Specialized primers and kits for identifying pediococcus pentosaceus

Abstract

The application discloses special primers and kits for identifying Pediococcus pentosaceus. The special primers for identifying Pediococcus pentosaceus disclosed by the application are composed of primers with names F3, B3, FIP, BIP, LF and LB, and the F3, B3, FIP, BIP, LF and LB are single-stranded DNAs shown in sequences 1-6 in the sequence listing. The special primers for identifying Pediococcus pentosaceus can detect Pediococcus pentosaceus, have the advantages of high sensitivity, strong specificity, simple operation, rapidity and the like, can amplify Pediococcus pentosaceus specific gene fragments by using a loop-mediated isothermal amplification method, fill the blank of the LAMP detection method of Pediococcus pentosaceus in beer, and are suitable for grass-roots application and on-site rapid detection in breweries.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and specifically to a special primer and kit for identifying Pediococcus pentosaceus. BACKGROUND

[0002] Pediococcus pentosaceus is a gram-positive coccus widely distributed in fermented plants and wort, and is a common beer spoilage bacteria. The presence of Pediococcus pentosaceus in beer will produce acid and buttery diacetyl, thereby causing beer spoilage. It is understood that there is a risk of Pediococcus pentosaceus contamination in each stage from malt preparation to beer brewing in beer production, and rapid detection of Pediococcus pentosaceus has become a major problem to be solved for quality control in breweries.

[0003] Common detection methods for Pediococcus pentosaceus include culture method and molecular biology method. After culture in NBB and MRS culture medium, the strain is qualitatively determined according to its colony morphological characteristics, peroxidase reaction and physiological and biochemical indicators. Although the operation is simple, the steps are tedious and time-consuming, and it is difficult to provide timely risk warning for breweries. Molecular biology methods, which rely on nucleic acid amplification for target identification, have higher detection efficiency. Common molecular biology methods mainly include gene chip technology, polymerase chain reaction and isothermal amplification technology. Gene chip technology is a comprehensive biological technology formed by the cross of molecular biology, bioinformatics, physics, computer science and other disciplines. It uses micro-mechanical technology to construct a microfluidic unit system on a solid chip, and realizes the qualitative detection of various contaminating bacteria by inoculating specific oligonucleotide probes with known sequences on the chip surface. Gene chip technology has high specificity and sensitivity, can realize high-throughput analysis and detection, but is prone to false positives, has high technical cost and cannot be widely promoted. Polymerase chain reaction (PCR) is a well-developed method in molecular biology. It can form new DNA strands by repeatedly changing temperature to split DNA double strands and hybridize primers. After nucleic acid amplification by PCR technology, the product can be judged by observing the length of the target band after gel electrophoresis, or the result can be judged by the change of fluorescence accumulation of probe and fluorescent reagent, greatly reducing the operation steps and saving manpower and time for enterprises. However, PCR technology relies on precise temperature control equipment and detection instruments, and has high requirements for laboratory environment and personnel quality, which is not conducive to the detection task of breweries at the grassroots level.

[0004] Isothermal amplification method is a new in vitro nucleic acid amplification method in recent years. Unlike PCR technology, isothermal amplification has low precision requirement for temperature change, does not require special instruments and equipment, greatly solves the problem of difficulty in application at the grassroots level, in addition, isothermal amplification has strong specificity, high sensitivity, can realize target amplification detection within 0.5h, greatly improves the detection efficiency, and has been widely used in the fields of pathogenic microorganisms, transgenic species, animal-derived component analysis and the like, and has been recognized by scholars and enterprises at home and abroad. SUMMARY

[0005] The technical problem to be solved by the present application is how to identify Pediococcus pentosaceus.

[0006] To solve the above technical problems, the present application first provides a primer set consisting of primers F3, B3, FIP, BIP, LF and LB;

[0007] The F3 is as follows (a1) or (a2):

[0008] (a1) a single-stranded DNA molecule shown in sequence 1 of the sequence listing;

[0009] (a2) a DNA molecule obtained by substituting, deleting and / or adding one or more nucleotides to sequence 1 and having the same function as sequence 1;

[0010] The B3 is as follows (a3) or (a4):

[0011] (a3) a single-stranded DNA molecule shown in sequence 2 of the sequence listing;

[0012] (a4) a DNA molecule obtained by substituting, deleting and / or adding one or more nucleotides to sequence 2 and having the same function as sequence 2;

[0013] The FIP is as follows (a5) or (a6):

[0014] (a5) a single-stranded DNA molecule shown in sequence 3 of the sequence listing;

[0015] (a6) a DNA molecule obtained by substituting, deleting and / or adding one or more nucleotides to sequence 3 and having the same function as sequence 3;

[0016] The BIP is as follows (a7) or (a8):

[0017] (a7) a single-stranded DNA molecule shown in sequence 4 of the sequence listing;

[0018] (a8) a DNA molecule obtained by substituting, deleting and / or adding one or more nucleotides to sequence 4 and having the same function as sequence 4;

[0019] The LF is as follows (a9) or (a10):

[0020] (a9) a single-stranded DNA molecule as shown in SEQ ID NO: 5 of the sequence listing;

[0021] (a10) a DNA molecule which is subject to substitution and / or deletion and / or addition of one or several nucleotides of SEQ ID NO: 5 and has the same function as SEQ ID NO: 5;

[0022] The LB is as follows (a11) or (a12):

[0023] (a11) a single-stranded DNA molecule as shown in SEQ ID NO: 6 of the sequence listing;

[0024] (a12) a DNA molecule which is subject to substitution and / or deletion and / or addition of one or several nucleotides of SEQ ID NO: 6 and has the same function as SEQ ID NO: 6.

[0025] The primer set is a primer set based on loop-mediated isothermal amplification.

[0026] The use of the primer set is as follows (b1) or (b2): (b1) identifying or assisting in identifying Pediococcus pentosaceus; (b2) detecting or assisting in detecting whether Pediococcus pentosaceus is contained in a sample to be tested.

[0027] In the primer set, the molar ratio of F3, B3, FIP, BIP, LF, and LB is 1:1:8:8:4:4, respectively.

[0028] The present application also provides any of the following uses of the primer set:

[0029] X1) preparing a reagent or kit for identifying or assisting in identifying Pediococcus pentosaceus;

[0030] X2) preparing a reagent or kit for detecting or assisting in detecting whether Pediococcus pentosaceus is contained in a sample to be tested;

[0031] X3) identifying or assisting in identifying Pediococcus pentosaceus;

[0032] X4) detecting or assisting in detecting whether Pediococcus pentosaceus is contained in a sample to be tested.

[0033] The present application also provides a reagent, which comprises the primer set.

[0034] The reagent can further comprise other reagents required for LAMP reaction. The other reagents can be Isothermal Master Mix (Zhejiang Zhongjing Experimental Authentication Co., Ltd., item number ISO-004).

[0035] The reagent can be composed of the primer set and Isothermal Master Mix. Each 11 μL of the reagent can comprise: outer primer F3 2.5 pmol, B3 2.5 pmol, inner primer FIP 20 pmol, BIP 20 pmol, loop primer LF 10 pmol, LB 10 pmol, Isothermal Master Mix 7.5 μL.

[0036] The application of the reagent in identifying or assisting in identifying P. pentosus also falls within the protection scope of the present application.

[0037] The application of the reagent in detecting or assisting in detecting whether the sample to be tested contains P. pentosus also falls within the protection scope of the present application.

[0038] The present application also provides a kit containing the primer set or the reagent; the kit is used for (b1) identifying or assisting in identifying P. pentosus or (b2) detecting or assisting in detecting whether the sample to be tested contains P. pentosus.

[0039] The application of the kit in identifying or assisting in identifying P. pentosus also falls within the protection scope of the present application.

[0040] The application of the kit in detecting or assisting in detecting whether the sample to be tested contains P. pentosus also falls within the protection scope of the present application.

[0041] The present application also provides a method for identifying or assisting in identifying P. pentosus, which comprises the following steps:

[0042] (1) extracting nucleic acid of the microorganism to be tested;

[0043] (2) using the primer set to perform loop-mediated isothermal amplification with the nucleic acid extracted in step (1) as a template; if the primer set can realize specific amplification with the extracted nucleic acid as a template, the microorganism to be tested is or is suspected to be P. pentosus; if the primer set cannot realize specific amplification with the extracted nucleic acid as a template, the microorganism to be tested is or is suspected to be non-P. pentosus.

[0044] The present application also provides a method for detecting whether the sample to be tested contains P. pentosus, which comprises the following steps:

[0045] (1) extracting nucleic acid of the sample to be tested;

[0046] (2) Using the nucleic acid extracted in step (1) as a template, loop-mediated isothermal amplification is performed using the primer set; if the primer set can achieve specific amplification using the extracted nucleic acid as a template, the sample to be tested contains or is a candidate to contain Pediococcus pentosaccharis; if the primer set cannot achieve specific amplification using the extracted nucleic acid as a template, the sample to be tested does not contain or is a candidate not to contain Pediococcus pentosaccharis.

[0047] In the above method, the sample to be tested can be a beer sample.

[0048] The reaction system for loop-mediated isothermal amplification using the primer set can be (12.5 μL): 7.5 μL IsothermalMaster Mix, 3.5 μL of the primer set, and 1.5 μL of the template solution to be tested. In the 3.5 μL primer set, the concentrations of each primer are: 2.5 pmol for outer primers F3 and B3, 2.5 pmol for inner primers FIP and BIP, and 10 pmol for loop primers LF and LB.

[0049] The reaction conditions for loop-mediated isothermal amplification using the primer set described above can be: 65℃ for 30 min.

[0050] The beneficial effects of this invention are as follows: (1) High specificity: Primers are designed based on six specific regions of the target gene, and the complementary base pairing is more stable and specific; (2) High sensitivity: For the amplification of certain pathogens, the template only needs a few copies, which is 1-2 orders of magnitude better than PCR sensitivity; (3) Fast and efficient: No preheating denaturation or repeated temperature cycling is required, which reduces time loss. In addition, the introduction of two loop primers further accelerates the reaction rate, so that nucleic acid amplification can be achieved in 0.5h under isothermal conditions, which greatly ensures the amplification efficiency; (4) Convenient product detection: When nucleic acid amplification is performed using LAMP technology, a large number of pyrophosphate ions are generated. When they combine with magnesium ions, they can generate a white magnesium pyrophosphate precipitate, which can be used to directly judge the product results. In addition, fluorescent reaction reagents can be used for real-time monitoring of target amplification, which is convenient and reliable; (5) Simple operation: No need for repeated physiological and biochemical experiments to verify, only a water bath is needed to realize the entire amplification process, which is highly operable.

[0051] This invention utilizes loop-mediated isothermal amplification (LAMP) technology to amplify specific gene fragments of Pediococcus pentosaceus, filling the gap in LAMP detection methods for Pediococcus pentosaceus in beer. It has advantages such as high specificity, simple operation, and speed and convenience, and can be used for beer quality monitoring. It is suitable for grassroots applications in breweries and rapid on-site detection. Attached Figure Description

[0052] Figure 1 This is the fluorescence amplification curve for specific detection in Example 2.

[0053] Figure 2 Fluorescence amplification curve for LAMP in Example 3.

[0054] Figure 3 Fluorescence amplification curve for real-time fluorescent PCR in Example 3.

[0055] Figure 4 Fluorescence amplification curve for LAMP reaction using three primer sets respectively in Comparative Example.

[0056] Figure 5 Melting curve for LAMP reaction using P1 primer set in Comparative Example. DETAILED DESCRIPTION

[0057] The present application will be further described in conjunction with the specific embodiments, and the examples given are only to illustrate the present application, but not to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application.

[0058] In the following examples, the experimental methods are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. In the following examples, the materials, reagents, instruments, etc. used, unless otherwise specified, can be obtained commercially. In the following examples, the quantitative tests were all set up in triplicate, and the results were averaged. In the following examples, unless otherwise specified, the 1st nucleotide of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last nucleotide is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0059] Example 1, Preparation of primer set for identifying P. pentosus

[0060] The primer set for identifying P. pentosus provided in this example consists of six primers, and the sequences are as follows:

[0061] Outer primer F3 (sequence 1 of the sequence listing): 5'-CGCAGCTTGAGAAACATCA-3';

[0062] Outer primer B3 (sequence 2 of the sequence listing): 5'-GGACGCACTTTAGCTTGT-3';

[0063] Inner primer FIP (sequence 3 of the sequence listing): 5'-ATGCTCACAAAGTTGAAATCGAATTTGTTTGCTCCATAGTTGTACTC-3';

[0064] Inner primer BIP (sequence 4 of the sequence listing): 5'- AGTCTGGACGGTAGCTACCTTTAGTGGTGTGACCTTAGCC-3';

[0065] Loop primer LF (sequence 5 of the sequence listing): 5'- CGTAACTCCCGTAAGGTTCTGG-3';

[0066] Loop primer LB (sequence 6 of the sequence listing): 5'- CACTTTAGTAGCGTTAATTCCAGCA-3'.

[0067] The molar ratio of the outer primer F3, the outer primer B3, the inner primer FIP, the inner primer BIP, the loop primer LF and the loop primer LB in the primer set for identifying Pediococcus pentosaceus is 1:1:8:8:4:4.

[0068] Example 2, specificity

[0069] The test bacteria were Pediococcus pentosaceus CICC21862, Pediococcus pentosaceus CICC22230, Lactobacillus brevis CICC20014, Lactobacillus casei CICC6117, Lactobacillus plantarum CICC20261, Lactobacillus acidophilus CICC6074, Lactobacillus delbrueckii CICC6047, Bifidobacterium longum CICC6069, Streptococcus thermophilus CICC6038, Lactococcus lactis CICC6242, Leuconostoc mesenteroides CICC20714, Bacillus subtilis CICC24713, Escherichia coli CICC10003 and Pseudomonas aeruginosa CICC10204. Each strain was a product of CICC, i.e. China Center of Industrial Culture Collection.http: / / sales.china-cicc.org / .

[0070] 1. Preparation of template solution

[0071] Take 10 5 CFU / mL of test bacteria, treat at 99℃ for 5 min, terminate the reaction at 4℃, collect the supernatant to obtain nucleic acid template solution of each bacteria.

[0072] 2. Perform LAMP reaction

[0073] Perform LAMP reaction using the primer set of Example 1, the reaction system (12.5 μL) of loop-mediated isothermal amplification: Isothermal Master Mix 7.5 μL, primer set 3.5 μL, template solution 1.5 μL. In 3.5 μL of primer set, the content of each primer is: outer primer F3 2.5 pmol, B3 2.5 pmol, inner primer FIP 20 pmol, BIP 20 pmol, loop primer LF 10 pmol, LB 10 pmol. Among them, Isothermal Master Mix: Zhongqing Inspection Certification Co., Ltd., item number ISO-004.

[0074] Set up sterile double distilled water instead of template solution as negative control.

[0075] Reaction conditions of loop-mediated isothermal amplification: 65℃, 30 min. The reaction was performed in Genie II real-time fluorescence detector.

[0076] The fluorescence amplification curve is shown in Figure 1 , Figure 1 Pediococcus pentosaceus 1 and Pediococcus pentosaceus 2 are Pediococcus pentosaceus CICC21862 and Pediococcus pentosaceus CICC22230 respectively. Both of the two strains of Pediococcus pentosaceus can produce fluorescence amplification curve, and other test bacteria cannot produce fluorescence amplification curve. The results show that the primer set of Example 1 has good specificity.

[0077] Example 3, Sensitivity

[0078] The test bacteria are Pediococcus pentosaceus CICC21862.

[0079] 1. Preparation of template solution

[0080] Determine the concentration of test bacteria suspension by plate count, dilute it with sterile physiological saline by 10 times gradient, and obtain bacteria concentration of 10 0 -10 5Bacterial suspension with CFU / mL gradient. Take the bacterial suspension, treat at 99℃ for 5 min, finally terminate the reaction at 4℃, collect the supernatant to obtain the nucleic acid template solution of P. pentosus with different concentrations.

[0081] 2. Perform LAMP reaction

[0082] Perform LAMP reaction with the primer set of Example 1, and the reaction system is the same as that of Example 2.

[0083] Set the sterile double distilled water instead of the template solution as the negative control.

[0084] Reaction condition: 65℃, 30 min. Perform the reaction in Genie II real-time fluorescence detector.

[0085] The fluorescence amplification curve is shown in Figure 2 .10 2 -10 5 The nucleic acid of the bacterial suspension with CFU / mL concentration can produce the fluorescence amplification curve, and the detection sensitivity is 10 2 CFU / mL. The nucleic acid of the bacterial suspension with CFU / mL and below can not produce the fluorescence amplification curve. It is shown that, after simple nucleic acid extraction, the detection sensitivity of the specific primer set for P. pentosus is 10 2 CFU / mL.

[0086] 3. Perform real-time fluorescence PCR

[0087] Reaction system (20 μL): TB Green Fast qPCR Mix (2X) 10 μL, ROX Reference Dye (50X) 0.4 μL, outer primer F3 and B3 of Example 1, template solution 2 μL, and the rest is water. In the reaction system, the content of each primer is: outer primer F3 8 pmol, B3 8 pmol.

[0088] Reaction condition: 95℃ 30 s; 95℃ 5 s, 60℃ 15 s, 45 cycles. Perform the reaction in ABI7900 real-time fluorescence detector, and the amplification result with Ct value ≤40 is determined as positive.

[0089] The fluorescence amplification curve is shown in Figure 3 .10 3 -10 5 The nucleic acid of the bacterial suspension with CFU / mL concentration can produce the fluorescence amplification curve, and the detection sensitivity is 10 3 CFU / mL. It is shown that, after the same nucleic acid extraction step, the LAMP detection method of the application has 10 times higher sensitivity than the real-time fluorescence PCR.

[0090] Example 4, preparation of LAMP detection reagent for detecting P. pentosus

[0091] Detection reagent: Isothermal Master Mix 7.5 μL, each primer of the primer set of Example 1. The content of each primer in 11 μL detection reagent is: outer primer F3 2.5 pmol, B3 2.5 pmol, inner primer FIP 20 pmol, BIP 20 pmol, loop primer LF 10 pmol, LB 10 pmol.

[0092] Method for using detection reagent: take 11 μL detection reagent, add 1.5 μL nucleic acid template of sample to be tested, and perform LAMP reaction on Genie II real-time fluorescence detector. Reaction condition: 65°C, 30 min. The reaction result is determined by fluorescence amplification curve.

[0093] Comparative example, effect comparison of different primer sets

[0094] The inventors also detected the detection effect of two other LAMP primer sets (P2 primer set, P3 primer set) on P. pentosus. The sequences of each primer set are shown in Table 1, and the detection steps are as follows:

[0095] Table 1

[0096]

[0097] The test bacteria are P. pentosus CICC21862.

[0098] 1, take 10 5 CFU / mL of test bacteria solution, treat at 99°C for 5 min, terminate the reaction at 4°C, collect the supernatant, and obtain the P. pentosus nucleic acid template solution.

[0099] 2, perform LAMP.

[0100] Three primer sets are used for LAMP reaction, and the reaction system (12.5 μL) of loop-mediated isothermal amplification is: Isothermal Master Mix 7.5 μL, primer set 3.5 μL, template solution 1.5 μL. In 3.5 μL primer set, the content of each primer is: outer primer F3 (P1-F3, P2-F3 or P3-F3 in Table 1) 2.5 pmol, B3 (P1-B3, P2-B3 or P3-B3 in Table 1) 2.5 pmol, inner primer FIP (P1-FIP, P2-FIP or P3-FIP in Table 1) 20 pmol, BIP (P1-BIP, P2-BIP or P3-BIP in Table 1) 20 pmol, loop primer LF (P1-LF, P2-LF or P3-LF in Table 1) 10 pmol, primer LB (P1-LB, P2-LB or P3-LB in Table 1) 10 pmol.

[0101] Reaction condition of loop-mediated isothermal amplification: 65℃, 30 min. The reaction was performed in Genie II real-time fluorescence detector.

[0102] Each primer group was set with sterile double distilled water instead of template solution as negative control.

[0103] The results of fluorescence amplification curve of three primer groups LAMP reaction were shown in Figure 4 No matter the amplification time or fluorescence value, P1 primer group was superior to other primer groups. The annealing temperature of P1 primer group was 86℃ (see Figure 5 ).

[0104] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In general, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the present application. Some basic features can be applied according to the scope of the following attached claims. SEQUENCE LISTING <110> Beijing Yanjing Beer Co., Ltd., China National Light Industry Inspection and Certification Co., Ltd. <120> Specialized primers and kits for identifying Pediococcus pentosaceus <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> DNA <213> Artificial sequence <400> 1 cgcagcttga gaaacatca 19 <210> 2 <211> 18 <212> DNA <213> Artificial sequence <400> 2 ggacgcactt tagcttgt 18 <210> 3 <211> 47 <212> DNA <213> Artificial sequence <400> 3 atgctcacaa agttgaaatc gaatttgttt gctccatagt tgtactc 47 <210> 4 <211> 40 <212> DNA <213> Artificial sequence <400> 4 agtctggacg gtagctacct ttagtggtgt gaccttagcc 40 <210> 5 <211> 22 <212> DNA <213> Artificial sequence <400> 5 cgtaactccc gtaaggttct gg 22 <210> 6 <211> 25 <212> DNA <213> Artificial sequence <400> 6 cactttagta gcgttaattc cagca 25

Claims

1. Primer set, consisting of primers named F3, B3, FIP, BIP, LF and LB; The F3 is the single-stranded DNA molecule shown in Sequence 1 of the sequence listing; B3 is the single-stranded DNA molecule shown in sequence 2 of the sequence listing; The FIP is a single-stranded DNA molecule as shown in sequence 3 of the sequence listing; The BIP is a single-stranded DNA molecule as shown in sequence 4 of the sequence listing; The LF is the single-stranded DNA molecule shown in sequence 5 of the sequence listing; The LB is the single-stranded DNA molecule shown in Sequence 6 of the sequence listing.

2. Any of the following applications of the primer set of claim 1: X1) Prepare reagents or kits for the identification or auxiliary identification of Pediococcus pentosacchari; X2) Prepare reagents or kits for detecting or assisting in the detection of whether Pediococcus pentosaccharis in the sample to be tested; X3) Identification or auxiliary identification of Pediococcus pentosaccharis; X4) Detection or auxiliary detection of whether Pediococcus pentosaccharis is present in the sample to be tested.

3. A reagent comprising the primer set of claim 1.

4. The reagent according to claim 3, characterized in that: The reagents also include other reagents required for the LAMP reaction.

5. The use of the reagent described in claim 3 or 4 in the identification or auxiliary identification of Pediococcus pentosaceus.

6. The use of the reagent according to claim 3 or 4 in detecting or assisting in the detection of whether a sample contains Pediococcus pentosaceus.

7. A kit containing the primer set of claim 1 or the reagent of claim 3 or 4, the kit being used for the following (b1) or (b2): (b1) to identify or assist in the identification of Pediococcus pentosacchari; (b2) to detect or assist in the detection of whether Pediococcus pentosacchari is present in a sample to be tested.

8. The use of the kit according to claim 7 in the identification or auxiliary identification of Pediococcus pentosaceus.

9. The use of the kit according to claim 7 in detecting or assisting in the detection of whether a sample contains Pediococcus pentosaceus.

10. A method for identifying or assisting in the identification of Pediococcus pentosaceus, comprising the following steps: (1) Extract nucleic acid from the microorganism to be tested; (2) Using the nucleic acid extracted in step (1) as a template, loop-mediated isothermal amplification is performed using the primer set described in claim 1; if the primer set can achieve specific amplification using the extracted nucleic acid as a template, the microorganism to be tested is or is a candidate for Pediococcus pentosaccharis; if the primer set cannot achieve specific amplification using the extracted nucleic acid as a template, the microorganism to be tested is or is a candidate for Pediococcus non-Pediococcus pentosaccharis.

11. A method for detecting whether a sample contains Pediococcus pentosaceus, comprising the following steps: (1) Extract nucleic acid from the sample to be tested; (2) Using the nucleic acid extracted in step (1) as a template, loop-mediated isothermal amplification is performed using the primer set described in claim 1; if the primer set can achieve specific amplification using the extracted nucleic acid as a template, the sample to be tested contains or is a candidate to contain Pediococcus pentosaccharis; if the primer set cannot achieve specific amplification using the extracted nucleic acid as a template, the sample to be tested does not contain or is a candidate not to contain Pediococcus pentosaccharis.

12. The method according to claim 11, characterized in that: The sample to be tested was a beer sample.

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