing to a target nucleic acid. As used herein, “specifically hybridizes” means that a probe, primer, or oligonucleotide recognizes and physically interacts (that is, base-pairs) with a substantially complementary nucleic acid (for example, a sample nucleic acid) under high stringency conditions, and does not substantially base pair with other nucleic acids. This invention takes a novel approach to heat resistant yeast and mold detection by providing a method for detecting all yeast and molds and combines it with a selective enrichment scheme plus a treatment that allows for only detecting viable organisms. The disclosed method is faster than those known in the art and more compatible with rapid production and shipping schedules in the beverage industry. The method steps include: 1. Obtaining a liquid sample known to be, or suspected of being, contaminated with a heat resistant yeast or mold cells. 2. Heat shocking the liquid sample at a temperature and length of time to kill heat-sensitive organisms therein. 3. Combining a liquid sample with enrichment medium in a vessel to propagate the microbial organisms therein. 4. Incubating the heat shocked cultured liquid under conditions conducive to propagating the surviving contaminating organisms. 5. Treating the heat shocked enriched liquid sample with a nucleic acid stain (e.g., a viability PCR reagent) to enable polymerase chain reaction (PCR) detection only of viable organisms that survived the heat treatment. 6 Detecting and analyzing amplicons produced by a variation of the PCR reaction (for example, real time PCR using fluorescence resonance energy transfer (FRET) based chemistry) or other methods of detecting specific DNA sequences. METHODS OF USE An example workflow according to the present invention is illustrated and described with reference to FIG.1A. As shown in FIG.1A, at step 102, a liquid sample known to be, or suspected of being, contaminated with a heat resistant yeast or mold cells and / or spores is obtained. In general, the liquid sample can be any liquid sample known to be, or suspected of being, contaminated with a heat-resistant yeast or mold cells and / or spores. In accordance with some embodiments of the present invention, the liquid sample is a juice or other beverage known to be, or suspected of being, contaminated with a heat resistant yeast and / or mold cells and / or spores is obtained. The method may include an enrichment step to enrich the heat resistant yeast or mold cells and / or spores in the sample while killing the heat sensitive organisms therein. In an embodiment of the disclosed method, as shown in FIG. 1A at step 104, the enrichment step begins by heat shocking the sample by heating the sample at a temperature and length of time sufficient to kill heat sensitive organisms therein. For example, in one embodiment, the liquid sample can be heated at a temperature of greater than 60°C for more than 10 minutes. In another embodiment, the liquid sample is heat shocked at a temperature of between about 60°C to about 80°C for about 10 minutes to about an hour. In another embodiment, the heat shock step is performed by heating the sample at a temperature of between about 60°C to about 80°C for about 10 minutes to about 30 minutes. In yet another embodiment, the heat shock step is performed by heating the sample at about 75°C for about 30 minutes. In some embodiments, the method can further be used to detect non-heat resistant (or heat sensitive) organisms that have survived the heat shock step and / or pasteurization. Optional, following the heat shock step, the sample may then be cooled prior to subsequent steps described herein. For example, in one embodiment, the heat shocked sample is cooled by incubating the sample (e.g., in a water bath) at a temperature of about 20°C to about 25°C for at least about 10 minutes. In another example, the cooling step may be performed for about 15 minutes. In another example, the cooling step may be performed for about 20 minutes. In some embodiments, the cooling step may be performed by housing the heat shocked sample in a refrigerated enclosure, ice bath, or placing the heat shocked sample on ice until it reaches a temperature of 25°C, 20°C, or lower. In some embodiments, the heat shocked sample may be stored at 25°C, 20°C, or lower until the next step in the disclosed method is to be performed. In some embodiments, the heat shocked sample may then be filtered, for example, through an 0.8 µM filter, after which the filtrate may be discarded. The heat shocked sample, or the filter if the heat shocked sample was filtered, may be combined with a growth medium in a container. The growth medium may be any that is sufficient for propagation of, or growth of, any heat resistant yeast and / or mold contained in the sample. In one embodiment, the growth medium is malt extract broth (MEB). In an example, the growth medium may be provided in a volume of about 200 mL to about 400 mL. In some embodiments the container may be a sealable bag, for example, a filter bag. Next, the Enrichment Step further comprises a growth step for the growth of heat sensitive yeast and / or mold that survived the heat shock step. As shown in step 106 and 108 of FIG.1A, a growth medium is added to the heat shocked liquid sample creating a culture mixture and the mixture incubated at a temperature and for a length of time sufficient to allow heat resistant yeast and / or mold to grow. In some embodiments, the mixture is incubated at a temperature and for a length of time sufficient to allow any surviving non-heat resistant organisms to grow. In still other embodiments, the liquid sample itself, or a filter liquid sample, may be combined with the growth medium prior to the heat shock step. The container housing the culture mixture comprising the heat shocked sample (or housing the filtered sample) and the growth medium may be incubated at a temperature and for a length of time that is conducive to culturing any live, heat resistant yeast and / or mold contained therein. In one embodiment, the culture mixture is incubated at a temperature between about 10°C to about 35°C for at least 36 hours. In another embodiment, the culture mixture is incubated at temperature between about 25°C to about 32°C for about 48 hours to about 96 hours. In yet another embodiment, the culture mixture is incubated at a temperature of 30°C ± 2°C for 72 ± 2 hours. In some embodiments, the step of incubating may be performed in an incubator. After the enrichment step, as shown at step 110 in FIG. 1A, a viability PCR reagent is added to the culture mixture. A “viability PCR reagent” is a molecular reagent comprising a molecule that is capable of selectively penetrating cells that have compromised or damaged cell walls and that are capable of irreversibly binding to DNA molecules therein. As one of skill in the art would readily understand, the use of a viability PCR reagent prevents DNA molecules from non-viable cells (or free DNA molecules released therefrom) from being amplified in a subsequent PCR reaction. In general, any known viability PCR reagent can be used in the practice of the present invention. In one embodiment, the viability PCR reagent is a photo-reactive) DNA-binding dye that upon photolysis with visible light becomes covalently attached to DNA. In some embodiments, the viability PCR reagent is a photo-reactive, fluorogenic nucleic acid stain. Examples of photo-activated, fluorogenic nucleic acid stains which may be used in this method include, propidium monoazide, PMAxx™ (an improved propidium monoazide, available from Biotium Inc., Fremont, California) and ethidium monoazide. In some embodiments, any heat resistant yeast and / or mold cells (or any surviving non-heat resistant organism therein) in the enriched sample may be concentrated prior to addition of the viability PCR reagent. In an example, the heat resistant yeast and / or mold cells may be concentrated by centrifuging the enriched sample and decanting the supernatant. The cells may be resuspended in a buffer, for example, a buffer designed to buffer the pH and condition the sample for a viability PCR reagent, thus creating a cell suspension including any heat resistant yeast and / or mold cells. In one embodiment, after addition of the viability PCR reagent to the culture mixture (or optionally to the re-suspended cells) the mixture is incubated to allow the viability PCR reagent to intercalate into DNA. As previously described, the viability PCR reagent binds only to DNA which is not contained within living cells thereby preventing subsequent amplification of the DNA. More specifically, the viability PCR reagent binds to free DNA that has been released by lysed cells or to DNA within cells having compromised or damaged cell walls. In general, this step is performed according to the instructions provided by the manufacturer or vendor of the viability PCR reagent. In some embodiments, this incubation is performed for about 10 minutes, for about 15 minutes, for about 20 minutes, for about 30 minutes or more at about room temperature. In some embodiments, the combination of viability PCR reagent and cell suspension is mixed during the incubation. In some embodiments the combination of viability PCR reagent and cell solution is transferred to a black opaque container prior to incubation to prevent light from prematurely activating the viability PCR reagent. If incubated in a black opaque container, the combination may be transferred to a clear container after the incubation step. As shown in step 110, in accordance with some embodiments, the viability PCR reagent is activated. For example, when a photo-reactive viability PCR reagent is used, the mixture may be exposed to a light source to activate the viability PCR reagent, thereby causing it to irreversibly bind to DNA molecules therein. In one embodiment, the mixture is exposed in a light emitting device, for example, an LED photolysis device, to photoactivate the viability PCR reagent. In another embodiment, the LED photolysis device emits light at one or more wavelengths of between 465-475 nm. In general, this photo-activation step is carried out in accordance with instructions provided by the manufacturer of the photo-reactive viability PCR reagent. In some embodiments, photo-activation may occur for about 5 minutes, for about 10 minutes, for about 15 minutes, for about 20 minutes, for about 30 minutes or more at room temperature. After the photo-activation step, as shown in FIG. 1A at step 112, the live heat resistant yeast and / or mold cells (or any surviving non-heat resistant organism therein) may again be concentrated, for example, by centrifugation. The concentrated heat resistant yeast and / or mold cells may then be resuspended in a solution suitable for subsequent steps in accordance with this invention. For example, in one embodiment, the heat resistant yeast and / or mold cells are resuspended in a solution or buffer suitable for an optional cell lysis step for lysis of remaining heat resistant yeast and / or mold cells. In another embodiment, the heat resistant yeast and / or mold cells can be resuspended in a buffer comprising a chelating agent to absorb cations that may inhibit subsequent DNA amplification (for example by PCR). In still another embodiment, the concentrated cells may be suspended in sterile water. Optionally, after the photo-activation step using the viability PCR reagent and resuspending the concentrated cells, the resuspended cells may be lysed using a digest step. In general, any known method in the art can be used to lyse heat resistant yeast and / or mold cells in the resuspended sample. In accordance with one embodiment, to digest the cells, the resuspended cells (or a sample thereof) may be incubated with an enzyme that is able to lyse / break down cell walls. Example, enzymes for use in this lysis step include lyticase and zymolase. Zymolase is a mixture of several different carbohydrases (i.e., β-1,3 glucan laminaripentaohydrolase, β-1,3 glucanase, protease, and mannase. In some embodiments, the resuspended cells are incubated with the enzyme at about 25°C to about 30°C. In some embodiments, the resuspended cells are incubated with the enzyme at about 30°C. In some embodiments, the resuspended cells are incubated with the enzyme at about room temperature. In some embodiments, the resuspended cells are incubated with the enzyme for about 20 minutes, for about 30 minutes, for about 60 minutes or more. In some embodiments, the resuspended cells and enzyme are incubated at a temperature of about 95°C. In some embodiments, the resuspended cells and enzyme are incubated at about 95°C for about 10 minutes. Finally, as shown at step 114 in FIG.1A, DNA molecules from the heat resistant yeast and / or mold cells are detected, and thereby detecting the presence of the heat resistant yeast and / or mold cells in the original liquid sample. In some embodiments, DNA molecules from non-heat resistant organisms that survived the heat shock step and / or a pasteurization step may also be detected. In general, any known means in the art for detecting DNA can be used, including PCR detection method, sequencing based detection methods, or any other known means. Non-limiting examples of PCR detection methods include emulsion PCR, multiplex PCR, long-range PCR, single-cell PCR, fast-cycling PCR, methylation-specific PCR (MSP), hot start PCR, high-fidelity PCR, rapid amplified polymorphic DNA analysis (RAPD), rapid amplification of cDNA ends (RACE), in situ PCR, differential display PCR, and bridge PCR (bPCR) amplification. Non-limiting examples of isothermal reactions include isothermal amplification is LOOP-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), thermophilic helicase-dependent amplification (tHDA), rolling-circle amplification (RCA),multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), nucleic acid sequence-based amplification (NASBA), self-sustained sequence reaction (3SR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), and bridge amplification. As one of skill in the art would readily understand, the PCR reaction includes at least one pair of primers that specifically hybridize to a target nucleic acid molecule derived from a heat resistant yeast or mold under amplification conditions conducive to producing an amplicon. In one embodiment, DNA molecules released from lysed heat resistant yeast and / or mold cells (or optionally DNA molecules released from lysed non-heat resistant organisms) are detected using a real time PCR reaction. For example, in one embodiment, the GENE-UP®system, a real-time PCR solution from bioMérieux can be used. In some embodiments, the real time PCR reaction may utilize detection probes to detect amplicons derived from heat resistant yeast and / or mold DNA molecules. In some embodiments, the PCR reaction can use FRET hybridization probes for detection of heat resistant yeast and / or mold DNA molecules. These FRET hybridization probes may consist of two different, short oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of the reaction cycle. In a non-limiting example, the first probe for the sample assay may be labeled at the 3' end with fluorescein; the second probe may be labeled at the 5' end with LC Red 640. FRET detection occurs only after the two probes come in close proximity from hybridizing to the template DNA. With reference to FIG. 1B, another example workflow for use with the GENE- UP®system (bioMérieux) is illustrated and described. As previously described, a liquid sample known to be, or suspected of being, contaminated with a heat resistant yeast or mold cells and / or spores is obtained. The liquid sample can be from a juice or other liquid beverage. As shown in FIG.1B at step 202, 100 mL of the liquid sample is transferred into a container (e.g., into filter bag) and heat shock in a hot water bath for 30 minutes at 75°C to kill heat sensitive organisms therein. Optionally, as previously described, the liquid sample can filtered before or after this heat shock step. Next, as shown at step 204 in FIG. 1B, 400 mL of a malt extract broth (MEB) growth medium is added to the container with the heat shocked sample, creating a culture mixture, that is mixed for 10 seconds and incubate for 72±2 hours at 30±2°C allowing heat resistant yeast and mold cells contained therein to divide and grow. After this incubation step, as shown at step 206 in FIG.1B, 500 µL of the post- growth culture mixture is transferred to a microcentrifuge tube, the mixture for 10 min at 3,000x g, the supernatant decanted and the cell pellet resuspended in 500 µL of a resuspension buffer. Next, as shown in FIG. 1B at step 208, the 500 µL resuspended cell sample is transferred to tube containing 100 µL of a viability PCR reagent (i.e., a photo-reactant DNA- binding dye), mixed and incubate at room temperature for 15 minutes. As shown at step 210 in FIG. 1B, the viability PCR reagent treated sample is photo-activated using a light source (e.g., a LED Photolysis device) for 15 minutes causing the causing the viability PCR reagent to irreversibly bind to DNA molecules therein. Next, as shown at step 212 in FIG.1B, the photo-activated sample is centrifuge sample for 10 min at 3,000g, the supernatant decanted and the cell pellet resuspend with 500 µL of Sample Diluent, thereby separating the heat resistant yeast and mold cells from the viability PCR reagent. At step 214 in FIG.1B, 50 µL of the resuspended cell sample is transferred to a digestion tube containing reagent for digesting the heat resistant yeast and / or mold cells. As shown in FIG.1B at step 216, the digestion tube is placed into the GENE-UP®system, and the digestion program is selected to digest (or lyse) the heat resistant yeast and / or mold cells. As shown in FIG.1B at step 218, the digestion tube is removed from the GENE- UP®system, 20 µL of digested sample transferred to a separate tube containing 80 µL of a sample diluent, and then 10 µL from the top of digestion tube is removed and transferred to a PCR tube. Finally, as shown in FIG.1B at step 220, the PCR tube is placed into the GENE- UP®System, the real-time PCR protocol for detection of heat resistant yeast and / or mold (the HRM protocol) selected, and after the protocol cycle completed, at step 222, results are analyzed for detection of heat resistant yeast and / or mold DNA molecules, and thereby, detection of heat resistant yeast and / or mold in the liquid sample. In some embodiments, as described for FIG.1A, non-heat resistant organisms that survived the heat shock step and / or a pasteurization step can also be detected using the real-time PCR protocol and the GENE-UP®System. KITS In some embodiments, kits are provided for usage with the methods described herein. The kit can include a detection device as described herein, a sample collector, sample tubes, reagent tubes, one or more buffers, enrichment medium (e.g., MEB), an instruction manual, a positive control, a negative control, PCR primers, PCR probes, a DNA polymerase, dNTPs, or any combination thereof. In some embodiments, the kit contains digest tubes which include an enzyme capable of lysing a cell wall. In some embodiments, the enzyme is lyticase. In another example, the enzyme is zymolase. In some embodiments, the kit may contain PCR tubes. In some embodiments, the PCR tubes contain one or more of a buffer, dNTPs, a DNA polymerase, and at least one pair of primers that specifically hybridize to a target nucleic acid molecule derived from a heat resistant yeast or mold. In some embodiments, the PCR tubes further contain FRET hybridization probes. These probes may consist of two different, short oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of the reaction cycle. In one embodiment, the first probe for the sample assay is labeled at the 3' end with fluorescein; the second probe is labeled at the 5' end with LC Red 640. Non-limiting examples of detectable labels include: 1,5 IAEDANS; 1,8-ANS; 2′-chloro-7′phenyl-1,4- dichloro-6-carboxyfluorescein (VIC); 3(4)-carboxyfluorescein dipivalate NHS ester; 3(4)- carboxyfluorescein dipivalate hydroxyhexyl carboxamide; 3(4)-carboxyfluorescein dipivalate hydroxyhexyl carboxamide phosphoramidite; 3(4)-carboxyfluorescein dipivaloyl-N- succinimidyl ester; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5- Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5-carboxyfluorescein dipivalate; 5- Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA (5- Carboxytetramethyirhodamine); 6-Carboxyfluorescein (6-FAM); 6-carboxyfluorescein dipivalate; 6-carboxynapthofluorescein; 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino- 4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9-Amino- 6-chloro-2-methoxyacridine; ABQ; ABY; Acid Fuchsin; ACMA (9-Amino-6-chloro-2- methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Alexa Fluor 350; Alexa Fluor 405; Alexa Fluor 430; Alexa Fluor 488; Alexa Fluor 500; Alexa Fluor 514; Alexa Fluor 532; Alexa Fluor 546; Alexa Fluor 555; Alexa Fluor 568; Alexa Fluor 594; Alexa Fluor 610; Alexa Fluor 633; Alexa Fluor 635; Alizarin Complexon; Alizarin Red; AMC; AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO 390; ATTO 425; ATTO 465; ATTO 488; ATTO 495; ATTO 520; ATTO 532; ATTO 550; ATTO 565; ATTO 590; ATM 594; ATTO 610; ATTO 611X; ATTO 620; ATTO 633; ATTO 635; ATTO 647; ATTO 647N; ATTO 655; ATTO 680; ATTO 700; ATTO 725; ATTO 740; ATTO-TAG CBQCA; ATTO-TAG FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; Blancophor SV; BOBO-1; BOBO-3; Bodipy 492 / 515; Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X; SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO-1; BO-PRO-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson; CAL Fluor Gold 540; CAL Fluor Orange 560; CAL Fluor Red590; CAL Fluor Red 610; CAL Fluor 635; Calcium Green; Calcium Green-1 Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; carboxyfluorescein diacetate; carboxyfluorescein diacetate succinimidyl ester; carboxyfluorescein dipivalate succinimide ester; carboxyfluorescein succinimidyl ester (CFSE); Carboxy-X-rhodamine (5-ROX); Cascade Blue; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coumarin Phalloidin; CPM Methylcoumarin; CTC; CTC Formazan; Cy2; Cy3.18; Cy3.5; Cy3; Cy5.1 8; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3′ DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD—Lipophilic Tracer; DiD (DiIC18(5)); DIDS; Dihydorhodamine 123 (DHR); DiI (DiIC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); dipivaloyl-3(4)-(N-(6’-hydroxyhexul))-carboxamide; DM-NERF (high pH); DNP; Dopamine; DTAF; DY-630-NHS; DY-635-NHS; DyLight 405; DyLight 488; DyLight 549; DyLight 633; DyLight 649; DyLight 680; DyLight 800; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (III) chloride; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein amidite (FAM); Fluorescein Diacetate; fluorescein diacetate 6-isothiocyanate; fluorescein dipivaloyl amidite; fluorescein phosphoramidite; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43; FM 4-46; Fura Red (high pH); Fura Red / Fluo-3; Fura-2; Fura- 2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow SGF; GeneBlazer (CCF2); Gloxalic Acid; Granular blue; Haematoporphyrin; HEX; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1; high calcium; Indo-1; low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JOE; JO- JO-1; JO-PRO-1; JUN; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein / Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow / Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin EBG; Oregon Green; Oregon Green 488-X; Oregon Green; Oregon Green 488; Oregon Green 500; Oregon Green 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; S65A; S65C; S65L; S65T; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6- methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine Extra; SUN; SYBR Green; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; TAMARA; Tetracycline; tetrachlorofluorescein (TET); Tetramethylrhodamine (TAMRA); Texas Red; Texas Red-X conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TRITC (tetramethylrodamine isothiocyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yamika Yellow; YO-PRO-1; YO-PRO-3; YOYO-1; or YOYO-3, or any combination thereof. In some embodiments, the probe additionally comprises a quencher of a detectable label, wherein the quencher of a detectable label is covalently bound to the probe. In some embodiments, the quencher is selected from the group consisting of: BBQ-650, BHQ-1, BHQ-2, BHQ-3, Black Hole Quencher (BHQ), Dabcyl, Eclipse, IBFQ, Iowa Black, TAMARA, TAMRA, ZEN, or ZEN Iowa Black, or any combination thereof. However, it is understood that these are non-limiting examples of quenchers. With respect to the kit, a positive control is a sample that is known to contain the analyte(s) that may be detected with the device present in the kit. In contrast the negative control, would not contain an analyte that can be detected by the kit. SYSTEMS In some embodiments, systems are provided for usage with the methods described herein. The system may include a kit, as described above, along with an instrument to run the assay. In at least one embodiment, the system as shown in Fig.2 may include at least one PCR reaction mixture housed in PCR tube 714. In certain embodiments, the PCR tube 714 may include a PCR reaction mixture configured to permit and / or effect amplification of a template nucleic acid. Certain illustrative embodiments may also include at least one sample block or chamber 716 configured to receive the at least one PCR tube 714. The PCR tube 714 may include any plurality of PCR tubes in individual, strip, plate, or other format, and, illustratively, may be provided as or received by a sample block or chamber 716. One or more embodiments may also include at least one sample temperature controlling device 718 and / or 720 configured to manipulate and / or regulate the temperature of the sample(s). Such a sample temperature controlling device may be configured to raise, lower, and / or maintain the temperature of the sample(s). In one example, sample controlling device 718 is a heating system and sample controlling device 720 is a cooling system. Illustrative sample temperature controlling devices include (but are not limited to) heating and / or cooling blocks, elements, exchangers, coils, radiators, refrigerators, filaments, Peltier devices, forced air blowers, handlers, vents, distributors, compressors, condensers, water baths, ice baths, flames and / or other combustion or combustible forms of heat, hot packs, cold packs, dry ice, dry ice baths, liquid nitrogen, microwave- and / or other wave-emitting devices, means for cooling, means for heating, means for otherwise manipulating the temperature of a sample, and / or any other suitable device configured to raise, lower, and / or maintain the temperature of the sample(s). The illustrative PCR system 700 also includes an optical system 710 configured to detect an amount of fluorescence emitted by the sample in PCR tube 714 (or a portion or reagent thereof). Such an optical system 710 may include one or more fluorescent channels, as are known in the art, and may simultaneously or individually detect fluorescence from a plurality of samples. At least one embodiment of the PCR system may further include a CPU 706 programmed or configured to operate, control, execute, or otherwise advance the heating system 718 and cooling system 720 to thermal cycle the PCR reaction mixture, illustratively while optical system 710 collects fluorescent signal. CPU 706 may then generate an amplification curve, a melting curve, or any combination, which may or may not be printed, displayed on a screen of the user terminal 704, or otherwise outputted. Optionally, a positive, negative, or other call may be outputted based on the amplification and / or melting curve for example on the screen of the user terminal 704. Optionally, only the calls are outputted, illustratively, one call for each target tested. The CPU 706 may include a program memory, a microcontroller or a microprocessor (MP), a random-access memory (RAM), and an input / output (I / O) circuit, all of which are interconnected via an address / data bus. The program memory may include an operating system such as Microsoft Windows®, OS X®, Linux®, Unix®, etc. In some embodiments, the CPU 706 may also include, or otherwise be communicatively connected to, a database or other data storage mechanism (e.g., one or more hard disk drives, optical storage drives, solid state storage devices, etc.). The database may include data such as melting curves, annealing temperatures, denaturation temperatures, and other data necessary to generate and analyze melting curves. The CPU 706 may include multiple microprocessors, multiple RAMS, and multiple program memories as well as a number of different types of I / O circuits. The CPU 706 may implement the RAM(s) and the program memories as semiconductor memories, magnetically readable memories, and / or optically readable memories, for example. The microprocessors may be adapted and configured to execute any one or more of a plurality of software applications and / or any one or more of a plurality of software routines residing in the program memory, in addition to other software applications. One of the plurality of routines may include a thermocycling routine which may include providing control signals to the heating system 718 and the cooling system 720 to heat and cool the sample in PCR tube 714 respectively, in accordance with the two-step PCR protocol. Another of the plurality of routines may include a fluorescence routine which may include providing control signals to the optical system 710 to emit a fluorescence signal and detect the amount of fluorescence scattered by the sample in PCR tube 714. Yet another of the plurality of routines may include a sample calling routine which may include obtaining fluorescence data (temperature, fluorescence pairs) from the optical system 710 during the in-cycle temperature adjusting segment for each of N cycles, generating a composite melting curve by combining the fluorescent data from each of the N cycles during the respective in-cycle temperature adjusting segments, analyzing the composite melting curve to make a positive or negative call, and displaying the composite melting curve, individual melting curve, and / or an indication of the call on the user terminal 704. In some embodiments, the CPU 706 may communicate with the user terminal 704, the heating system 718, the cooling system 720, the optical system 710, and the sample block 716 over a communication network 722-732 via wired or wireless signals and, in some instances, may communicate over the communication network via an intervening wireless or wired device, which may be a wireless router, a wireless repeater, a base transceiver station of a mobile telephony provider, etc. The communication network may be a wireless communication network such as a fourth- or third-generation cellular network (4G or 3G, respectively), a Wi-Fi network (802.11 standards), a WiMAX network, a wide area network (WAN), a local area network (LAN), the Internet, etc. Furthermore, the communication network may be a proprietary network, a secure public Internet, a virtual private network and / or some other type of network, such as dedicated access lines, plain ordinary telephone lines, satellite links, combinations of these, etc. Where the communication network comprises the Internet, data communication may take place over the communication network via an Internet communication protocol. Still further, the communication network may be a wired network where data communication may take place via Ethernet or a Universal Serial Bus (USB) connection. In some embodiments, the CPU 706 may be included within the user terminal 704. In other embodiments, the CPU 706 may communicate with the user terminal 704 via a wired or wireless connection (e.g., as a remote server) to display individual melting curves, composite melting curves, calls, etc. on the user terminal 704. The user terminal 704 may include a user interface, a communication unit, and a user-input device such as a “soft” keyboard that is displayed on the user interface of the user terminal 704, an external hardware keyboard communicating via a wired or a wireless connection (e.g., a Bluetooth keyboard), an external mouse, or any other suitable user-input device in addition to the CPU 706 or another CPU similar to the CPU 706. In other embodiments, other systems for detecting amplicons using temperature- cycling amplification reactions may be used. Without wishing to be bound by theory, an amplicon can be produced by amplifying a nucleic acid molecule through many means, and the amplicon can be detected through many means. Non-limiting examples of DNA amplification reactions include polymerase chain reaction (PCR), isothermal amplification, ligase chain reaction (LCR), and rolling circle replication (RCR). Non-limiting examples of PCR reactions include emulsion PCR, real time PCR (RT-PCR), multiplex PCR, long-range PCR, single-cell PCR, fast-cycling PCR, methylation-specific PCR (MSP), hot start PCR, high-fidelity PCR, rapid amplified polymorphic DNA analysis (RAPD), rapid amplification of cDNA ends (RACE), in situ PCR, differential display PCR, and bridge PCR (bPCR) amplification. Non-limiting examples of isothermal reactions include isothermal amplification is LOOP-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), thermophilic helicase- dependent amplification (tHDA), rolling-circle amplification (RCA),multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), nucleic acid sequence- based amplification (NASBA), self-sustained sequence reaction (3SR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), and bridge amplification. EXAMPLES Various aspects of the present disclosure are illustrated with reference to the following non-limiting examples. Example 1- Assay for the Detection of Heat Resistant Molds in Select Matrices The objective of this study was to examine the ability of the method disclosed herein to detect a variety of the claimed target heat resistant mold strains and to distinguish those strains from relevant non-target strains (heat sensitive strains). Test Organisms The following challenge organisms were used in this study: Table 1: Exclusivity Cultures No. Strain ID

[0002] 21 ATCC 42693 Aspergillus caesellus 22 NRRL 238 versicolor No. Strain ID

[0003] 19 HRM 82 Aspergillus nidulans (Emericella nidulans) 20 M1-5 amstelodami (Eurotium amstelodami) Exclusivity cultures are those known to be non-heat resistant strains or sensitive to heat. Inclusivity cultures are those known to be heat resistant strains. Inocula Preparation For bacteria inocula: Cultures were grown on Standard Methods Agar (SMA) at 35°C for 48 hours. Acetic-acid bacteria and lactic-acid bacteria were enumerated by Acetobacter Agar and MRS Agar (microaerophilic) for 72 hours, respectively. Plates were swabbed by sterile cotton swabs wetted with sterile 0.1% Peptone Water and suspended into the same solution. Before inoculating, the inoculum was adjusted to the right concentration by enumeration using dilution plating. For yeast inocula: Cultures were grown on Malt Extract agar (MEA) at 25°C for 5 days. Plates were swabbed by sterile cotton swabs wetted with sterile 0.1% Peptone Water and suspended into the same solution. Before inoculating, the inoculum was adjusted to the right concentration by enumeration using dilution plating. For mold inocula: Cultures were grown on Malt Extract agar (MEA) at 25°C for 5 days. Plates were swabbed by sterile cotton swabs wetted with sterile 0.1% Peptone water supplemented with 0.5% tween 80 and suspended into the same solution. Before inoculating, the inoculum was adjusted to the right concentration by enumeration using dilution plating. For the heat resistant mold inocula: The heat resistant mold ascospore inocula were prepared as follows: 1. Heat resistant mold was inoculated on Malt Extract agar (MEA). Plates were incubated at 30°C for 45-60 days. 2. Ascospores were harvested from the MEA plates using a buffer containing 0.05% Tween 80 to ensure good emulsification of spores. 3. Inocula were filtered through sterile glass wool to eliminate any hyphae that was present. 4. The inoculum was examined under the microscope to confirm the presence of single ascospores. 5. The inoculum was diluted using the same dilution buffer until the appropriate cfu / ml was achieved. 6. Before plating the ascospore inoculum for enumeration, an aliquot was heat- shocked for 30 min at 75 C. The heat-shock conditions for A. pseudoglaucus were 15 min at 60°C. The inocula were diluted and plated on MEA. 7. After the inoculum had reached the target concentration, the inocula was stored at 4°C until needed. Exclusivity / Inclusivity Methodology Heat resistant mold inclusivity and exclusivity strains were tested as pure cultures, at a concentration of approximately 1 x 103cfu (approx.1000 x Limit of Detection). Each culture was processed according to the following sampling procedures. 1. Add 1 x 103cfu of inoculum to 10 mL of MEB in a 15 mL conical tube. 2. Heat shock for 30 min at 75°C. a. 15 min at 60°C was used for xerophilic Aspergillus species (former Eurotium species). b. All inclusivity and exclusivity strains were heat shocked, enriched, and processed through full heat shock process and viability treatment. 3. Cool for 10 min at room temperature. 4. Incubate heat shocked samples for 72 hours. at 30°C. 5. Homogenize the samples in the 15 mL conical tubes. 6. Add 500 µL of each sample to a 1.5 mL clear microcentrifuge tube. 7. Centrifuge the samples for 10 min. at 3,000 x g. 8. Decant the supernatants leaving any pellets. 9. Add 500 µL buffer and homogenize. 10. Transfer 500 µL of buffered samples to black opaque microcentrifuge tubes containing PMAxx™ viability reagent. 11. Incubate the buffered samples and viability reagent at room temp for 15 minutes while mixing on tube rotator. 12. Transfer 600 µL of each of the buffered samples to a 1.5 mL clear microcentrifuge. 13. Incubate samples in a light box for 15 min (LED output wavelength 465-475 nm). 14. Centrifuge samples at 3000 x g for 10 minutes, decant the supernatants, and resuspend the pellets in 500 uL PCR-grade water. 15. Add 50 µL of each of the resuspended pellets to a digest tube containing buffer and lyticase, incubate at about 37°C for about 30 minutes. An optional incubation at 95°C for 10 minutes can be included to inactivate the lyticase. 16. Add 10 µL of each of the digested samples to a PCR tube, each of which includes a DNA polymerase, a buffer, dNTPs, PCR primers, and probes for amplifying DNA from microbes. Run PCR program in the GENE-UP® thermocycler. Results and Discussion Exclusivity study testing for non-heat resistant microorganisms. The results of the exclusivity study are shown in Table 4. All of the non-HRM cultures tested by the GENE-UP PRO HRM assay were negative for the detection of HRM species and gave no false HRM positive results. Inclusivity study testing for heat resistant mold species. The results of the inclusivity study are shown in Table 5. Many of the heat resistant mold cultures (36 / 46) tested by the disclosed assay were positive for the detection of heat resistant species and gave no false negative results. However, there were some cultures (10 / 46) that were not detected using this assay (underlined on Table 3 and 5). Of these 10 strains, 6 failed to grow on reference plates, indicating that no viable spores were in the samples, matching PCR results. This may have been because these inocula did not have viable ascospores due to long term storage under refrigeration. Paecilomyces lagunculariae showed PCR inhibition in one run (Table 3). Two strains, A. fischeri and Monascus pilosus, were undetected due to inhibition of PCR. These two strains were successfully detected at the bioMerieux facility in Philadelphia using ascospore solutions supplied by BCN Labs. They also detected several Paecilomyces species at this facility. Table 6 has the list of all the strains that were tested by bioMerieux in their internal inclusivity test. Table 3. Negative results for the inclusivity study. Accession Organism Result No. M1-60 Aspergillus fischeri (N. fischeri) Inhibited 2X CB5-58 Aspergillus hiratsukae (N. hiratsukae) Negative*** CB3-10 Monascus ***Was not detected by PCR but was detected on the reference plates. Conclusions None of the 30 cultures of bacteria, yeasts and molds tested in this study using the disclosed method gave positive results for heat resistant organisms (false positive). Of the heat resistant mold culture tested 36 / 46 gave positive results. However, 10 cultures gave negative results. Of these 10 strains, 6 failed to grow on reference plates, indicating that no viable spores were in the samples, matching PCR results. These inocula may not have had viable ascospores due to long term storage under refrigeration. Two strains, A. fischeri and Monascus pilosus, were undetected due to inhibition of PCR. Aspergillus hiratsukae showed typical growth on the reference plates but was not detected even though the PCR internal control was successful (no inhibition). Table 4. Exclusivity study testing for non-heat resistant microorganisms. Accession Organism Result VTT E-65 Pediococcus damnosus VTT Pediococcus claussenii Negative 032355 M3-61 Penicillium griseofulvum Negative Accession Organism Result No. X * CB3-10 Monascus pilosus Negative, Inhibited** M1-27 Paecilomyces fulvus (B. fulva) Positive

[0004] *Not detected on the reference plates. ***Was not detected by PCR but was detected on the reference plates. Table 6: Biomerieux Internal Inclusivity Test Strain ID Gene UP HRM Result NA fulvus +

[0005] M2-33 Rasamsonia brevistipitata + M4-30 Talaromyces trachyspermus + were compared to the Compendium methodology (Rico-Munoz et al., Compendium of Methods for the Microbiological Examination of Foods (CMMEF), 2015, chapter 22, “Detection and Enumeration of Heat-Resistant Molds (referred to herein as “the Compendium” or “the Compendium method”)) in two studies: Study 1: Each matrix (juice concentrate) was tested individually with a specific heat resistant mold ascospore inoculation. Study 2: Each matrix inoculated was tested in the presence of a competitive microorganism. Matrices: The following juice concentrates were provided by Ocean Spray® in pails (cranberry and apple juice) or zip-tied bags in a cardboard box (grapefruit and grape juice): 1. Cranberry juice concentrate 2. Concord grape juice concentrate 3. Grapefruit juice concentrate 4. Apple juice concentrate Since there was a contaminated batch of grapefruit and grape juice concentrate, concentrates were heat-treated before testing (75°C, 15 min). Test organisms: The following challenge organisms were assessed: 1. Paecilomyces variotii (syn. Byssochlamys spectabilis) CB15-20 / 21 2. Paecilomyces fulvus (syn. Byssochlamys fulva) M1-27 3. Aspergillus pseudoglaucus (syn. Eurotium pseudoglaucum) CB3-1 4. Paecilomyces niveus (syn. Byssochlamys nivea) M2-61 Heat Resistant Mold Ascospore Inocula Preparation: The ascospore inocula were prepared as follows: Heat resistant molds were inoculated on Malt Extract agar (MEA). Plates were incubated at 30°C for 45-60 days. Ascospores were harvested from the MEA plates using a buffer containing 0.05% Tween 80 to ensure good emulsification of spores. The inoculum was examined under the microscope to confirm the presence of single ascospores. The inoculum was diluted using the same dilution buffer until the appropriate cfu / ml was achieved. Before plating the ascospore inoculum for enumeration, an aliquot was heat-shocked for 30 min at 75 C. The heat-shock conditions for A. pseudoglaucus were 15 min at 60°C. The inocula were diluted and plated on MEA. After the inoculum had reached the target concentration, the inocula was stored at 4°C until needed. Matrix Testing Methodology: The heat resistant mold ascospores recovery from each inoculated matrix was tested according to the disclosed method and according to the Compendium Methodology as follows: A. Disclosed method of enrichment and sampling procedures a. Heat-shock and enrichment: i. Each inoculum of the specific heat resistant mold ascospores was added to 100 mL of the matrix in a 36 oz filter bag to get final concentrations as specified in Tables 7 and 8. ii. The inoculated matrix was heat shocked for 30 min at 75°C (or for 15 min at 60°C for Aspergillus pseudoglaucus in grapefruit samples). iii. The inoculated matrix was cooled for 10 min at room temperature. iv. After cooling, 400mL of Malt Extract Broth (MEB) was added to each bag. 1. i. For cranberry juice concentrate: a. Filter the matrix through a 0.8 μm removable filter funnel. b. Add filter to a 24-36 oz filter bag. c. Add 200 mL of MEB to the bag. v. Incubate enrichments for 72 h at 30°C. b. Reaction with viability PCR reagent: i. Homogenize 400 mL MEB filter bag. ii. Add 500 µL sample to 1.5 mL clear microcentrifuge tube. iii. Centrifuge for 10 min. at 3,000 x g. iv. Decant supernatant leaving any pellet. v. Add 500 µL buffer and homogenize. vi. Transfer 500 µL of buffered sample to black microcentrifuge tube containing PMAxx™ viability reagent. vii. Incubate at room temperature for 15 minutes while mixing on tube rotator. viii. Transfer 600 µL to a 1.5 mL clear microcentrifuge. ix. Incubate in a light box for 15 minutes (LED output wavelength 465-475 nm). x. Centrifuge samples at 3000 x g for 10 min and resuspend in 500 uL PCR-grade water. xi. Dilute sample by adding 20 µL into 80 µL PCR-grade water. xii. Add 50 µL to digest tube containing buffer and lyticase, incubate at 37°C for about 30 minutes. An optional incubation at 95°C for 10 minutes can be included to inactivate the lyticase. xiii. Add 10 µL to PCR tube, run PCR program. xiv. Confirm results by spread plating 0.1 mL of the MEB broth onto MEA at 30°C for up to 7 days. B. Compendium methodology For all the juice concentrates, the centrifugation method was followed (see below). a. The sample was vigorously mixed before analyzing. b. 25 to 50 g of sample were weighed into sterile centrifuge bottles for a total of 100 g of sample being tested. c. 0.1% peptone buffer was added up to the 200 mL mark. d. The above steps were repeated for all sample bottles. The contents were shaken vigorously to mix. e. The bottles were centrifuged at least 3,000 x g for 15 min. f. The supernatants were poured off and the pellets were resuspended by adding sterile 0.1% peptone buffer up to the 200-mL mark. If there was no pellet, all but 20-30 ml of supernatant was poured off and then, buffer was added up to the 200 ml mark. g. The bottles were centrifuged for a second time and the supernatant was discarded as above. The pellet was resuspended in a 10-30 mL sterile peptone buffer and the aliquots were poured into a sterile sample bag (4-5 oz). The bottles were rinsed with another 10-30 mL sterile buffer and added to the sample bag. The bags were closed to ensure that no air was present. h. The bags were placed in a large metal mesh basket laid horizontally with a weight on top of them. They were submerged in the basket in an 80°C water bath for 35 min. After this time, they were cooled down quickly to 45-55°C (using an ice bath). i. The content in the bag was mixed well and then, the entire volume was distributed into petri dishes (no more than 5 mL per plate). At least 20 ml of potato dextrose agar (PDA) or MEA with antibiotics was poured in the plates, mixed well. j. After solidifying, the plates were inserted upright inside a loosely closed bag to prevent dehydration and then, incubated at 30°C + 1.0°C for up to 21 days. Plates were examined weekly for the presence of heat resistant mold colonies. Most heat resistant molds colonies are visible within 7-10 days; however, heat- injured, or debilitated ascospores may require additional time to form visible colonies. k. Total colony counts were reported as presumptive heat resistant mold ascospore count per weight of the matrix used. l. All presumptive heat resistant mold colonies were confirmed using PCR / DNA sequencing as well as macro- and micro-morphological characteristics. Results and Discussion of Study 1 Experimental results of Study 1 are presented in Table 8 of this application. Differences among matrices: Since each of the four juices inoculated by different heat resistant mold inocula, conclusions cannot be made for the differences among matrices. Differences among heat resistant mold cultures: All the heat resistant mold species used in this experiment were detected by both methods except for A. pseudoglaucus that was not detected by any of the methods. At the current heating conditions used in the Compendium methodology (75-80°C and 30-35 min), A. pseudoglaucus would be difficult to be recovered since this method is not designed to detect less heat resistant species such as these xerophilic Aspergillus species (former Eurotium species). The Compendium method did not give any false positive samples for the uninoculated samples. The method disclosed herein used a lower heat shock temperature and time (65°C and 15 min). This disclosed method was not able to recover A. pseudoglaucus. The method gave two false positive results of the uninoculated samples and five false positive results of the inoculated samples since none of those samples were confirmed as having an A. pseudoglaucus contamination. Pink yeast was isolated from these samples which may have interfered with the PCR results. Differences between the disclosed method and the Compendium methods Cranberry juice concentrate In the case of cranberry juice concentrate inoculated with P. variotii, both the disclosed method and the Compendium methods could detect the absence (negative result) or presence (positive result) of this heat resistant mold ascospores for the uninoculated and inoculated samples. The Compendium method was able to give counts of 1-51 ascospores per 100 g of concentrate. Concord grape juice concentrate For the concord grape juice concentrate inoculated with P. fulvus, both methods could detect each of the three negative samples of the uninoculated controls. The disclosed method, including the real-time PCR reaction, could detect 5 / 5 of the inoculated samples. The Compendium method detected 4 / 5 of the positive samples. This method gave counts between 2 and 5 ascospores per 100 g which indicates that the inoculation level was very low, possibly <1 ascospore in one of the samples. Grapefruit juice concentrate When the grapefruit juice concentrate was inoculated with A. pseudoglaucus, the Compendium method did not detect the presence of this heat resistant mold in any of the inoculated samples. This may have been due to the lower heat resistance of this mold ascospores that could have not survived the heat-shock of 80°C for 35 min given to the concentrate. This method did not give any false positive samples in the case of the uninoculated samples. When the disclosed method was used, the method gave two false positive samples for the uninoculated samples. In the case of the inoculated samples, the method gave positive results for the five samples; however, these positive results were not confirmed to be A. pseudoglaucus but, rather, pink yeast. The inoculum of this heat resistant mold was contaminated with pink yeast that may have interfered with the PCR results. Apple juice concentrate. In the case of the apple juice concentrate inoculated with P. niveus, both methods detected the uninoculated samples as negative. The Compendium method detected all the inoculated samples as positive. The disclosed method detected four of the five inoculated samples as positive. The MEA confirmation of the samples detected as negative by the disclosed method gave a positive result for P. niveus. The Compendium method gave counts in the inoculated samples from 19-25 ascospores per 100 g. Results and Discussion of Study 2: Experimental results of Study 2 are presented in Table 9 of this application. Detection of the competitive microorganisms: None of the competitive microorganisms used in this study were detected by any of the two methods used. Differences among heat resistant mold cultures: As was the case in Study 1, none of the methods could detect A. pseudoglaucus. Differences between methods: Cranberry juice concentrate In cranberry juice concentrate inoculated with P. variotii, both the disclosed method and the Compendium method detected the absence (negative result) and presence (positive result) of this heat resistant mold ascospores for the uninoculated and inoculated samples respectively. The Compendium method gave counts of 2-15 ascospores per 100 g of concentrate. Concord grape juice concentrate In concord grape juice concentrate inoculated with P. fulvus, both the disclosed method and the Compendium methods detected the absence (negative result) or presence (positive result) of this heat resistant mold ascospores for the uninoculated and inoculated samples respectively. The Compendium method gave counts of 4 ascospores per 100 g of concentrate in the three inoculated samples. Grapefruit juice concentrate When the grapefruit juice concentrate was inoculated with A. pseudoglaucus, both the disclosed method and the Compendium methods detected the absence (negative result) of this heat resistant mold in the samples. Neither method detected the presence (positive result) of this heat resistant mold ascospores for the inoculated samples. A pink yeast was isolated from the confirmation plates from the disclosed method and by the Compendium method. Apple juice concentrate. In apple juice concentrate inoculated with P. niveus, both the disclosed method and the Compendium methods detected the absence (negative result) and presence (positive result) of this heat resistant mold ascospores for the uninoculated and inoculated samples respectively. The Compendium method gave counts of 49-57 ascospores per 100 g of concentrate. Conclusions: Both methods can detect the presence of the high heat resistance Paecilomyces species which are commonly found in spoiled fruit juices, juice containing beverages, and other beverages such teas, energy drinks, and sport drinks. None of the methods were able to detect the less heat resistant mold A. pseudoglaucus. The disclosed method gave positive results for the inoculated samples but upon confirmation, the culture isolated was not A. pseudogalucus but a pink yeast that could have caused an interference in the assay. In the case of the three Paecilomyces species, neither method gave any false positive results of the uninoculated or inoculated samples. The Compendium method did not detect P. fulvus in one of the inoculated samples of the concord grape juice concentrate which may have been due to the low level of inoculation of these samples. The disclosed method did not detect P. niveus in one of the inoculated samples of apple juice concentrate giving a false negative result since the confirmation plates had P. niveus growing on them. None of the competitive microorganisms were detected by any of the methods. Results in the four matrices could not be compared since they were inoculated with different heat resistant mold species. The Compendium method gave counts of ascospores per 100 g. Even though many specifications for fruit juice concentrates are set at < 1 ascospore per 100 g, the risk to the final product may be different if the numbers found in the ingredients are less than five (<5) ascospores per 100 g compared to more than five (>5). The disclosed method was much faster than the Compendium method. Example 3 The following example is a method of testing juice samples for heat resistant mold using in the GENE-UP® Thermocycler to perform real-time PCR. The method is for the enrichment and presumptive detection of viable heat resistant mold species from juice concentrate and raw material samples. The method couples the advantages of the real-time format with a streamlined sampling protocol that requires no DNA purification, complicated end user manipulations, or data analysis. The GENE-UP® Thermocycler detects fluorescence at several wavelengths (channels) to allow multitarget detection in the same reaction vessel. The fluorescent signal of the target is recorded in channel 640, while the fluorescent signal for an internal amplification control is recorded in channel 705. The software automatically interprets the results for both fluorescence channels and determines the sample result based on the outcome of the control. Both the method for detecting the target and the internal amplification control utilize dual Fluorescence Resonance Energy Transfer (FRET) hybridization probes. These probes consist of two different, short oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of the reaction cycle. The first probe for the sample assay is labeled at the 3' end with fluorescein; the second probe is labeled at the 5' end with LC Red 640. FRET occurs only after the two probes come in close proximity from hybridizing to the template DNA. The resulting fluorescent signal from the FRET interaction forms a real-time amplification curve thereby enabling the GENE-UP® Thermocycler to detect the amplified target. After the PCR cycling program finishes, the PCR product(s) are melted to determine the presence of the target DNA. The software interprets data for each sample and gives a positive, negative, or inhibited result. Sample Preparation Juice Concentrate Sampling and Enrichment 1. Transfer 100 mL of Juice Concentrate to two 36 oz filter bags each. 2. Heat shock the samples: a. Sample A: 30 min at 75°C, cool for 10 min at 20-25°C. b. Sample B: 15 min at 60°C, cool for 10 min at 20-25°C. 3. Transfer 400 mL of MEB to the sample bags. 4. Seal bag, and briefly (5-10 seconds) massage bag. 5. Place bag into 30°C ± 2°C incubator and incubate samples for 72 ± 2 hours. 6. Proceed to Viability Sample Prep. Cranberry Juice Concentrate Sampling and Enrichment 1. Transfer 100 mL of Juice Concentrate to two 36 oz filter bags each. 2. Heat shock – a. Sample A: 30 min at 75°C, cool for 10 min at 20-25°C. b. Sample B: 15 min at 60°C, cool for 10 min at 20-25°C. 3. Filter the sample through a removable 0.8 µM filter funnel and discard the filtrate. 4. Transfer the filter to 200 mL of MEB in a 36 oz filter bag. 5. Seal bag, and briefly (5-10 seconds) massage bag. 6. Place bag into 30°C ± 2°C incubator and incubate samples for 72 ± 2 hours. 7. Proceed to Viability Sample Prep. Viability Sample Preparation 1. Dilute 20mM PMAxx™ stock reagent 400X in dH2O (to a concentration of 50 uM) and dispense 100 uL of solution to black opaque microcentrifuge tubes (one for each sample to be tested). . Transfer 500 μL of enriched sample to a clear microcentrifuge tube. . Centrifuge microcentrifuge tube with sample for 10 minutes at 3000 x g. . Decant supernatant and resuspend the pellet in 500 μL of Buffer Q provided with the PMAxx™ product. . Transfer 500 μL from each resuspended pellet to one of the black opaque microcentrifuge tubes containing 100 μL of 50 uM viability solution. . Incubate the black opaque microfuge tubes for 15 minutes at room temperature using an end-over-end mixer or manually invert tube periodically (3-5x) within the 15-minute incubation time. . Transfer entire sample volume from each of the black opaque tube microcentrifuge tubes to a clear microcentrifuge tube and incubate the sample for 15 minutes in an LED photolysis device (LED output wavelength 465-475 nm). . Centrifuge clear microcentrifuge tubes for 10 minutes at 3000 x g. . Decant supernatant and resuspend pellet in 500 μL of buffer conducive to lyticase activity 0. Proceed to Sample Digest. Sample Digest . Transfer 50 μL from each of the 1.5 mL clear microcentrifuge tube to thawed Digest tube containing lyticase. . Place Digest Tube into GENE-UP®Thermocycler using the PCR Tube Holder plate. . Incubate the samples with the lyticase enzyme in the GENE-UP®Thermocycler for about 30 minutes at 37°C and 10 minutes at 95°C. . Upon completion of digest reaction, remove Digest tube(s) from thermocycler. . Proceed to Final Setup for PCR section. Results and Interpretation Interpret results according to the following table: Target Channel IC Final Result Interpretation + + +Presumptive Positive for HRM-+ -Presumptive Negative for HRMetail with reference to certain preferred embodiments thereof, other versions are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description and the preferred versions contained within this specification. The embodiments are now described with reference to the following examples. These examples are provided for the purpose of illustration only and the embodiments should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results. Example 4: Real-Time PCR Assay for the Detection of Heat Resistant Mold in Juice Concentrate Matrices This study is an example method of testing juice samples for heat resistant mold using the GENE-UP® Thermocycler (bioMérieux, Inc., Salt Lake City, Utah) to perform real- time PCR. The method is for the enrichment and detection of viable heat resistant mold species from juice concentrate and raw material samples. The method couples the advantages of the real-time format with a streamlined sampling protocol that requires no DNA purification, complicated end user manipulations, or data analysis. If a heat resistant mold (HRM) ascospore gets into a final product after it has undergone pasteurization or similar hot fill processing, it can become activated and slowly grow over time and spoil the product long after it has left the manufacturer. The GENE-UP® Thermocycler detects fluorescence at several wavelengths (channels) to allow multitarget detection in the same reaction vessel. The fluorescent signal of the target is recorded in channel 640, while the fluorescent signal for an internal amplification control is recorded in channel 705. The software automatically interprets the results for both fluorescence channels and determines the sample result based on the outcome of the control. The assay utilizes dual FRET hybridization probes. These probes consist of two different, short oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of the reaction cycle. The first probe for the sample assay is labeled at the 3' end with fluorescein; the second probe is labeled at the 5' end with LC Red 640 or Cy5. FRET occurs only after the two probes come in close proximity from hybridizing to the template DNA. The resulting fluorescent signal from the FRET interaction, which forms a real-time amplification curve, indicates whether target amplification has occurred. After the amplification finishes, a melt curve analysis is done, and positivity is determined if the obtained temperature of dissociation is within a defined positivity window. A. Materials Target strains tested were acquired from BCN Research Laboratories and Ocean Spray Inc. for use during HRM assay development. Non-target organisms were from the BioMérieux (Philadelphia) Strain library. B. PCR Formulation and Optimization PCR formulation optimization was centered around three key approaches, to choose the primer and probe concentration and base reagents that produced the most robust amplification of HRM species, to choose the reagents that would work best for lyophilization, and to limit and eliminate the amplification of off-target DNA. The PCR targets a wide range of yeast and mold species therefore it is especially prone to false positives caused by DNA contamination from individual PCR components and the environment. Early master mix formulations had very high false positive rates for this reason. To remove the endogenous DNA that could be present in the master mix, we decided to use a HL-DNAse treatment strategy. A HL-DNAse treatment was used on the base PCR reagent and inactivated prior to lyophilization. C. Thermal Cycling Program and Formulation Optimization Thermal cycling program and formulation assessment against A. lacinosis (representative of high sensitivity strains) and B. nivea (representative of reduced sensitivity strains) gDNA dilutions, deionized water and Verilflow sample diluent (VSD) negative controls. In this study, PCR reagents treated with HL-DNAse at 0.5 U / reaction and 1 U / reaction, and a modified thermal cycling program and a traditional thermal cycling program were compared to determine the final configuration for the assay. Puried A. lacinosis and B. nivea gDNA were diluted to approximately 250 fg / μL, which is equivalent to 8 copies / μL in 1mL of deionized water.1:2 dilutions were prepared at 4 copies / μL through 0.625 copies / μL by adding 500 μL of both gDNA dilutions separately to 500 μL of deionized water.10μL of each dilution point, 16 water no template controls (NTCs) and 16 Verilfow sample diluent (VSD) NTCs were run on two formulations of lyophilized HRM PCR reagent (Form.17.4 and Form.20.3) in duplicate and run on two thermal cycling programs (HRM3 and YnM3s). Both formulations and programs produced robust amplification of target gdna at the correct melt temperature. Increased sensitivity was observed using the traditional cycling program (YnM v0.1). Although more false positives, specifically on VSD NTC samples, were observed with the YnM v0.1 cycling program, there was a greater difference between melt height values on true positive samples and false positive samples. For this reason, we decided to move forward with the traditional PCR program with a reduction of cycle number, denaturation time and annealing time. Additionally, a melt height limit of 20 -dF / dt was added as a parameter to the knowledge base to remove false positive calls while maintaining sensitivity at 1-2 copies of gDNA per PCR reaction. We also found that the mastermix prepared with 2X HL-DNAse more consistently reduced the false positive rate in both water and VSD NTCs. However, we decided to use sterile water in the final resuspension step during sampling since running samples with VSD alone added to false positive rates. D. Sampling and Viability Protocol and Optimization Sampling protocols were vetted and decided by first assessing the best growth conditions for HRM species. The juice concentrate sample volume of 100g for testing was determined based on the Compendium method. We maintained the same heat shock protocol from the Compendium method to differentiate between HRM and non-HRM species, focusing on 75°C for 30 minutes to culture the most heat resistant strains. We tested various media formulations and ratios before landing on Blakeslee’s formula of malt extract broth (MEB) at 1:5 sample to media. Using these enrichment conditions, we were able to demonstrate robust growth of HRM species in 72 hours at 30°C. Post-enrichment sampling volumes were assessed from 50 mL to 500 μL. Due to the high background yeast and mold DNA present in some juice concentrate samples, the 500 μL sampling volume demonstrated the most robust recovery without introducing FPs. Viability protocol optimization began with testing protocols validated for other assays. We tested inoculated and uninoculated juice concentrate enrichment samples using the viability protocol developed for the beer and cannabis assays. We found that the concentration of PMAxx™ (Biotium, Fremont, California) solution and incubation times used for those assays were too low and were not effective in removing background DNA contamination in the enrichment samples. We determined that a final concentration of 50µM PMAxx™ in the sample with both incubation steps increased to 15 minutes each, was needed to remove non-viable organism DNA from the enrichment samples. Sampling and Viability Protocols Juice Concentrate Sampling and Viability Protocol: 1. Transfer 100mL of sample into filter bag, heat shock in hot water bath for 30 minutes at 75°C, remove and let cool to 20-25°C for 10 minutes. 2. Add 400mL MEB to sample bag, homogenize 15 seconds then enrich for 72 hours at 30±2°C. 3. Homogenize 15 seconds, transfer 500µL to 1.5mL microcentrifuge tube, centrifuge sample for 10 minutes at 3,000x g. 4. Decant supernatant, resuspend pellet in 500µL Buffer Q. 5. Transfer 500µL of buffered sample to black tube containing 100µL of thawed viability PCR solution, mix and incubate on tube rotator at room temperature for 15 minutes. 6. Transfer 600µL of sample to clear microcentrifuge tube and place on LED photolysis device for 15 minutes. 7. Centrifuge sample for 10 minutes at 3,000x g, decant supernatant, resuspend pellet with 500µL sterile water, and vortex to fully homogenize pellet. 8. Load thawed digest tubes on PCR plate with lid and spin in plate spinner for 30 seconds. 9. Pipette 50µL of buffered sample into thawed digest tube. 10. Spin PCR plate with lid on in plate spinner for 30 seconds. 11. Load the plate into the GENE-UP®, remove lid, select and run digest program. 12. Remove plate from GENE-UP®, transfer 10µL from top of digest tube with optical caps and spin plate for 30 seconds. 13. Load the plate into the GENE-UP®, remove lid, select “HRM” program and set up run. 14. Review and report results. Cranberry Juice Concentrate Sampling and Viability Protocol: 1. Transfer 100mL of sample into filter bag, heat shock in hot water bath for 30 minutes at 75°C, remove and let cool to 20-25°C for 10 minutes. 2. Filter 100mL heat shocked sample through 0.8µM membrane filter and discard filtrate. 3. Remove filter and add to enrichment bag containing 200mL MEB to sample bag, homogenize 15 seconds to mix and then enrich for 72 hours at 30±2°C. 4. Homogenize enrichment bag, transfer 500µL to 1.5mL microcentrifuge tube, centrifuge sample for 10 minutes at 3,000x g. 5. Decant supernatant, resuspend pellet in 500µL Buffer Q. 6. Transfer 500µL of buffered sample to black tube containing 100µL of thawed viability PCR solution, mix and incubate on tube rotator at room temperature for 15 minutes. 7. Transfer 600µL of sample to clear microcentrifuge tube and place on LED photolysis device for 15 minutes. 8. Centrifuge sample for 10 minutes at 3,000x g, decant supernatant, resuspend pellet with 500µL sterile water, and vortex to fully homogenize pellet. 9. Load thawed digest tubes on PCR plate with lid and spin in plate spinner for 30 seconds. 10. Pipette 50µL of buffered sample into thawed digest tube. 11. Spin PCR plate with lid on in plate spinner for 30 seconds. 12. Load the plate into the GENE-UP®, remove lid, select and run digest program. 13. Remove plate from GENE-UP®, transfer 10µL from top of digest tube with optical caps and spin plate for 30 seconds. 14. Load the plate into the GENE-UP®, remove lid, select “HRM” program and set up run. 15. Review and report results. Sampling and Viability Protocol for Samples Prone to PCR Inhibition: 1. Split 100mL of sample into two 50mL conical tubes, heat shock in hot water bath for 30 minutes at 75°C, remove and let cool to 20-25°C for 10 minutes. 2. Spin 50mL tubes at 1000x g for 10 minutes, decant supernatant, resuspend pellet with 10mL MEB broth and pool the resuspension. 3. Add 400mL MEB to sample bag and incubate for 72 hours at 30±2°C. 4. Transfer 500µL to 1.5mL microcentrifuge tube, centrifuge sample for 10 minutes at 3,000x g. 5. Decant supernatant, resuspend pellet in 500µL Buffer Q. 6. Transfer 500µL of buffered sample to black tube containing 100µL of thawed viability PCR solution, mix and incubate on tube rotator at room temperature for 15 minutes. 7. Transfer 600µL of sample to clear microcentrifuge tube and place on LED photolysis device for 15 minutes. 8. Centrifuge sample for 10 minutes at 3,000x g, decant supernatant, resuspend pellet with 500µL sample diluent. 9. Pipette 50µL of sample into thawed digest tube on PCR plate. 10. Spin PCR plate with lid on in plate spinner for 10 seconds. 11. Load the plate into the GENE-UP®, remove plate lid, select and run digest program. 12. Remove plate from GENE-UP®, transfer contents of digest tube to 5 µM filter column, spin tube for 5 minutes at 500x g. 13. Remove and discard filter, transfer 20µL of filtrate to a new tube containing 80µL GENE-UP®sample diluent. 14. Transfer 10µL of sample to thawed PCR tube by piercing through blue septum cap. 15. Remove blue septum caps with DECAP-UP tool and replace with clear optical caps using Capping Tool. 16. Spin PCR plate with lid on in plate spinner at full speed for 10 seconds. 17. Load the plate into the GENE-UP®, remove lid, select “HRM” program and set up run. 18. Review and report results. E. Performance in Matrix – Post-Enrichment Matrix enrichment performance analysis against B. fulva inoculations at 0 cfu, 1 cfu, and 10 cfu per 100 g of concord grape and cranberry juice concentrate. Deionized water was used as a negative control. In this study, inoculated and uninoculated samples were processed using the protocol detailed above to analyze the recovery and detection of low concentrations of B. fulva ascospores in a juice concentrate sample. Colony picks of B. fulva cultures on plates were serially diluted to approximately 10 cfu / mL (based on enumeration plate counts) in MEB media.100 μL of the 10 and 100 cfu / mL dilutions were added to 55 oz filtered enrichment bags containing 100g of concord grape juice and cranberry juice concentrate. 100 μL of MEB media was also added to the concord grape juice concentrate as an NTC. All bags were heat shocked and processed via the protocols described above. After a 1:5 dilution in water and 50 μL digest, 10 μL of each sample and 1 water NTC were run on the finalized HRM PCR reagent (Form. 20) and run on the finalized thermal cycle program (YnMT2 for R&D use). GENE-UP®HRM Concord Grape Juice Concentrate Enrichment results (640 and 710 Channel, melt curves on YnMT2 v0.1). FIG 3A: The melt curves shown at 302 and 304 show the positive results from inoculated concord grape juice samples (at 1 cfu (302) and 10 cfu (304)). The melt curves at 306 show the negative results of an uninoculated concord grape sample and a water only NTC. FIG.3B: The melt curves shown at 308 show a positive result on the IC for all samples, indicating that the PCR is functioning properly. Results indicate accurate and robust detection of viable B. fulva in matrix post-enrichment and sampling. GENE-UP®HRM Cranberry Juice Concentrate Enrichment results (640 and 710 Channel, melt curves on YnMT2 v0.1). FIG.4A: The melt curves shown at 402 and 404 show the positive results from inoculated cranberry juice concentrate samples (at 1 cfu (402) and 10 cfu (404)). The melt curves shown at 406 show the negative results of an uninoculated cranberry juice concentrate sample. FIG 4B: The melt curves shown at 408 show a positive result on the IC for all samples, indicating that the PCR is functioning properly. Results indicate accurate and robust detection of viable B. fulva in matrix post-enrichment and sampling. We observed robust detection of all inoculated juice concentrate samples that were tested within the appropriate TM range and melt height. The matrix and water NTC tested negative. We concluded from this study and other juice studies (data not shown) that were run internally that the assay can successfully detect 1 cfu of HRM species in juice concentrate matrices post-enrichment. F. Performance in Matrix – Post-Enrichment with Inhibition An enrichment protocol was implemented to combat growth and PCR inhibition caused by matrices such as red grapefruit juice concentrate. Matrix enrichment performance analysis against A. chevalieri inoculations at 0 cfu, 1 cfu, and 10 cfu per 100 g of grapefruit juice concentrate. Deionized water was used as a negative control. In this study, inoculated and uninoculated samples were processed using the protocol detailed in figure 11 in section 5.3 to analyse the recovery and detection of low concentrations of A. chevalieri ascospores in a red grapefruit juice concentrate sample. Colony picks of A. chevalieri cultures on plates were serially diluted to approximately 1-10 cfu / mL (counts not confirmed due to lack of growth on plates) in MEB media.50 μL of the 1-10 and 10-100 cfu / mL dilutions were added to 250 mL tubes containing 100 g of red grapefruit juice concentrate. 100 μL of MEB media was also added to the red grapefruit juice concentrate as an NTC. All tubes were heat shocked and processed via the protocol outlined above. After a 50 μL digest, the digest sample was filtered through a 5 µm centrifugal filter, and diluted 1:5 in water.10 μL of each diluted filtrate samples were run on the finalized HRM PCR reagent (Form.20) and run on the finalized thermal cycle program (YnMT2 for R&D use). GENE-UP®HRM Red Grapefruit Juice Concentrate Enrichment results (640 and 710 Channel, melt curves on YnMT2 v0.1). FIG. 5A: The melt curves shown at 502 and 504 show the positive results from inoculated red grapefruit juice concentrate samples (at 1-10 cfu (502) and 10-100 cfu (504)). The melt curves shown at 506 show the negative results of an uninoculated red grapefruit sample. FIG. 5B: The melt curves shown at 508 show a positive result on the IC for all samples, indicating that the PCR is functioning properly. Results indicate accurate and robust detection of viable A. chevalieri in matrix post-enrichment and sampling. We observed robust detection of both inoculated juice concentrate samples that were tested within the appropriate TM range and melt height. The inhibition protocol resolved both growth and PCR failure. The matrix NTC tested negative. Inclusivity and Exclusivity Results An analysis of heat resistant mold strains acquired from USDA, BCN, ATCC, and Ocean Spray using the GENE-UP® Heat Resistant Mold sampling method and PCR assay. A colony pick of each strain was added to 10mL of MEB media in 2 sets of tubes. One set was heat treated at 75°C for 30 min and one set was heat treated at 65°C for 15 min. The cultures were incubated at 30°C for 72 hours.500 µL of each sample was viability treated and processed via the standard HRM workflow and digestion. Strains were tested by transferring 10 µL of digested samples to GENE-UP® Heat Resistant Mold PCR reagent tubes for amplification and qPCR analysis. All of the tested HRM strains, with the exception of Eurotium herbarium, were detected using the GENE-UP® Heat Resistant Mold sampling method and PCR assay. Each strain was plated on MEA to confirm viability post enrichment (data not shown). Recovery of the E. herbarium strain M14 could not be confirmed on PCR or plates (denoted in the table with **). To determine inclusivity for this strain, gDNA was isolated from a colony pick and tested via GENE-UP® Heat Resistant Mold PCR. Table 11: GENE-UP®Heat Resistant Mold compiled inclusivity panel analysis, against ascospores of heat resistant mold species.

[0006] Inclusivity Panel No. Strain ID Gene UP HRM Result An analysis of non-heat resistant yeast and mold strains using the GENE-UP® Heat Resistant Mold sampling method and PCR assay. A colony pick of each strain was added to 10mL of MEB media in 15 mL conical tubes. The tubes were heat treated at 75°C for 30 min. The cultures were incubated at 30°C for 72 hours.500 µL of each sample was viability treated and processed via the standard HRM workflow and digestion. Strains were tested by transferring 10 µL of digested samples to GENE-UP® Heat Resistant Mold PCR reagent tubes for amplification and qPCR analysis. All of the tested non-HRM strains were not detected using the GENE-UP® Heat Resistant Mold sampling method and PCR assay. Each strain was plated on MEA to confirm viability post enrichment (data not shown). Table 12: GENE-UP®Heat Resistant Mold compiled Exclusivity panel analysis, against non- heat resistant yeast and mold species. Exclusivity Panel No. Strain ID Gene UP HRM Result , we have provided sufficient evidence that the GENE-UP®HRM assay provides specific and robust detection of viable HRM species in under 96 hrs from juice concentrate matrices.

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Claims

1. CLAIMS We claim:

1. A method for detecting heat resistant yeast or mold cells in a liquid sample, the method comprising: a. obtaining a liquid sample suspected of being contaminated with a heat resistant yeast or mold cells and / or spores; b. heat shocking the liquid sample to kill heat sensitive organisms; c. adding a growth medium to the liquid sample to create a culture mixture; d. incubating the culture mixture at a temperature that allows the heat resistant yeast or mold cells to divide and / or grow; e. adding a viability PCR reagent to the culture mixture; f. separating the heat resistant yeast or mold cells from the viability PCR reagent and the culture mixture; and g. detecting DNA from the heat resistant yeast or mold cells, and thereby detecting the presence of heat resistant yeast or mold in the liquid sample.

2. The method of claim 1, wherein the step of adding a growth medium to the liquid sample to create a culture mixture is performed before the step of heat shocking the liquid sample.

3. The method of claim 1, wherein the step of adding a growth medium to the liquid sample to create a culture mixture is performed after the step of heat shocking the liquid sample.

4. The method of claim 1, wherein the step of heat shocking comprises placing the liquid sample in an environment comprising a temperature of at least about 60°C.

5. The method of claim 1, wherein the step of heat shocking comprises placing the liquid sample in an environment comprising a temperature of at least about 75°C.

6. The method of claims 4 or 5, wherein the liquid sample is maintained in the environment for at least about 15 minutes.

7. The method of claims 4 or 5, wherein the liquid sample is maintained in the environment for at least about 30 minutes.

8. The method of claim 1, further comprising the step of cooling the liquid sample after heat shocking the liquid sample and prior to incubating the culture mixture.

9. The method of claim 8, wherein the step of cooling comprises exposing the liquid sample to a temperature of between about 45°C and about 55°C.

10. The method of claim 8, wherein the step of cooling is performed for about 10 minutes.

11. The method of claim 1, wherein the culture mixture is incubated in step (d) at a temperature between about 10°C and about 35°C.

12. The method of claim 11, wherein the culture mixture is incubated in step (d) at a temperature between about 30°C to about 35°C.

13. The method of claim 11, wherein the culture mixture is incubated in step (d) at a temperature of about 30 ±2°C.

14. The method of claim 1, wherein incubating the culture mixture in step (d) is performed for at least 48 hours.

15. The method of claim 1, wherein incubating the culture mixture in step (d) is performed for at least 72 hours.

16. The method of claim 1, wherein incubating the culture mixture in step (d) is performed for about 72±2 hours.

17. The method of claim 1, wherein the viability PCR reagent added in step (e) comprises a fluorogenic nucleic acid stain.

18. The method of claim 17, wherein the fluorogenic nucleic acid stain comprises PMAxx™, propidium monoazide, or ethidium monoazide.

19. The method of any one of claims 17-18 further comprising the step of photoactivating the fluorogenic nucleic acid stain after mixing the heat resistant yeast or mold cells with the fluorogenic nucleic acid stain.

20. The method of claim 18, wherein the step of photoactivating the fluorogenic nucleic acid stain comprises exposing the viability PCR reagent to a light source.

21. The method of claim 1, wherein the step of separating the heat resistant yeast or mold cells from the viability PCR reagent and the culture mixture comprises centrifuging the viability PCR reagent and the culture mixture to pellet the heat resistant yeast or mold cells, removing a supernatant and optionally resuspending the pellet.

22. The method of claim 1, further comprising a step of digesting the heat resistant yeast or mold cells after the step of separating the contaminant cells from the viability PCR reagent and the culture mixture.

23. The method of claim 22, wherein the step of digesting comprises reacting the heat resistant yeast or mold cells with an enzyme capable of digesting cell walls.

24. The method of claim 22, wherein the step of digesting comprises reacting the heat resistant yeast or mold cells with lyticase or zymolase.

25. The method of claims 23 or 24, further comprising the step of incubating the heat resistant yeast or mold cells at about 95°C for about 10 minutes.

26. The method of claim 1, further comprising the step of detecting DNA in the heat resistant yeast or mold cells.

27. The method of claim 26, wherein the step of detecting DNA comprises performing a polymerase chain reaction (PCR), isothermal amplification, or ligase chain reaction (LCR) reaction.

28. The method of claim 27, wherein DNA is detected using fluorescent DNA probes.

29. The method of claim 28, wherein the DNA probes comprise FRET hybridization probes for detection of heat resistant yeast and / or mold DNA molecules.

30. The method of claim 1, wherein the liquid is a beverage.

31. The method of claim 1, wherein the liquid is a juice.

32. The method of claim 31, wherein the juice has a pH of below 7.0.

33. The method of and one of claims 1-32, wherein the method further comprises detecting non-heat resistant (or heat sensitive) organisms that survived the heat shock step and / or pasteurization 34. A kit, comprising: an enzyme capable of digesting cell walls; at least one pair of primers, each pair of primers comprising: a first primer that specifically hybridizes to a target nucleic acid molecule derived from a heat resistant yeast or mold and a second primer that specifically hybridizes to a complement of the target nucleic acid molecule under amplification conditions capable of producing an amplicon, and a viability PCR reagent.

35. The kit of claim 34, further comprising at least one of the following: a DNA polymerase, deoxynucleotide triphosphates, a buffer, and at least one probe.

36. The kit of claim 35, wherein the at least one probe comprises a sequence that is substantially homologous to or substantially complementary to a sequence of the target nucleic acid.

37. The kit of claim 34, wherein the at least one pair of primers and at least one of a DNA polymerase, deoxynucleotide triphosphates, a buffer, and at least one probe are provided in each of at least one PCR tube.

38. A system, comprising: a kit of any one of claims 34-37; and an instrument configured to perform a DNA amplification assay.

39. The system of claim 38, further comprising at least one PCR tube, wherein each of the at least one PCR tube comprises at least one of the following: a DNA polymerase, deoxynucleotide triphosphates, a buffer, and a probe.

40. The system of claim 38, wherein the kit comprises the kit of claim 39.

41. The system of claim 38, wherein the instrument comprises a sample temperature controlling device.

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