Composition and method for detecting heat-resistant and acid-resistant bacteria in food

Abstract

The present disclosure provides compositions, methods, and kits for detecting guaiacol-producing microorganisms, in particular heat resistant and acid resistant bacteria (TAB), in food. In one example, a method comprises: (a) contacting a set of oligonucleotides with a sample, where the set of oligonucleotides comprises: a first set of primers targeting a nucleic acid of a guaiacol-producing microorganism; and a second set of primers, the second set of primers being targeted to a nucleic acid of alicyclic bacillus acidoterrestris, the first set of primers being a nucleic acid of alicyclic bacillus acidoterrestris, and the second set of primers being a nucleic acid of alicyclic bacillus acidoterrestris. (b) amplifying the DNA in the sample with the set of oligonucleotides under a multiplex polymerase chain reaction (PCR); and (c) determining the presence of the PCR product of step (b), wherein the presence of the PCR product in the sample indicates that the sample is contaminated by both the guaiacol-producing microorganism and the Alicyclobacillus acidoterrestris.

Description

[0001] This application was filed as a PCT international patent application on July 7, 2023, and claims the benefit and priority of U.S. Provisional Patent Application Serial No. 63 / 358,966 filed on July 7, 2022, the entire disclosure of which is incorporated by reference in its entirety.

[0002] Submission of sequence listing

[0003] The sequence listing associated with this application is submitted in electronic format via EFS-Web and is hereby incorporated by reference into this specification in its entirety.

[0004] introduction

[0005] Microorganisms (e.g., bacteria) may cause food (e.g., beverages) to spoil during or after manufacturing. Some microorganisms may cause one or more of several undesirable effects (e.g., unpleasant odors and unpleasant tastes), rendering the food unsafe or inedible due to the presence of the off-flavors. The inability to accurately and quickly detect the presence of microorganisms that cause food spoilage may increase the risk of food spoilage. Challenges to the rapid and accurate detection of microorganisms that cause food spoilage may include, for example, the lengthy duration of traditional microbiological methods for detecting microorganisms. These traditional methods may take an average of at least five (5) days to complete.

[0006] Especially, the heat-resistant and acid-resistant bacteria (TAB) strain (member of the Alicyclobacillus) is heat-resistant, acid-resistant, and can survive pasteurization in the processing of beverages and the ingredients for preparing beverages, thereby causing corruption and producing guaiacol-a compound characterized by its medical, phenolic or preservative peculiar smell. Acid terrestrial Alicyclobacillus is the species most relevant to the generation of guaiacol, although other species (for example acidic Alicyclobacillus, grass Alicyclobacillus) can also produce this molecule. Therefore, the corruption caused by Alicyclobacillus is the major problem of the beverage industry. Although TAB is not pathogenic for people, the pollution during processing and production can cause the significant economic loss of the beverage industry.

[0007] Therefore, there is a need for improved compositions and methods for detecting food spoilage microorganisms, particularly TAB. Summary of the invention

[0008] Aspects of the present disclosure may be embodied in various exemplary and non-limiting forms. In particular, this summary is intended only to illustrate various embodiments of the present disclosure and is not intended to limit the scope of the disclosure.

[0009] The present disclosure provides a solution for effectively and accurately detecting TAB in food samples by using polymerase chain reaction (PCR) technology. The provided solution can be used to determine: whether the organism is an isolate of the genus Alicyclobacillus (genus level detection); whether the organism is Alicyclobacillus acidoterrestris (species level determination); and / or whether the organism has a gene that causes guaiacol production.

[0010] In some aspects, the present disclosure provides a method for detecting spoilage microorganisms in food samples. In one example, the method includes: (a) contacting a set of oligonucleotides with a sample, wherein the set of oligonucleotides comprises: a first primer set, the first primer set targeting a nucleic acid of a microorganism producing guaiacol; and a second primer set, the second primer set targeting a nucleic acid of Bacillus acidoterrestris; (b) amplifying DNA in the sample with the set of oligonucleotides under a multiplex polymerase chain reaction (PCR); and (c) determining the presence of the PCR product of step (b), wherein the presence of the PCR product in the sample indicates that the sample is contaminated by both the microorganism producing guaiacol and Bacillus acidoterrestris.

[0011] In another example, a method for detecting spoilage microorganisms in a food sample comprises: (a) contacting a set of oligonucleotides with the sample, wherein the set of oligonucleotides comprises: a first primer set that targets nucleic acid of a guaiacol-producing microorganism; a second primer set that targets nucleic acid of Alicyclobacillus acidoterrestris; and a third primer that targets nucleic acid of a 16S rRNA gene common to the genus Alicyclobacillus; (b) amplifying DNA in the sample using the set of oligonucleotides under a multiplex polymerase chain reaction (PCR); and (c) determining the presence of a PCR product of step (b), wherein the presence of the PCR product in the sample indicates that the sample is contaminated with a guaiacol-producing microorganism, a specific species of Alicyclobacillus acidoterrestris, and the genus Alicyclobacillus.

[0012] In some aspects, the present disclosure provides systems and assay kits for spoilage microorganisms in food samples. In one example, the system comprises a set of oligonucleotides for multiplex PCR, wherein the set of oligonucleotides comprises: a first primer set that targets nucleic acids of microorganisms that produce guaiacol; and a second primer set that targets nucleic acids of Alicyclobacillus acidoterrestris.

[0013] In another example, a system comprises a set of oligonucleotides for multiplex PCR, wherein the set of oligonucleotides comprises: a first primer set that targets nucleic acid of a guaiacol-producing microorganism; a second primer set that targets nucleic acid of Alicyclobacillus acidoterrestris, and a third primer set that targets nucleic acid of a 16S rRNA gene common to the genus Alicyclobacillus.

[0014] In yet another example, a kit for detecting spoilage microorganisms in a food sample comprises: a first primer set that targets nucleic acid of a microorganism that produces guaiacol; and a second primer set that targets nucleic acid of Bacillus acidoterrestris. In some embodiments, the kit further comprises: instructions for providing a method comprising: (a) contacting the set of oligonucleotides with a sample; (b) amplifying DNA in the sample with the set of oligonucleotides under a multiplex polymerase chain reaction (PCR); and (c) determining the presence of a PCR product of step (b), wherein the presence of a PCR product in the sample indicates that the sample is contaminated with a microorganism that produces guaiacol and a specific species of Bacillus acidoterrestris.

[0015] In another example, a kit for detecting spoilage microorganisms in a food sample comprises: a first primer set targeting nucleic acid of a guaiacol-producing microorganism; a second primer set targeting nucleic acid of acidoterrestris alicyclobacillus, and a third primer set targeting nucleic acid of a 16S rRNA gene common to the genus Alicyclobacillus. In some embodiments, the kit further comprises: instructions for providing a method comprising: (a) contacting the set of oligonucleotides with a sample; (b) amplifying DNA in the sample using the set of oligonucleotides under a multiplex polymerase chain reaction (PCR); and (c) determining the presence of a PCR product of step (b), wherein the presence of a PCR product in the sample indicates that the sample is contaminated by a guaiacol-producing microorganism, a specific species of acidoterrestris alicyclobacillus, and alicyclobacillus.

[0016] In some embodiments, the guaiacol-producing gene encodes vanillin decarboxylase (vdc). In some embodiments, the guaiacol-producing gene encoding vanillin decarboxylase (vdc) has at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO:51.

[0017] In some embodiments, the second primer set targets the rpoB gene and / or the gyrB gene of Bacillus acidoterrestris. In some embodiments, the rpoB gene comprises a nucleic acid sequence having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 9-14. In some embodiments, the gyrB gene comprises a nucleic acid sequence having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 15-26.

[0018] In some embodiments, the 16S rRNA gene comprises a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence set forth in SEQ ID NOs:27-50.

[0019] In some embodiments, the first primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 1-2.

[0020] In some embodiments, the second primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 3-6.

[0021] In some embodiments, the third primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs:7-8.

[0022] In some embodiments, the first primer set hybridizes to a nucleic acid of a gene that produces guaiacol. In some embodiments, the second primer set hybridizes to a nucleic acid of Bacillus acidoterrestris. In some embodiments, the third primer set hybridizes to a nucleic acid of a 16S rRNA gene.

[0023] In some embodiments, the second primer set comprises at least one: a forward primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 3 and 5.

[0024] In some embodiments, the second primer set comprises at least one: a reverse primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs:4 and 6.

[0025] In some embodiments, the third primer set comprises at least one: a forward primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, the third primer set comprises at least one: a reverse primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO: 8.

[0026] In some embodiments, each primer of the first primer set has a length of no more than 35 nucleotides. In some embodiments, each primer of the second primer set has a length of no more than 35 nucleotides. In some embodiments, each primer of the third primer set has a length of no more than 35 nucleotides.

[0027] In some embodiments, the spoilage microorganisms detected by the method include at least one of: Alicyclobacillus acidocalordi, Alicyclobacillus contaminating, Alicyclobacillus disulfide oxidizing, Alicyclobacillus fastidious, Alicyclobacillus ferrooxidans, Alicyclobacillus hesperidin, Alicyclobacillus fruiti, Alicyclobacillus saccharide, Alicyclobacillus Sendai, Alicyclobacillus Shizuoka, Alicyclobacillus resistant, Alicyclobacillus vulcani, Alicyclobacillus tengchong, Alicyclobacillus acidoterrestris, Alicyclobacillus acidic, Alicyclobacillus cycloheptyl, and Alicyclobacillus grassi.

[0028] In some embodiments, the food is a beverage, drink, juice or smoothie.

[0029] In some embodiments, the PCR is quantitative PCR or real-time PCR.

[0030] In some embodiments, the multiplex PCR reaction takes about 5 days or less, about 4 days or less, about 3 days or less, about 2 days or less, or about 1 day or less. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 shows an example of gene sequence alignment of SEQ ID NOs: 9-14.

[0032] FIG. 2 shows an example of gene sequence alignment of SEQ ID NOs: 15-26.

[0033] FIG. 3 shows an example of gene sequence alignment of SEQ ID NOs: 27-50.

[0034] Figure 4A The structure of the vdc region according to the present disclosure is shown as well as the locations of genetic elements and selected test primers.

[0035] Figure 4B Gel electrophoresis of PCR amplicons obtained by Bur5 / 6 primers (SEQ ID NO: 1-2) is shown. Red marked strains are guaiacol producers; white marked strains do not produce guaiacol (Doehler test kit).

[0036] Figure 5Shown are the gel electrophoresis results of a multiplex 2 PCR reaction for guaiacol production and specific for Alicyclobacillus acidoterrestris using primer combinations having the sequences set forth in SEQ ID NOs: 1-6.

[0037] Figure 6 Shown are gel electrophoresis results of multiplex 3PCR reactions using primer combinations with sequences listed in SEQ ID NOs: 1-8. Alicyclobacillus acidoterrestris (AT) showed 3 bands - a species-specific band (gyrB); a band producing guaiacol (Bur5 / 6); and a genus-specific band (AGES). Non-AT strains showed a single band (AGES).

[0038] Brief Description of Sequence Listing

[0039] SEQ ID NO: 1 discloses the primer Bur5 sequence (5'-3') targeting the vanillin decarboxylase (vdc) region of Alicyclobacillus acidoterrestris: CCGACGTGATGCTCAARGAGCGCA.

[0040] SEQ ID NO: 2 discloses the primer Bur6 sequence (5'-3') targeting the vanillin decarboxylase (vdc) region of Alicyclobacillus acidoterrestris: GTSGCRTCGAGAATCATCTTGTG.

[0041] SEQ ID NO: 3 discloses the forward primer sequence (5'-3') of the rpoB gene: CCGTCGTGAGGACGTTATCGCGGTGCCG.

[0042] SEQ ID NO: 4 discloses the reverse primer sequence (5'-3') of the rpoB gene: CGCAACGTCACCGCGAACAAGTCGG.

[0043] SEQ ID NO: 5 discloses the forward primer sequence (5'-3') of the gyrB gene: GTTCCCAAGTACGATTTGAAAGTGG.

[0044] SEQ ID NO: 6 discloses the reverse primer sequence (5'-3') of the gyrB gene: GGTTGCTCTCGCTCCCCTTAATC.

[0045] SEQ ID NO: 7 discloses the forward primer sequence (5'-3') of AGES: GTYRCTCGGGRAGAGCG.

[0046] SEQ ID NO: 8 discloses the reverse primer sequence (5'-3') of AGES: CGCCGRCAGTCACCTGT.

[0047] SEQ ID NO:9 discloses the sequence of the rpoB gene of Alicyclobacillus acidocaldarius.

[0048] SEQ ID NO: 10 discloses the sequence of the rpoB gene of Alicyclobacillus mali.

[0049] SEQ ID NO: 11 discloses the sequence of the rpoB gene of Alicyclobacillus acidoterrestris ATCC_49025.

[0050] SEQ ID NO: 12 discloses the sequence of the rpoB gene of Alicyclobacillus acidophilus.

[0051] SEQ ID NO: 13 discloses the sequence of the rpoB gene of Bacillus saccharolyticus.

[0052] SEQ ID NO: 14 discloses the sequence of the rpoB gene of Alicyclobacillus hesperidinus.

[0053] SEQ ID NO: 15 discloses the sequence of the gyrB gene of contaminating Alicyclobacillus sp. ACON_DSM17975.

[0054] SEQ ID NO: 16 discloses the sequence of the gyrB gene of Alicyclobacillus acidoterrestris AT_NBRC106287.

[0055] SEQ ID NO: 17 discloses the sequence of the gyrB gene of Alicyclobacillus acidoterrestris AT_ATCC49025.

[0056] SEQ ID NO: 18 discloses the sequence of the gyrB gene of Alicyclobacillus acidoterrestris AT_ATCC49025_2.

[0057] SEQ ID NO: 19 discloses the sequence of the gyrB gene of Alicyclobacillus acidoterrestris AT_OR3.

[0058] SEQ ID NO: 20 discloses the sequence of the gyrB gene of Alicyclobacillus hesperidinus AH_DSM12489.

[0059] SEQ ID NO: 21 discloses the sequence of the gyrB gene of Alicyclobacillus sp. ASach_DSM17974.

[0060] SEQ ID NO: 22 discloses the sequence of the gyrB gene of Alicyclobacillus acidophilus AP_NBRC100859.

[0061] SEQ ID NO: 23 discloses the sequence of the gyrB gene of Alicyclobacillus acidophilus AP_undefined.

[0062] SEQ ID NO: 24 discloses the sequence of the gyrB gene of Alicyclobacillus acidocaldarius AC_DSM446.

[0063] SEQ ID NO: 25 discloses the sequence of the gyrB gene of Alicyclobacillus Sendai AS_NBRC100866.

[0064] SEQ ID NO: 26 discloses the sequence of the gyrB gene of Alicyclobacillus Sendai AS_undefined.

[0065] SEQ ID NO: 27 discloses the sequence of 16S rRNA of Bacillus subtilis 168.

[0066] SEQ ID NO: 28 discloses the sequence of 16S rRNA of G. stearothermophilus R-32640.

[0067] SEQ ID NO: 29 discloses the sequence of 16S rRNA of Desulfuronidase Bacillus stibiiarsenate (D. stibiiarsenatis) MLFW-2.

[0068] SEQ ID NO:30 discloses the sequence of 16S rRNA contaminating Alicyclobacillus sp. 3-A191.

[0069] SEQ ID NO:31 discloses the sequence of 16S rRNA contaminating Alicyclobacillus sp. NBRC103102.

[0070] SEQ ID NO: 32 discloses the sequence of 16S rRNA of Alicyclobacillus Sendai JCM1181.

[0071] SEQ ID NO: 33 discloses the sequence of 16S rRNA of Alicyclobacillus Sendai NBRC100866.

[0072] SEQ ID NO: 34 discloses the sequence of 16S rRNA of Alicyclobacillus Sendai NTAP-1.

[0073] SEQ ID NO: 35 discloses the sequence of 16S rRNA of A. acidocaldarius rittmannii DSM11297.

[0074] SEQ ID NO: 36 discloses the sequence of 16S rRNA of Alicyclobacillus acidocaldarius subsp. ritanya MR1.

[0075] SEQ ID NO: 37 discloses the sequence of 16S rRNA of A. acidocaldariusacidocaldarius DSM446.

[0076] SEQ ID NO: 38 discloses the sequence of 16S rRNA of Alicyclobacillus acidocaldarius 104-1A.

[0077] SEQ ID NO: 39 discloses the sequence of 16S rRNA of Bacillus acidocaldarius subsp. acidocaldarius_ATCC27009.

[0078] SEQ ID NO:40 discloses the sequence of 16S rRNA of Alicyclobacillus acidocaldarius NBRC15652.

[0079] SEQ ID NO:41 discloses the sequence of 16S rRNA of Alicyclobacillus acidophilus NBRC100859.

[0080] SEQ ID NO: 42 discloses the sequence of 16S rRNA of Alicyclobacillus acidophilus TA-67.

[0081] SEQ ID NO: 43 discloses the sequence of 16S rRNA of Bacillus saccharolyticus NBRC103105.

[0082] SEQ ID NO: 44 discloses the sequence of 16S rRNA of Bacillus glycosaminoglycans RB718.

[0083] SEQ ID NO: 45 discloses the sequence of 16S rRNA of Alicyclobacillus hesperidinus DSM12489.

[0084] SEQ ID NO: 46 discloses the sequence of 16S rRNA of Alicyclobacillus hesperidinus FR-11.

[0085] SEQ ID NO: 47 discloses the sequence of 16S rRNA of Alicyclobacillus acidoterrestris DSM3922.

[0086] SEQ ID NO:48 discloses the sequence of 16S rRNA of Alicyclobacillus acidoterrestris ATCC49025.

[0087] SEQ ID NO: 49 discloses the sequence of 16S rRNA of Alicyclobacillus fastidious NBR103109.

[0088] SEQ ID NO: 50 discloses the sequence of 16S rRNA of Alicyclobacillus fastidious S-TAB.

[0089] SEQ ID NO:51 discloses the sequence of the vdc region of a guaiacol-producing microorganism. DETAILED DESCRIPTION

[0090] According to the method described herein, a sample is obtained from a test material, such as a sample of a beverage, a juice or other food product. The sample is processed to extract any polynucleotide in the sample, particularly a polynucleotide from a target microorganism that may be present in the material. After extracting and processing according to methods described herein and / or otherwise known in the art, the sample is processed with a composition comprising at least one of the following components: a forward primer oligonucleotide, a reverse primer oligonucleotide, and / or a labeled oligonucleotide probe, wherein the composition targets a specific region in the target organism genome. The sample is then processed according to polymerase chain reaction (PCR), particularly multiple PCR amplification methods. Primers are used to carry out initial amplification of the PCR product. The labeled probe is combined with the target sequence in the PCR product to indicate the presence of contaminating microorganisms, and the PCR product corresponds to the target region in the genomic DNA of the contaminating bacteria. Therefore, the combination of this composition and PCR technology ensures specific, rapid and sensitive detection of the presence of target bacteria. By using multiplex PCR, the protocols provided in the present disclosure allow for efficient and accurate determination of whether an organism is an isolate of the genus Alicyclobacillus (genus level detection), whether the organism is Alicyclobacillus acidoterrestris (species level determination), and whether the isolate possesses the gene that leads to guaiacol production.

[0091] Primer selection

[0092] Primers are selected in the conservative region shown in the gene sequence comparison (e.g., Fig. 1-Fig. 3) to increase the fragment of the appropriate size for optimal detection. A primer is located at each end of the sequence to be amplified. The length of such primers will be generally between 10 and 35 nucleotides, and has a preferred length between 18 and 22 nucleotides. The length of the minimum sequence that can be amplified is about 50 nucleotides (e.g., a forward and reverse primer of 20 nucleotides in length, and its position in the sequence is separated by at least 10 nucleotides). Longer sequences can be amplified. Preferably, the length of the amplified sequence is a length of 75 to 250 nucleotides, and the length for PCR determination is between 75 to 150 nucleotides.

[0093] One type of primer used herein is a "forward primer" and is located at the left end of the region to be amplified. The sequence of the forward primer is identical to a region in the top strand of the DNA (when double-stranded DNA is depicted using the convention in which the top strand is shown with polarity in the 5' to 3' direction). The sequence of the forward primer is such that it hybridizes with a DNA strand that is complementary to the top strand of the DNA.

[0094] Another type of primer used herein is a "reverse primer", and it is located at the right end of the region to be amplified. The sequence of the reverse primer is such that its sequence is complementary to the region in the DNA top strand, that is, the sequence of the reverse primer is the reverse complementary sequence of the sequence in the region in the DNA top strand. The reverse primer hybridizes with the DNA top strand.

[0095] PCR primers should also be selected according to a plurality of other conditions. In certain embodiments, the length of PCR primers used herein is 10 to 30 nucleotides, to minimize hybridization with more than one region in the template. In certain embodiments, primers with long strings of single bases are avoided. In certain embodiments, primers used herein have a G+C percentage content between 40% and 60%. In an embodiment, the G+C percentage content at the 3' end of the primer is higher than the G+C percentage content at the 5' end of the primer. In an embodiment, primers do not contain a sequence that can hybridize with another sequence in the primer. In an embodiment, a plurality of primers used in the same PCR reaction do not hybridize with each other. Although PCR primers are preferably selected according to the above suggestions, these primers do not necessarily need to meet these conditions. Other primers can work, but the possibility that they produce good results is lower.

[0096] One of the available multiple computer programs can be used to select PCR primers that can be used to amplify the DNA in a given sequence. Such a program selects the best primers for amplifying a given sequence (i.e., such a program selects primers based on the above conditions plus other conditions that can maximize the functionality of the PCR primers). A computer program is the Genetics Computer Group (GCG recently became Accelrys) analysis package, which has a routine for selecting PCR primers. There are also several websites that can be used to select the best PCR primers to amplify the input sequence. One such website is http: / / alces.med.umn.edu / rawprimer.html. Another such website is http: / / www-genome.wi.mit.edu / cgi-bin primer / primer3_www.cgi.

[0097] Gene sequence alignment

[0098] As used herein, gene sequence alignment refers to a technique or process for comparing and detecting the similarity between two or more sequences.Gene sequence alignment is a powerful tool for identifying regions of similarity from a plurality of gene sequences, which regions of similarity may be the result of functional relationships, and therefore contribute to primer design.The comparison of a target gene or a target gene can be carried out by any commercial or non-commercial algorithm or analysis package.The example of an alignment algorithm includes Serial Cloner program, ClustalW etc.

[0099] Preparation of oligonucleotide primers and / or probes

[0100] Oligonucleotide primers and / or probes according to the present disclosure can be prepared in a variety of ways. One way to prepare these oligonucleotides is to use a commercially available nucleic acid synthesizer to synthesize them. There are multiple such synthesizers and are well known to those skilled in the art. Although other chemical substances can be used, many such synthesizers use phosphoramidite chemistry. Phosphoramidite chemistry utilizes DNA phosphoramidite nucleosides (commonly referred to as monomers) to synthesize DNA chains or oligonucleotides. Such monomers are modified with dimethoxytrityl (DMT) protecting groups (at the 5'-end), 3'-phosphite groups protected by b-cyanoethyl, and may also include other modifiers for protecting reactive primary amines in heterocyclic structures (to prevent branching or other undesirable side reactions during the building-up process).

[0101] In order to prepare the oligonucleotide of specific sequence, add phosphoramidite nucleoside one by one on the 3'-5' direction of oligonucleotide, start from the post containing 3' nucleoside temporarily fixed on solid support.By simply handling with acid [dichloroacetic acid (DCA) or trichloroacetic acid (TCA) in dichloromethane (DCM)], the 5'-trityl group of 3' nucleoside of cracking immobilization begins synthesis, to produce reactive 5'-hydroxyl group.The next monomer activated by tetrazolium is coupled to available 5'-hydroxyl, and the resulting phosphite bond is oxidized to phosphate by processing with iodine (in THF / pyridine / H2O solution).A base is described above and added in oligonucleotide.Each base added is carried out to other circulation.The final oligonucleotide added does not have 5' phosphoric acid.When synthesis is completed, oligonucleotide is discharged and deprotected (removing the blocking group on the nucleotide) from support by ammonium hydroxide.Usually, the oligonucleotide up to 150-180 base length can be effectively synthesized using nucleic acid synthesizer by this method. In order to prepare oligonucleotides longer than 100 bases, two single-stranded oligonucleotides complementary along their length portions can be synthesized, annealed to form duplexes, and then connected to a plasmid vector. Once a plasmid containing a duplex connected has been formed, other oligonucleotide duplexes can be connected to the plasmid, adjacent to the duplex previously connected, to form a longer sequence. Oligonucleotide duplexes can also be sequentially connected to each other, to form a long, specific sequence, and then this single long sequence is cloned into a plasmid vector.

[0102] As used herein, "nucleotide sequence" or "nucleic acid" includes "polynucleotide", "oligonucleotide" and "nucleic acid molecule", and generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, synthetic or obtained (e.g., isolated and / or purified) from a natural source, which may contain natural, non-natural or altered nucleotides, and which may contain natural, non-natural or altered internucleotide bonds, such as phosphoramidate bonds or phosphorothioate bonds, instead of the phosphodiester found between the nucleotides of unmodified oligonucleotides. It is generally preferred that the present nucleic acid does not contain any insertions, deletions, inversions and / or substitutions. However, as discussed herein, in some cases, it is appropriate that the nucleic acid contains one or more insertions, deletions, inversions and / or substitutions.

[0103] The present nucleic acids can be constructed based on chemical synthesis (as discussed above) and / or enzymatic ligation reactions using procedures known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, New York 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, 1994. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed after hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-1-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylquenoside, inosine, N- 6 -Isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 Substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl quercetin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil and 2,6-diaminopurine.

[0104] Isolation of DNA from samples

[0105] Separate or extract DNA from the microbial cells contained in the test sample. For example, DNA extraction can be performed using any of a number of commercially available kits for such purposes. One such kit (Sigma Aldrich Genomic DNA Extraction Kit) is available from Sigma Aldrich in St. Louis. DNA extraction can contain a paper filter to which cells are applied. By treating the paper filter as described by the manufacturer, most of the cell components are retained in the paper filter, and the DNA is released into the aqueous solution. The DNA in the solution can then be added to multiple enzymatic amplification reactions as discussed below. There are other commercially available kits for extracting DNA from cells. However, in order to obtain satisfactory DNA, it is not necessary to use commercial kits. Standard methods well known to those skilled in the art have been disclosed in the scientific literature. Such methods generally involve lysing cells and removing cell components other than nucleic acids by precipitation or by extraction with organic solvents. Enzymatic treatment using proteases and ribonucleases can be used to remove proteins and RNA, respectively. DNA is then typically precipitated from the sample using alcohol.

[0106] PCR

[0107] As used herein, the term "PCR" refers to various forms of PCR known in the art, including but not limited to quantitative PCR, reverse transcriptase PCR, real-time PCR, hot start PCR, long PCR, LAPCR, multiplex PCR, touchdown PCR, etc. A variety of methods can be used to determine whether a PCR product has been produced. One way to determine whether a PCR product has been produced in a reaction is to analyze a portion of the PCR reaction by agarose gel electrophoresis. For example, a horizontal agarose gel of 0.6% to 2.0% agarose is prepared, and a portion of the PCR reaction mixture is electrophoresed by an agarose gel. After electrophoresis, the gel is stained with ethidium bromide. When the gel is observed during illumination with ultraviolet light, the PCR product is visible. By comparing with a standardized size marker, it is determined whether the PCR product has the correct expected size.

[0108] The PCR program is preferably performed in a manner that allows the amount of the PCR product to be quantified. Such a "quantitative PCR" program typically involves comparing the amounts of the PCR products produced in different PCR reactions. Many such quantitative PCR programs and variations thereof are well known to those skilled in the art. However, an inherent property of such a program is the ability to determine the relative amount of the target sequence in the template amplified in the PCR reaction.

[0109] One specific method of quantitative PCR used to quantify the number of copies of a sequence within a PCR template is a modification of standard PCR known as "real-time PCR." Real-time PCR utilizes a thermal cycler (i.e., an instrument that provides the temperature changes required for a PCR reaction to occur) that contains a fluorometer (i.e., an instrument that measures fluorescence). In one type of real-time PCR, the reaction mixture also contains a reagent whose incorporation into the PCR product can be quantified and the quantification of the reagent indicates the number of copies of the sequence in the template.

[0110] Basic multiplex PCR methods and the considerations necessary for their successful execution are known in the art and are readily applicable to the methods described herein, wherein the ability to effectively separate and detect amplicons of different sizes from different known targets allows detection of multiple (e.g., 2, 3, 5, 10, 20, 50 or more) target signals in a single reaction. Multiplex PCR generally requires that interactions between primers specific for different targets be minimized to reduce artifacts, i.e., one attempts to avoid any two primers that may be used in the reaction from hybridizing to each other rather than to their respective target molecules. Commonly available software packages allow analysis and prediction of primer-primer interactions for a given primer set.

[0111] In some aspects, the present disclosure provides a method for detecting spoilage microorganisms in a sample, the method comprising: (a) contacting a set of oligonucleotides with the sample, wherein the set of oligonucleotides comprises: a first primer set that targets a nucleic acid of a guaiacol-producing microorganism; and a second primer set that targets a nucleic acid of Bacillus acidoterrestris; (b) amplifying DNA in the sample with the set of oligonucleotides under a multiplex polymerase chain reaction (PCR); and (c) determining the presence of a PCR product of step (b), wherein the presence of the PCR product in the sample indicates that the sample is contaminated with both the guaiacol-producing microorganism and Bacillus acidoterrestris.

[0112] In some embodiments, the set of oligonucleotides further comprises a third primer set targeting a nucleic acid of a 16S rRNA gene common to Alicyclobacillus, and wherein the presence of the PCR product in the sample indicates that the sample is contaminated with a guaiacol-producing microorganism, a specific species of Alicyclobacillus acidoterrestris, and Alicyclobacillus.

[0113] In some embodiments, the guaiacol-producing gene encodes vanillin decarboxylase (vdc). In certain embodiments, the guaiacol-producing gene encoding vanillin decarboxylase (vdc) has at least 75% identity to the nucleic acid sequence listed in SEQ ID NO: 51, as shown in Table 1.

[0114] Table 1. Target genes encoding vanillin decarboxylase (vdc).

[0115]

[0116] In some embodiments, the second primer set targets the rpoB gene and / or the gyrB gene of Bacillus acidoterrestris. In certain embodiments, the rpoB gene comprises a nucleic acid sequence having at least 75% identity to any one of the nucleic acid sequences listed in SEQ ID NOs: 9-14, as shown in Table 2. In certain embodiments, the gyrB gene comprises a nucleic acid sequence having at least 75% identity to any one of the nucleic acid sequences listed in SEQ ID NOs: 15-26, as shown in Table 2.

[0117] Table 2. rpoB gene and / or gyrB gene of Alicyclobacillus acidoterrestris.

[0118]

[0119]

[0120] In some embodiments, the Alicyclobacillus consensus 16S rRNA gene comprises a nucleic acid sequence that is at least 75% identical to any one of the nucleic acid sequences listed in SEQ ID NOs: 27-50, as shown in Table 3.

[0121] Table 3. Common 16S rRNA genes among Alicyclobacillus species.

[0122]

[0123]

[0124] In some embodiments, the first primer set hybridizes to a nucleic acid or a fragment thereof of a gene producing guaiacol. In some embodiments, the first primer set comprises at least one primer that hybridizes to a nucleic acid or a fragment thereof of a gene sequence listed in SEQ ID NO:51. In certain embodiments, the first primer set comprises at least one primer that has at least 75% identity to a nucleic acid sequence listed in SEQ ID NO:1-2, as shown in Table 4.

[0125] Table 4. Primers targeting the guaiacol-producing gene, vanillin decarboxylase (vdc).

[0126]

[0127] In some embodiments, the second primer set hybridizes with a nucleic acid or fragment thereof of Alicyclobacillus acidoterrestris. In some embodiments, the second primer set comprises at least one primer that hybridizes to a nucleic acid common to two or more gene sequences listed in SEQ ID NO:9-14. In some embodiments, the second primer set comprises at least one primer that hybridizes to a nucleic acid common to two or more gene sequences listed in SEQ ID NO:15-26. In some embodiments, the second primer set comprises at least one primer that hybridizes to an alignment product of the gene sequences listed in SEQ ID NO:9-14. In some embodiments, the second primer set comprises at least one primer that hybridizes to an alignment product of the gene sequences listed in SEQ ID NO:15-26. Examples of alignment products of the gene sequences of SEQ ID NO:9-14 are shown in Figure 1. Examples of alignment products of the gene sequences of SEQ ID NO:15-26 are shown in Figure 2. In certain embodiments, the second primer set comprises at least one primer that has at least 75% identity to the nucleic acid sequences listed in SEQ ID NO:3-6, as shown in Table 5.

[0128] In some embodiments, the second primer set comprises at least one: a forward primer having at least 75% identity to the nucleic acid sequences set forth in SEQ ID NOs: 3 and 5. In some embodiments, the second primer set comprises at least one: a reverse primer having at least 75% identity to the nucleic acid sequences set forth in SEQ ID NOs: 4 and 6.

[0129] Table 5. Primers for species-specific target (Alicyclobacillus acidoterrestris).

[0130]

[0131] In some embodiments, the third primer set hybridizes to nucleic acids of 16S rRNA genes. In some embodiments, the third primer set comprises at least one primer that hybridizes to nucleic acids common to two or more gene sequences listed in SEQ ID NOs: 27-50. In some embodiments, the second primer set comprises at least one primer that hybridizes to the alignment products of the gene sequences listed in SEQ ID NOs: 27-50. Examples of gene sequence alignment products of SEQ ID NOs: 27-50 are shown in Figure 3. In certain embodiments, the third primer set comprises at least one primer that has at least 75% identity to the nucleic acid sequences listed in SEQ ID NOs: 7-8, as shown in Table 6.

[0132] In some embodiments, the third primer set comprises at least one: a forward primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, the third primer set comprises at least one: a reverse primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO: 8.

[0133] Table 6. Primers targeting genus-specific (16S rRNA common to Alicyclobacillus, Bacillus, Desulfuribacillus, and Geobacillus).

[0134]

[0135] In some embodiments, each primer of the first primer set has a length of no more than 35 nucleotides. In some embodiments, each primer of the second primer set has a length of no more than 35 nucleotides. In some embodiments, each primer of the third primer set has a length of no more than 35 nucleotides.

[0136] In some embodiments, a primer or nucleic acid according to the present disclosure comprises a sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any nucleic acid described herein.

[0137] In some embodiments, the determining step further comprises comparing the presence of one or more PCR products in at least one test sample with the absence of one or more PCR products in a negative sample lacking microbial nucleic acid, wherein the detection of the complex indicates the presence of one or more Alicyclobacillus microorganisms. In some embodiments, the method comprises determining a signal background level produced by a marker in a negative sample lacking microbial nucleic acid, and comparing the signal background level with the signal level detected in the test sample. A higher or lower signal level in the test sample compared to the signal level measured in the negative sample may indicate the presence of one or more microorganisms.

[0138] In an embodiment, the method optionally includes comparing the amount of one or more PCR products having a certain sequence in at least one test sample with the amount of a gene having a similar or identical sequence to one or more PCR product sequences from a negative sample lacking microbial nucleic acid, wherein an increase in the amount of the gene sequence from at least one test sample indicates the presence of one or more microorganisms. In this regard, if the amount of one or more PCR products detected in the sample does not exceed the amount of the gene sequence detected in a negative sample known to lack microbial nucleic acid, the sample is negative for food spoilage microorganisms. If the amount of one or more PCR products detected in the sample exceeds the amount of the gene sequence detected in a negative sample known to lack microbial nucleic acid, the sample is positive for food spoilage microorganisms.

[0139] In some embodiments, the spoilage microorganisms detected by the method include at least one of: Alicyclobacillus acidocalordi, Alicyclobacillus contaminating, Alicyclobacillus disulfide oxidizing, Alicyclobacillus fastidious, Alicyclobacillus ferrooxidans, Alicyclobacillus hesperidin, Alicyclobacillus fruiti, Alicyclobacillus saccharide, Alicyclobacillus Sendai, Alicyclobacillus Shizuoka, Alicyclobacillus resistant, Alicyclobacillus vulcani, Alicyclobacillus tengchong, Alicyclobacillus acidoterrestris, Alicyclobacillus acidic, Alicyclobacillus cycloheptyl, and Alicyclobacillus grassi.

[0140] In some embodiments, the multiplex PCR reaction takes about 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less.

[0141] The method may include detecting microorganisms in any food. The food may be, for example, any one or more of dairy products; fats, oils and fat emulsions; edible ices (including, for example, sherbet and sorbet); fruits and vegetables (including, for example, mushrooms and fungi, roots and tubers, beans and legumes, and aloe); seaweed; nuts and seeds; confectionery; cereals and cereal products; baked goods (such as bread); meat and meat products (including, for example, poultry and game); fish and fish products (including mollusks, crustaceans and echinoderms); eggs and egg products; sweeteners, including, for example, honey and sugar (solid or liquid); salts, spices, soups, sauces, salads, protein products; foods for specific nutritional purposes; and beverages (such as water, juices, drinks, beer and wine). In a preferred embodiment, the food is a beverage, typically containing juice.

[0142] The method may include obtaining a sample of the food to be tested in any suitable manner and culturing the microorganisms in the sample. For example, when the food is juice, the sample may be any one or more of juice concentrate, pasteurized juice, unpasteurized juice, other juice, or any additional additives or ingredients of the juice. Additional additives and ingredients of the juice may include, for example, any one or more of water (e.g., condensed water and / or water added to the final juice product), soluble additives, and sweeteners (e.g., sugar (solid or liquid)).

[0143] The method can include cultivating microorganisms in any suitable culture medium known in the art. The culture medium can be selected according to the properties of the food to be tested and the microorganism. Exemplary culture mediums can include yeast-sucrose-glucose (YSG) culture medium, potato dextrose culture medium, and alicyclobacillus broth (BAT) culture medium.

[0144] These microorganisms can be cultivated at any suitable temperature known in the art and for any suitable duration. The cultivation temperature and duration can be selected according to the nature of the food to be tested and the microorganism. For example, these microorganisms can be cultivated at a temperature of about 40°C to about 60°C, preferably about 45°C to about 50°C. These microorganisms can be cultivated for about 1 day to about 14 days, or about 1 day to about 7 days, or about 1 day to about 3 days, or about 1 day to about 2 days.

[0145] The method can include extracting nucleic acid from microorganisms in any suitable manner known in the art. The nucleic acid can be RNA and / or DNA. The scheme for extracting nucleic acid can be selected according to the properties of food to be tested, microorganisms and nucleic acid known in the art. Preferably, nucleic acid is extracted in any manner that lyses gram-positive bacteria and gram-negative bacteria, and the manner reclaims the DNA of a testable amount without using polymerase chain reaction (PCR) inhibitors. Nucleic acid extraction can be performed using any of a variety of commercially available nucleic acid extraction kits according to the manufacturer's instructions.

[0146] In some embodiments, a method for detecting spoilage microorganisms in a food sample comprises: (a) contacting a set of oligonucleotides with the sample, wherein the set of oligonucleotides comprises: a first primer set targeting nucleic acid of a guaiacol-producing microorganism; a second primer set targeting nucleic acid of Alicyclobacillus acidoterrestris; and a third primer targeting nucleic acid of a 16S rRNA gene common to the genus Alicyclobacillus; (b) amplifying DNA in the sample using the set of oligonucleotides under a multiplex polymerase chain reaction (PCR); and (c) determining the presence of a PCR product of step (b), wherein the presence of the PCR product in the sample indicates that the sample is contaminated with a guaiacol-producing microorganism, a specific species of Alicyclobacillus acidoterrestris, and the genus Alicyclobacillus.

[0147] In some aspects, the disclosure provides a system or assay kit comprising one or more nucleic acid sets for specific detection of spoilage microorganisms in food samples. The assay kit may comprise one or more nucleic acid sets for detecting any one or more guaiacol-producing microorganisms, in combination with any one or more Alicyclobacillus species described herein, and / or in combination with any genus-specific set described herein. In some embodiments, the system or assay kit may further comprise any one or more DNA polymerases, buffers, deoxyribonucleotide triphosphates (dNTPs), Mg ++ Etc. The DNA polymerase is not limited and can be any DNA polymerase suitable for qPCR or PCR. Exemplary DNA polymerases include Thermus aquaticus (Taq) polymerase and Pyrococcus furiosus (Pfu) polymerase. The buffer is not limited and can be any buffer suitable for PCR or qPCR. Exemplary buffers include combinations of KCl, Tris and MgCl2 and combinations of (NH4)2SO4, Tris-HCl, MgCl2, β-mercaptoethanol and EDTA.

[0148] In certain embodiments, the disclosure provides a kind of support comprising any one of nucleic acid as herein described or nucleic acid set fixed on the support.In certain embodiments, the support comprises the sample to be tested fixed on the support, and one or more nucleic acids as herein described or one or more nucleic acid sets are applied to the support.The support can be any support suitable for fixing this nucleic acid.The support can further comprise detectable label.The label can be any label suitable for detecting the complex of this nucleic acid and microbial nucleic acid.Exemplary detectable label can comprise any one or more of radioactive label, non-radioactive label, fluorescent label and chemiluminescent label.

[0149] In some embodiments, a system for detecting spoilage microorganisms in food samples comprises a set of oligonucleotides for multiplex polymerase chain reaction (PCR), the set of oligonucleotides comprising: a first primer set targeting nucleic acid of a microorganism producing guaiacol; and a second primer set targeting nucleic acid of Alicyclobacillus acidoterrestris. In some embodiments, the set of oligonucleotides further comprises: a third primer set targeting nucleic acid of a 16S rRNA gene common to Alicyclobacillus genus.

[0150] In some embodiments, a kit for detecting spoilage microorganisms in a food sample comprises: a first primer set that targets nucleic acid of a guaiacol-producing microorganism; and a second primer set that targets nucleic acid of Alicyclobacillus acidoterrestris.

[0151] In some embodiments, the kit further comprises: instructions for providing a method comprising: (a) contacting the set of oligonucleotides with a sample; (b) amplifying DNA in the sample using the set of oligonucleotides under a multiplex polymerase chain reaction (PCR); and (c) determining the presence of the PCR product of step (b), wherein the presence of the PCR product in the sample indicates that the sample is contaminated with a guaiacol-producing microorganism and a specific species of Alicyclobacillus acidoterrestris.

[0152] In some embodiments, the kit further comprises: a third primer set, the third primer set targeting nucleic acid of the 16S rRNA gene common to the genus Alicyclobacillus. In some embodiments, the instructions further provide: the presence of a PCR product in the sample indicates that the sample is contaminated by guaiacol-producing microorganisms, specific species of Alicyclobacillus acidoterrestris and Alicyclobacillus spp.

[0153] The term "about" is used in conjunction with a numerical value to include normal variations in measurements as would be expected by one skilled in the art and should be understood to have the same meaning as "approximately" and encompass typical margins of error, such as ±10% of the stated value. The term "about" also encompasses different amounts due to different equilibrium conditions of the composition resulting from a particular initial composition. Whether or not modified by the term "about", the claims include equivalents to the quantities.

[0154] As used herein, the term "isolated" means having been removed from its natural environment. As used herein, the term "purified" means to increase purity, wherein "purity" is a relative term and is not necessarily considered absolute purity. For example, the purity can be at least about 50%, can be greater than 60%, 70% or 80%, 90% or can be 100%.

[0155] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "a compound" includes having two or more compounds that are the same or different from each other. It should also be noted that the term "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise.

[0156] For the sake of brevity and conciseness, any range of values ​​stated in this specification contemplates all values ​​within that range and should be interpreted as supporting claims reciting any sub-ranges having endpoints with actual numerical values ​​within the specified range in question. By way of hypothetical illustrative example, the disclosure of a range from 1 to 5 in this specification should be considered supporting claims to any of the following ranges: 1-5; 1-4; 1-3; 1-2; 2-5; 2-4; 2-3; 3-5; 3-4; and 4-5.

[0157] The term "substantially free of" may refer to any component that the composition of the present disclosure lacks or mainly lacks. When referring to "substantially free of", it is intended that the component is not intentionally added to the composition of the present disclosure. The use of the term "substantially free of" a certain component allows trace amounts of the component to be included in the composition of the present disclosure because they are present in another component. However, it should be recognized that when a composition is referred to as "substantially free of" a certain component, only trace amounts or trace amounts of the component will be allowed. In addition, the term if a composition is referred to as "substantially free of" a certain component, if the component is present in trace amounts or trace amounts, it should be understood that it will not affect the effectiveness of the composition. It should be understood that if a component is not explicitly included in this article or it is not stated in this article that it may be included, the composition of the present disclosure may be substantially free of the component. Similarly, the explicit inclusion of a component allows its explicit exclusion, thereby allowing the composition to be substantially free of explicitly stated components.

[0158] The methods, compositions and kits of the present disclosure may comprise, consist essentially of, or consist of the components of the present disclosure as well as other components described herein. As used herein, "consisting essentially of" means that the methods and compositions may include additional steps, components or ingredients, but only if the additional steps, components or ingredients do not materially change the basic and novel features of the claimed methods and compositions.

[0159] Examples

[0160] Certain embodiments of the present disclosure are further described with reference to the following experiments and examples. These experiments, examples and samples are intended to illustrate the present disclosure only, and are not intended to limit or restrict the scope of the present disclosure in any way, and should not be construed as providing conditions, parameters, reagents or starting materials that must be specifically utilized in order to practice the technology of the present disclosure.

[0161] Example 1 - Multiplex PCR Assay

[0162] A multiplex PCR assay for the detection of guaiacol production and Alicyclobacillus species was developed, with a strain library taken from the ASL culture collection. A total of 65 strains belonging to 10 Alicyclobacillus species were analyzed. All strains were grown on BAT plates for 5 days at 45°C under aerobic conditions. All strains were tested for guaiacol production by using the Doehler Guaiacol Kit. All strains were tested by MALDI-TOF analysis or 16S PCR sequencing to confirm the assigned species. To isolate DNA, the Sigma-Aldrich Genomic DNA Extraction Kit was used, and the presence of DNA was confirmed by gel electrophoresis.

[0163] To further develop a PCR test for detecting genes that produce guaiacol, a literature search provided primers that detect the presence of the gene encoding vanillin decarboxylase (vdc), an enzyme involved in guaiacol anabolism (Dekowska et al., 2018). Although variations in primers and amplicon length were tested, the best results were obtained with the Bur5 / 6 primers.

[0164] All strains producing guaiacol produced positive PCR results with a 1500 bp amplicon, except for the strain Bacillus acidicola B360, which was found to have a negative phenotype but a positive genotype for guaiacol production. This has been observed in other studies, where the strain Bacillus fastidious DSM17978, which is known to be a non-guaiacol producer, showed a band corresponding to the vdc gene (Wang et al., 2021). In the present study, other strains of this species showed results consistent with the phenotypic assay results, such as Figure 4A-4B as shown in .

[0165] In order to develop a pair of primers that provide a band specific for Alicyclobacillus acidoterrestris (the most common and important producer of guaiacol), an alignment of genes (e.g., rpoB and gyrB) was performed. Sequences of related species were downloaded from the NCBI database, and ClustalW was used as an algorithm for alignment.

[0166] Initially, primers were designed against the rpoB gene, which encodes the beta subunit of the bacterial RNA polymerase. However, a single band was detected in only 75% of the strains of A. acidoterrestris. In response, primers were designed based on an alignment of the gyrB gene, which encodes the beta subunit of DNA gyrase. All A. acidoterrestris strains gave a positive reaction, while all non-A. acidoterrestris strains gave a negative reaction.

[0167] In the next step, the multiplexing of these two reactions (for guaiacol production and specific for Alicyclobacillus acidoterrestris) was combined. The conditions were optimized and the results showed that both reactions were successfully performed in a single tube, such as Figure 5 as shown in .

[0168] After this, the sequences of the 16S rRNA gene were downloaded to include representatives of all 10 Alicyclobacillus species tested and closely related genera (Bacillus, Desulfobacillus and Geobacillus). Degenerate primers were designed based on the alignment performed as described above. The primers were named AGES F (SEQ ID NO: 7) and AGESR (SEQ ID NO: 8), respectively.

[0169] Additional testing was performed to multiplex three primer sets [Bur5 / 6 (SEQ ID NOs: 1-2), gyrB (SEQ ID NOs: 5-6), and AGES F / R (SEQ ID NOs: 7-8)] to determine if all three targets would be detected. Bands were observed in the lanes indicating that the isolate tested was an Alicyclobacillus genus, indicating that the AGES primer set successfully identified a member of the genus Alicyclobacillus. The isolate identified in the table as Alicyclobacillus acidoterrestris was also determined to be guaiacol positive by culture-based biochemical testing. When the gel was imaged, the isolate identified as Alicyclobacillus acidoterrestris exhibited three bands: one band indicating that it belonged to the genus Alicyclobacillus, one band indicating that the isolate had the gene that causes guaiacol production, and one band indicating that the organism was a member of the species Alicyclobacillus acidoterrestris, as shown in FIG. Figure 6 as shown in .

[0170] The above results support that these primer sets showed no amplification of bands nonspecific to the isolates tested and that multiplexing the primers tested resulted in results that were both inclusive and exclusive for the target attribute.

[0171] Non-patent literature

[0172] CONNOR,C.J.,LUO,H.,GARDENER,B.B.&WANG,H.H.2005.Development of a real-time PCR-based system targeting the 16S rRNA gene sequence for rapiddetection of Alicyclobacillus spp.in juice products.Int JFood Microbiol,99,229-35.

[0173] DEKOWSKA,A.,NIEZGODA,J.&SOKOLOWSKA,B.2018.Genetic Heterogeneity ofAlicyclobacillus Strains Revealed by RFLP Analysis of vdc Region and rpoBGene.Biomed Res Int,2018,9608756.

[0174] LI,H.,CHEN,H.,LIU,B.,CAI,R.,JIANG,N.,YUE,T.&WANG,Z.2021.Establishmentof quantitative PCR assays for the rapid detection of Alicyclobacillusspp.that can produce guaiacol in apple juice.Int J Food Microbiol,109329.

[0175] LUO,H.,YOUSEF,A.E.&WANG,H.H.2004.Areal-time polymerase chainreaction-based method for rapid and specific detection of spoilageAlicyclobacillus spp.in apple juice.Lett Appl Microbiol,39,376-82.

[0176] WANG,Z.,YUE,T.,YUAN,Y.,ZHANG,Y.,GAO,Z.&CAI,R.2021.Targeting thevanillic acid decarboxylase gene for Alicyclobacillus acidoterrestrisquantification and guaiacol assessment in apple juices using real timePCR.International Journal of Food Microbiology,338,109006.

[0177] All publications, patents, and patent applications mentioned in the specification are indicative of the levels of those skilled in the art to which the present disclosure pertains.

[0178] The following numbered clauses define additional example aspects and features of the disclosure:

[0179] 1. A method for detecting spoilage microorganisms in a food sample, the method comprising:

[0180] (a) contacting a set of oligonucleotides with the sample, wherein the set of oligonucleotides comprises:

[0181] a first primer set that targets nucleic acid of a guaiacol-producing microorganism; and

[0182] a second primer set, the second primer set targeting nucleic acid of Alicyclobacillus acidoterrestris;

[0183] (b) amplifying DNA in the sample using the set of oligonucleotides under a multiplex polymerase chain reaction (PCR); and

[0184] (c) determining the presence of the PCR product of step (b), wherein the presence of the PCR product in the sample indicates that the sample is contaminated with both a guaiacol-producing microorganism and Alicyclobacillus acidoterrestris.

[0185] 2. The method of clause 1, wherein the set of oligonucleotides further comprises a third primer set targeting a nucleic acid of the 16S rRNA gene common to the genus Alicyclobacillus, and wherein the presence of the PCR product in the sample indicates that the sample is contaminated with a guaiacol-producing microorganism, the specific species Alicyclobacillus acidoterrestris, and the genus Alicyclobacillus.

[0186] 3. The method of any one of clauses 1-2, wherein the guaiacol-producing gene encodes vanillin decarboxylase (vdc).

[0187] 4. The method of clause 3, wherein the guaiacol-producing gene encoding vanillin decarboxylase (vdc) has at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO:51.

[0188] 5. The method of any one of clauses 1 to 4, wherein the second primer set targets the rpoB gene and / or the gyrB gene of Alicyclobacillus acidoterrestris.

[0189] 6. The method of clause 5, wherein the rpoB gene comprises a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence set forth in SEQ ID NOs: 9-14.

[0190] 7. The method of any one of clauses 5-6, wherein the gyrB gene comprises a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence set forth in SEQ ID NOs: 15-26.

[0191] 8. The method of any one of clauses 2 to 7, wherein the 16S rRNA gene comprises a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence set forth in SEQ ID NOs: 27-50.

[0192] 9. The method of any one of clauses 1 to 8, wherein the first set of primers comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO: 1-2.

[0193] 10. The method of any one of clauses 1-9, wherein the second set of primers comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 3-6.

[0194] 11. The method of any one of clauses 1-10, wherein the third primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 7-8.

[0195] 12. The method of any one of clauses 1-11, wherein the first set of primers hybridizes to the nucleic acid of the guaiacol-producing gene.

[0196] 13. The method of any one of clauses 1 to 12, wherein the second primer set hybridizes to the Alicyclobacillus acidoterrestris nucleic acid.

[0197] 14. The method of any one of clauses 1 to 13, wherein the third primer set hybridizes to nucleic acid of the 16S rRNA gene.

[0198] 15. The method of any one of clauses 1 to 14, wherein the second primer set comprises at least one: a forward primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 3 and 5.

[0199] 16. The method of any one of clauses 1 to 15, wherein the second primer set comprises at least one: a reverse primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 4 and 6.

[0200] 17. The method of any one of clauses 2 to 16, wherein the third primer set comprises at least one: a forward primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO:7.

[0201] 18. The method of any one of clauses 2 to 17, wherein the third primer set comprises at least one: a reverse primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO:8.

[0202] 19. The method of any one of clauses 1 to 18, wherein each primer of the first set of primers has a length of no more than 35 nucleotides.

[0203] 20. The method of any one of clauses 1 to 19, wherein each primer of the second set of primers has a length of no more than 35 nucleotides.

[0204] 21. The method of any one of clauses 2-20, wherein each primer of the third set of primers has a length of no more than 35 nucleotides.

[0205] 22. The method of any one of clauses 1 to 21, wherein the spoilage microorganisms detected by the method comprise at least one of: Alicyclobacillus acidocaldarius, Alicyclobacillus contaminating, Alicyclobacillus disulfide oxidizing, Alicyclobacillus fastidious, Alicyclobacillus ferrooxidans, Alicyclobacillus hesperidinus, Alicyclobacillus fruiti, Alicyclobacillus sacchari, Alicyclobacillus Sendai, Alicyclobacillus Shizuoka, Alicyclobacillus resistant, Alicyclobacillus vulcanus, Alicyclobacillus tengchong, Alicyclobacillus acidoterrestris, Alicyclobacillus acidic, Alicyclobacillus cycloheptyl, and Alicyclobacillus grassi.

[0206] 23. The method of any one of clauses 1-22, wherein the multiplex PCR reaction requires about 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less.

[0207] 24. The method of any one of clauses 1-23, wherein the food is a beverage.

[0208] 25. The method of any one of clauses 1-24, wherein the food is juice.

[0209] 26. The method of any one of clauses 1 to 25, wherein the PCR is quantitative PCR.

[0210] 27. The method of any one of clauses 1 to 26, wherein the PCR is real-time PCR.

[0211] 28. A method for detecting spoilage microorganisms in a food sample, the method comprising:

[0212] (a) contacting a set of oligonucleotides with the sample, wherein the set of oligonucleotides comprises:

[0213] a first primer set that targets nucleic acid of a guaiacol-producing microorganism;

[0214] a second primer set that targets nucleic acid of Alicyclobacillus acidoterrestris; and

[0215] a third primer, the primer targeting nucleic acid of the 16S rRNA gene common to the genus Alicyclobacillus;

[0216] (b) amplifying DNA in the sample using the set of oligonucleotides under a multiplex polymerase chain reaction (PCR);

[0217] (c) determining the presence of the PCR product of step (b), wherein the presence of the PCR product in the sample indicates that the sample is contaminated with a guaiacol-producing microorganism, the specific species Alicyclobacillus acidoterrestris, and the Alicyclobacillus genus.

[0218] 29. The method of clause 28, wherein the first set of primers comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 1-2.

[0219] 30. The method of any of clauses 28-29, wherein the second set of primers comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 3-6.

[0220] 31. The method of any of clauses 28-30, wherein the third set of primers comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 7-8.

[0221] 32. The method of any one of clauses 28 to 31, wherein each primer of the first set of primers has a length of no more than 35 nucleotides.

[0222] 33. The method of any one of clauses 28 to 32, wherein each primer of the second set of primers has a length of no more than 35 nucleotides.

[0223] 34. The method of any one of clauses 28 to 33, wherein each primer of the third set of primers has a length of no more than 35 nucleotides.

[0224] 35. A system for detecting spoilage microorganisms in a food sample, the system comprising a set of oligonucleotides for use in a multiplex polymerase chain reaction (PCR), wherein the set of oligonucleotides comprises:

[0225] a first primer set that targets nucleic acid of a guaiacol-producing microorganism; and

[0226] A second primer set, wherein the second primer set targets nucleic acid of Alicyclobacillus acidoterrestris.

[0227] 36. The system of clause 35, wherein the set of oligonucleotides further comprises: a third primer set that targets nucleic acid of a 16S rRNA gene common to the genus Alicyclobacillus.

[0228] 37. The system of any of clauses 35-36, wherein the first set of primers comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 1-2.

[0229] 38. The system of any of clauses 35-37, wherein the second set of primers comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 3-6.

[0230] 39. The system of any of clauses 36-38, wherein the third set of primers comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 7-8.

[0231] 40. A kit for detecting spoilage microorganisms in a food sample, the kit comprising:

[0232] a first primer set that targets nucleic acid of a guaiacol-producing microorganism; and

[0233] A second primer set, wherein the second primer set targets nucleic acid of Alicyclobacillus acidoterrestris.

[0234] 41. The kit of clause 40, further comprising: instructions for providing a method comprising:

[0235] (a) contacting the set of oligonucleotides with the sample;

[0236] (b) amplifying DNA in the sample using the set of oligonucleotides under a multiplex polymerase chain reaction (PCR);

[0237] (c) determining the presence of the PCR product of step (b), wherein the presence of the PCR product in the sample indicates that the sample is contaminated with a guaiacol-producing microorganism and the specific species Alicyclobacillus acidoterrestris.

[0238] 42. The kit of any one of clauses 40-41, wherein the first set of primers comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO: 1-2.

[0239] 43. The kit of any one of clauses 40-42, wherein the second primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 3-6.

[0240] 44. The kit of any one of clauses 40-43, further comprising: a third primer set targeting nucleic acid of the 16S rRNA gene common to the genus Alicyclobacillus.

[0241] 45. The kit of clause 44, wherein the third primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 7-8.

[0242] 46. ​​The kit of any one of clauses 44-45, wherein the instructions further provide that the presence of a PCR product in the sample indicates that the sample is contaminated with a guaiacol-producing microorganism, the particular species Alicyclobacillus acidoterrestris and the Alicyclobacillus genus.

[0243] Although only exemplary embodiments of the present disclosure are described in detail above, those skilled in the art will readily appreciate that many modifications are possible without departing substantially from the novel teachings and advantages of the present invention. Therefore, all such modifications are intended to be included within the scope of the present disclosure as defined in the following claims.

Claims

1. A method for detecting spoilage microorganisms in a food sample, the method comprising: (a) contacting a set of oligonucleotides with the sample, wherein the set of oligonucleotides comprises: a first primer set that targets nucleic acid of a guaiacol-producing microorganism; as well as a second primer set, the second primer set targeting nucleic acid of Alicyclobacillus acidoterrestris; (b) amplifying DNA in the sample using the set of oligonucleotides under a multiplex polymerase chain reaction (PCR); as well as (c) determining the presence of the PCR product of step (b), wherein the presence of the PCR product in the sample indicates that the sample is contaminated with both a guaiacol-producing microorganism and Alicyclobacillus acidoterrestris.

2. The method of claim 1, wherein the set of oligonucleotides further comprises a third primer set targeting a nucleic acid of the 16S rRNA gene common to the genus Alicyclobacillus, and wherein the presence of the PCR product in the sample indicates that the sample is contaminated with a guaiacol-producing microorganism, the specific species Alicyclobacillus acidoterrestris, and the genus Alicyclobacillus.

3. The method of any one of claims 1-2, wherein the guaiacol-producing gene encodes vanillin decarboxylase (vdc).

4. The method of claim 3, wherein the guaiacol-producing gene encoding vanillin decarboxylase (vdc) has at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO:

51.

5. The method of any one of claims 1-4, wherein the second primer set targets the rpoB gene and / or the gyrB gene of Alicyclobacillus acidoterrestris.

6. The method of claim 5, wherein the rpoB gene comprises a nucleic acid sequence having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 9-14.

7. The method of any one of claims 5-6, wherein the gyrB gene comprises a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence set forth in SEQ ID NOs: 15-26.

8. The method of any one of claims 2-7, wherein the 16S rRNA gene comprises a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence set forth in SEQ ID NOs: 27-50.

9. The method of any one of claims 1-8, wherein the first primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO: 1-2.

10. The method of any one of claims 1-9, wherein the second primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 3-6.

11. The method of any one of claims 1-10, wherein the third primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs:7-8.

12. The method of any one of claims 1-11, wherein the first primer set hybridizes to the nucleic acid of the guaiacol-producing gene.

13. The method of any one of claims 1-12, wherein the second primer set hybridizes to the nucleic acid of the Alicyclobacillus acidoterrestris.

14. The method of any one of claims 1-13, wherein the third primer set hybridizes to nucleic acid of the 16S rRNA gene.

15. The method of any one of claims 1-14, wherein the second primer set comprises at least one: a forward primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 3 and 5.

16. The method of any one of claims 1-15, wherein the second primer set comprises at least one: a reverse primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 4 and 6.

17. The method of any one of claims 2-16, wherein the third primer set comprises at least one: a forward primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO:

7.

18. The method of any one of claims 2-17, wherein the third primer set comprises at least one: a reverse primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO:

8.

19. The method of any one of claims 1-18, wherein each primer of the first set of primers has a length of no more than 35 nucleotides.

20. The method of any one of claims 1-19, wherein each primer of the second primer set has a length of no more than 35 nucleotides.

21. The method of any one of claims 2-20, wherein each primer of the third primer set has a length of no more than 35 nucleotides.

22. The method of any one of claims 1 to 21, wherein the spoilage microorganisms detected by the method comprise at least one of: Alicyclobacillus acidocaldarius, Alicyclobacillus contaminating, Alicyclobacillus disulfide oxidizing, Alicyclobacillus fastidious, Alicyclobacillus ferrooxidans, Alicyclobacillus hesperidin, Alicyclobacillus fruiti, Alicyclobacillus saccharide, Alicyclobacillus Sendai, Alicyclobacillus Shizuoka, Alicyclobacillus resistant, Alicyclobacillus vulcanus, Alicyclobacillus tengchong, Alicyclobacillus acidoterrestris, Alicyclobacillus acidic, Alicyclobacillus cycloheptyl, and Alicyclobacillus grassi.

23. The method of any one of claims 1-22, wherein the multiplex PCR reaction takes about 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less.

24. The method of any one of claims 1-23, wherein the food is a beverage.

25. The method of any one of claims 1-24, wherein the food is juice.

26. The method of any one of claims 1-25, wherein the PCR is quantitative PCR.

27. The method of any one of claims 1-26, wherein the PCR is real-time PCR.

28. A method for detecting spoilage microorganisms in a food sample, the method comprising: (a) contacting a set of oligonucleotides with the sample, wherein the set of oligonucleotides comprises: a first primer set that targets nucleic acid of a guaiacol-producing microorganism; a second primer set, the second primer set targeting nucleic acid of Alicyclobacillus acidoterrestris; as well as a third primer, the primer targeting nucleic acid of the 16S rRNA gene common to the genus Alicyclobacillus; (b) amplifying DNA in the sample using the set of oligonucleotides under a multiplex polymerase chain reaction (PCR); (c) determining the presence of the PCR product of step (b), wherein the presence of the PCR product in the sample indicates that the sample is contaminated with a guaiacol-producing microorganism, the specific species Alicyclobacillus acidoterrestris, and the Alicyclobacillus genus.

29. The method of claim 28, wherein the first set of primers comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 1-2.

30. The method of any one of claims 28-29, wherein the second primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 3-6.

31. The method of any one of claims 28-30, wherein the third primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs:7-8.

32. The method of any one of claims 28-31, wherein each primer of the first set of primers has a length of no more than 35 nucleotides.

33. The method of any one of claims 28-32, wherein each primer of the second primer set has a length of no more than 35 nucleotides.

34. The method of any one of claims 28-33, wherein each primer of the third primer set has a length of no more than 35 nucleotides.

35. A system for detecting spoilage microorganisms in a food sample, the system comprising a set of oligonucleotides for use in a multiplex polymerase chain reaction (PCR), wherein the set of oligonucleotides comprises: a first primer set that targets nucleic acid of a guaiacol-producing microorganism; and A second primer set, wherein the second primer set targets nucleic acid of Alicyclobacillus acidoterrestris.

36. The system of claim 35, wherein the set of oligonucleotides further comprises: a third primer set targeting nucleic acid of a 16S rRNA gene common to the genus Alicyclobacillus.

37. The system of any of claims 35-36, wherein the first set of primers comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 1-2.

38. The system of any one of claims 35-37, wherein the second primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 3-6.

39. The system of any one of claims 36-38, wherein the third primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 7-8.

40. A kit for detecting spoilage microorganisms in a food sample, the kit comprising: a first primer set that targets nucleic acid of a guaiacol-producing microorganism; and A second primer set, wherein the second primer set targets nucleic acid of Alicyclobacillus acidoterrestris.

41. The kit of claim 40, further comprising: instructions for providing a method comprising: (a) contacting the set of oligonucleotides with the sample; (b) amplifying DNA in the sample using the set of oligonucleotides under a multiplex polymerase chain reaction (PCR); (c) determining the presence of the PCR product of step (b), wherein the presence of the PCR product in the sample indicates that the sample is contaminated with a guaiacol-producing microorganism and the specific species Alicyclobacillus acidoterrestris.

42. The kit of any one of claims 40-41, wherein the first primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO: 1-2.

43. The kit of any one of claims 40-42, wherein the second primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs: 3-6.

44. The kit of any one of claims 40-43, further comprising: a third primer set targeting nucleic acid of the 16S rRNA gene common to the genus Alicyclobacillus.

45. The kit of claim 44, wherein the third primer set comprises at least one primer having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NOs:7-8.

46. ​​The kit of any one of claims 44-45, wherein the instructions further provide that the presence of a PCR product in the sample indicates that the sample is contaminated with a guaiacol-producing microorganism, the specific species Alicyclobacillus acidoterrestris, and the Alicyclobacillus genus.

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