Method, primer pair and kit for detecting bacillus coagulans strain

Abstract

The invention discloses a method, a primer pair and a kit for detecting a bacillus coagulans strain, and belongs to the field of microbiological detection. The nucleotide sequences of the primer pair are respectively as shown in SEQ ID NO. 1 and SEQ ID NO. 2. The detection method comprises the following steps: extracting genome DNA of a sample to be detected as a template, and performing PCR amplification by taking primer pairs with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2 as primers; and detecting whether a single amplification product exists at the 593 bp position in the PCR amplification product or not. The detection method disclosed by the invention is short in detection time, low in detection cost and reliable in detection result, and can be applied to detection of bacillus coagulans in food.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganism detection, and relates to a method for detecting Bacillus coagulans strains, a primer pair and a kit. BACKGROUND

[0002] Bacillus coagulans is a common gram-positive bacterium, and its cell morphology is long rod-shaped, and it can form chain arrangement or cluster aggregation on culture medium. The colony on solid culture medium is white, round, and slightly raised. For growth characteristics, the bacterium is facultative anaerobic, and the optimum growth temperature range is 35-50 DEG C, and the optimum pH value is 5.5-7.0. It can ferment glucose, sucrose, maltose and the like to produce L(+) -lactic acid, and does not produce gas; meanwhile, it secretes extracellular enzymes such as amylase, protease, lipase and cellulase. It has strong spore formation ability, can survive in extreme environment, and has a wide adaptation range to temperature and pH value, and generally grows better at a temperature below 50 DEG C and under neutral to alkaline conditions. The spore has strong stress resistance: survival rate > 96 % after 100 DEG C for 10 min; and activity is not lost after 90 DEG C water bath for 10 min. In the field of food, Bacillus coagulans is also one of the important causes of food poisoning. It exists in various foods such as rice, noodles, vegetables and meat products, and thus can cause foodborne disease outbreak. Once Bacillus coagulans contaminates food, it is likely to cause food poisoning events, especially when the food lacks proper processing (such as heat sterilization process) and improper storage conditions. The toxin produced thereby can cause acute gastrointestinal symptoms such as diarrhea, abdominal pain and vomiting, etc., which has brought great trouble to people's life and health. At present, the detection method for Bacillus coagulans mainly refers to GB4789.14-2014 'National Food Safety Standard Food Microbiological Examination Bacillus cereus' and GB / T26428-2010 'National Food Safety Standard Detection of Bacillus subtilis in feed microbial preparation'. However, the two traditional detection methods are time-consuming, cumbersome and require high technical requirements for operators. It usually takes 3-5 days to get the result. As can be seen from the above, the traditional detection method for Bacillus coagulans in food has low detection efficiency and high detection cost. SUMMARY

[0003] The present application aims to provide a method for detecting Bacillus coagulans strains, a primer pair and a kit. The method has fast detection speed, low cost, high sensitivity, good specificity and accurate and reliable results.

[0004] In a first aspect, the present application provides a primer pair for specifically amplifying Bacillus coagulans strains, wherein the primer pair comprises a primer gyrB-L with a nucleotide sequence as shown in SEQ ID NO. 1 and a primer gyrB-R with a nucleotide sequence as shown in SEQ ID NO. 2.

[0005] The sequence amplified by the primer pair is a gyrB gene, which is a gene encoding a DNA gyrase B subunit.

[0006] In a second aspect, the present application provides a kit for specifically amplifying Bacillus coagulans strains, wherein the kit comprises the primer pair for specifically amplifying Bacillus coagulans strains.

[0007] The target amplification bacteria of the kit are Bacillus coagulans.

[0008] The kit for specifically amplifying Bacillus coagulans strains comprises a primer gyrB-L with a nucleotide sequence as shown in SEQ ID NO. 1 and a primer gyrB-R with a nucleotide sequence as shown in SEQ ID NO. 2.

[0009] Further, the kit further comprises dNTP, PCR buffer, Mg 2+ , Taq DNA polymerase and ddH2O.

[0010] Further, the kit further comprises a genomic DNA extraction reagent.

[0011] In a third aspect, the present application provides a method for detecting Bacillus coagulans strains based on the primer pair, comprising the following steps:

[0012] Step (1) extracting genomic DNA of a sample to be detected, using the extracted genomic DNA as a template, using a primer gyrB-L with a nucleotide sequence as shown in SEQ ID NO. 1 and a primer gyrB-R with a nucleotide sequence as shown in SEQ ID NO. 2, and performing PCR amplification;

[0013] Step (2) detecting whether a single amplification product exists at a position of 593 bp in the PCR amplification product of step (1), if a single amplification product exists at the position of 593 bp, it indicates that the sample to be detected contains Bacillus coagulans strains, and if a single amplification product does not exist at the position of 593 bp, it indicates that the sample to be detected does not contain Bacillus coagulans.

[0014] Further, in step (1), the sample to be detected is a conventional sample to be detected in the art, preferably food and feed.

[0015] Further, in step (1), the extraction method of genomic DNA is a conventional extraction method in the art; preferably, it is CTAB method.

[0016] Further, in step (1), the reaction system of PCR amplification is a conventional reaction system in the art; preferably, it is 1×PCR reaction buffer, 12.5 mmol / L Mg 2+ , 0.25 mmol / L dNTP, 0.2 μM primer gyrB-L, 0.2 μM primer gyrB-R, Taq enzyme 0.04 U / μL, and 13.18 ng / μL of genomic DNA of the sample to be detected.

[0017] Further, in step (1), the reaction procedure of PCR amplification is a conventional reaction procedure in the art; preferably, it is 5 min of pre-denaturation at 94℃, and then the following cycles, each cycle having the procedure of 30 s of denaturation at 94℃, 30 s of annealing at 60℃, and 30 s of extension at 72℃; a total of 30 cycles; after the cycles, 10 min of extension at 72℃, cooling to 12℃, and ending.

[0018] Further, in step (2), the detection method is a conventional detection method in the art, and the amplification product can be observed, for example, gel electrophoresis detection; preferably, it is 1.5%-2.0% agarose gel electrophoresis detection; most preferably, it is 2.0% agarose gel electrophoresis detection.

[0019] Beneficial effects: the detection method of the present application detects Bacillus coagulans, and the detection time is only 24 hours, which improves the detection efficiency; it is simple and easy to operate, and has low detection cost; the detection result is reliable, the result determination is simple, and whether it is Bacillus coagulans can be determined specifically. The present application provides a simple, rapid and sensitive method for detecting Bacillus coagulans, and has great application value for judging whether food or feed contains Bacillus coagulans. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 2 is a result map of primer verification by 2.0% agarose gel electrophoresis experiment on the PCR product in Example 1. Lanes 1-7 are Bacillus coagulans CICC25162, Bacillus coagulans CICC21736, Bacillus coagulans MFSC1626, Bacillus coagulans MFSC1947, Bacillus coagulans MFSC1948, Bacillus coagulans MFSC2020, Bacillus coagulans MFSC2021, N: ddH2O (negative control sample); M is DL2000 bp DNA Marker.

[0021] Figure 2The electrophoretogram of the primer specificity experiment verified by 2.0% agarose gel electrophoresis of the PCR product in Example 2 is shown in the figure. Lanes 1-19 are as follows: S. aureus M3223, L. monocytogenes M3261, E. faecalis ATCC8459, Salmonella ATCC14028, C. sakazakii ATCC29544, B. lactis HN019, B. licheniformis M2132, L. plantarum CICC6240, G. thermoleucosporus M3304, B. subtilis ATCC6633, B. thuringiensis CICC21708, L. acidophilus CICC6075, L. curvatus M3001, L. fructivorans M0462, L. bulgaricus M2214, B. coagulans CICC25162, B. coagulans CICC21736, B. coagulans MFSC1626, B. coagulans MFSC1947, N: ddH2O (negative control sample); M is DL2000 bp DNA Marker.

[0022] Figure 3 The electrophoretogram of the primer sensitivity experiment verified by 2.0% agarose gel electrophoresis of the PCR product in Example 3 is shown in the figure. Lanes 1-9 are as follows: 263.6 ng / PCR, 26.36 ng / PCR, 2.636 ng / PCR, 263.6 pg / PCR, 26.36 pg / PCR, 2.636 pg / PCR, 263.6 fg / PCR, 26.36 fg / PCR, 2.636 fg / PCR; N: ddH2O (negative control sample); M is DL2000 bp DNA Marker.

[0023] Figure 4 The electrophoretogram of the PCR product after PCR detection of the 8 actual samples in Example 4 is shown in the figure. Lanes 1-8 are as follows: feed sample 1, feed sample 2, feed sample 3, feed sample 4, food sample 1, food sample 2, food sample 3, food sample 4, N: ddH2O (negative control); M is 2000 bp DNA Marker. DETAILED DESCRIPTION

[0024] The present application is further illustrated by the following specific examples and figures, but the present application is not limited in the scope of the examples. The experimental methods not specified in the following examples are selected according to conventional methods and conditions, or according to the instructions of the commercial products. The room temperature refers to 10-30℃.

[0025] The standard strains used in the embodiments of the application are purchased from China Industrial Microbial Culture Collection Center, and the isolated strains are preserved in the Food Science Center Laboratory of Merieux Technologies (Shanghai) Co., Ltd.; the culture medium is purchased from Beijing Land Bridge Technology Co., Ltd.; and the reagent is purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0026] Example 1: Synthesis of primers for detecting Bacillus coagulans and PCR detection

[0027] I. Primer synthesis

[0028] Primers (synthesized by Shanghai Sangon Biological Engineering Technology Service Co., Ltd.) capable of PCR amplifying the conserved sequence in the gene sequence encoding the B subunit of DNA gyrase of Bacillus coagulans are synthesized, and the primer sequences are as follows:

[0029] gryB-L: 5'-AAAAGCACGCGAACTGACAC-3' (SEQ ID NO: 1);

[0030] gryB-R: 5'-TCGTCTCCCAAAGCTGTTCC-3' (SEQ ID NO: 2).

[0031] II. PCR detection

[0032] Using the above primers, the genomic DNA of the standard strains Bacillus coagulans CICC25162 and Bacillus coagulans CICC21736 is used as a template to establish and optimize the PCR reaction system and reaction program, and it is found that the following reaction system and reaction program can obtain a single amplification product of about 593 bp.

[0033] The genomic DNA of Bacillus coagulans CICC25162 and Bacillus coagulans CICC21736 includes the steps of collecting bacterial cells and extracting genomic DNA by CTAB method, and the specific steps are as follows:

[0034] 1. Collecting bacterial cells: Bacillus coagulans CICC25162 and Bacillus coagulans CICC21736 were inoculated into 5 mL of Tryptic Soy Broth (TSB) liquid medium, respectively, and incubated at 37°C for 2 h. Then, 1 mL of bacterial solution was taken and placed in a 1.5 mL centrifuge tube, and then centrifuged at 10000 r / min for 5 min to collect the bacterial cells. The bacterial cells were suspended with sterile double-distilled water, washed by centrifugation, and then 456 μL of sterile TE buffer (10 mM) was added to obtain a bacterial cell suspension.

[0035] 2. Extracting genomic DNA by CTAB method:

[0036] (1) Add 24 μL lysozyme, 37 ℃ water bath or metal bath 1-2 h; then add 53 μL 10% SDS (sodium dodecyl sulfate), 60 ℃ incubation for 15 min; then add 87 μL 5 M NaCl, 69 μL 1% CTAB (cetyltrimethylammonium bromide), 68 ℃ incubation for 15 min;

[0037] (2) Add phenol-chloroform-isoamyl alcohol (25:24:1, v / v / v) 550 μL, stand, 12,000 r / min centrifugation for 5 min;

[0038] (3) Take 300-500 μL supernatant, add equal volume (300-500 μL) chloroform-isoamyl alcohol (24:1, v / v), 12,000 r / min centrifugation for 5 min, take 200-400 μL supernatant, add 400-800 μL anhydrous ethanol, -20 ℃ for 30 min-120 min;

[0039] (4) 10,000 r / min centrifugation for 5 min, discard the supernatant, add 70% ethanol for washing, 12,000 r / min centrifugation for 2 min, discard the supernatant, and dry at room temperature. Add 50-100 μL sterile deionized water or sterile TE (10 mM) solution to dissolve the DNA, and store at -20 ℃ for use as a template for subsequent PCR reaction system.

[0040] The PCR reaction system is: 1×PCR reaction buffer, 10-15 mmol / L Mg 2+ , 0.2-0.3 mmol / L dNTP, 0.1-0.3 μM primer gyrB-L, 0.1-0.3 μM primer gyrB-R, Taq enzyme 0.01-0.1 U / μL, DNA template 10-100 ng / μL.

[0041] The PCR amplification program is: 92-95 ℃ pre-denaturation for 3-6 min, and then start the following cycle, the program of each cycle is: 92-95 ℃ denaturation for 20-40 s, 58-63 ℃ annealing for 20-40 s, 68-74 ℃ extension for 20-40 s; a total of 30-35 cycles; after the cycle, 70-74 ℃ extension for 8-10 min, cooling to 4-15 ℃, and end.

[0042] Through preliminary experiments, it is found that when the annealing temperature is 55-60 ℃, the yield of the amplification product is the highest, and the electrophoresis band is the most obvious and clear, and the PCR reaction system is: 1×PCR reaction buffer, 12.5 mmol / L Mg 2+, 0.25 mmol / L dNTP, 0.2 μM primer gyrB-L, 0.2 μM primer gyrB-R, Taq enzyme 0.04 U / μL, DNA template 13.18 ng / μL. The yield of the amplification product of about 593 bp was the highest, and the PCR amplification procedure was as follows: pre-denaturation at 94 ℃ for 5 min, and then the following cycles were started, each cycle was as follows: denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 30 s, extension at 72 ℃ for 30 s; the cycles were 30 in total; after the cycles, extension at 72 ℃ for 10 min, cooling to 12 ℃, and ending.

[0043] The PCR amplification product was detected by agarose gel electrophoresis, and the electrophoresis result is shown in Figure 1 , Figure 1 The agarose gel electrophoresis experiment of the PCR product in Example 1 verified the primer result map. Lanes 1 to 7 were Bacillus coagulans CICC25162, Bacillus coagulans CICC21736, Bacillus coagulans MFSC1626, Bacillus coagulans MFSC1947, Bacillus coagulans MFSC1948, Bacillus coagulans MFSC2020, Bacillus coagulans MFSC2021, N: ddH2O (negative control sample); M was DL2000 bp DNA Marker.

[0044] From Figure 1 , it can be seen that the PCR amplification products of the standard strains Bacillus coagulans CICC25162 and Bacillus coagulans CICC21736 all exist single amplification bands at the position of 593 bp.

[0045] Example 2: Evaluation of the specificity of detecting Bacillus coagulans

[0046] (1) Acquisition of the genomic DNA template of the strain

[0047] One Bacillus coagulans standard strain CICC25162 and one Bacillus coagulans standard strain CICC21736, one Bacillus coagulans isolated strain MFSC1626 and one Bacillus coagulans isolated strain MFSC1947, and 15 other negative control strains (as shown in Table 1) were selected, the bacterial bodies were collected according to the steps of Example 1, and the genomic DNA was extracted by the CTAB method, which was used as the PCR reaction template for detecting Bacillus coagulans.

[0048] (2) PCR detection of whether it is Bacillus coagulans

[0049] 2 μL of the DNA solution (the DNA concentration was 13.18 ng / μL) of each strain obtained in step (1) was taken as the PCR reaction template, and was added to the PCR reaction system for amplification reaction.

[0050] PCR reaction system: 1 x PCR reaction buffer, 12.5 mmol / L Mg 2+ , 0.25 mmol / L dNTP, 0.2 μM primer gyrB-L, 0.2 μM primer gyrB-R, Taq enzyme 0.04 U / μL, DNA template 13.18 ng / μL, and sterile deionized water as the negative control of the reaction.

[0051] PCR reaction program: 94 ℃ pre-denaturation for 5 min, and then start the following cycle, each cycle program: 94 ℃ denaturation for 30 s, annealing temperature 60 ℃, annealing time 30 s, 72 ℃ extension for 30 s, a total of 30 cycles, after the cycle 72 ℃ extension for 10 min, cooling to 12 ℃, end.

[0052] The PCR amplification product was detected by agarose gel electrophoresis to determine whether a single amplification band existed at the position of 593 bp. If a single amplification band existed at the position of 593 bp, it was marked as “+”, that is, it was judged to be Bacillus coagulans; otherwise, it was marked as “-”. The results are shown in Table 1, and the electrophoresis results are shown in Figure 2 .

[0053] Table 1 Strains used for specificity evaluation and test results

[0054]

[0055] Figure 2 The figure of the primer specificity experiment verified by 2.0% agarose gel electrophoresis of the PCR product in Example 2. Lanes 1-19 are: Staphylococcus aureus M3223, Listeria monocytogenes M3261, Enterococcus faecalis ATCC8459, Salmonella ATCC14028, Cronobacter sakazakii ATCC29544, Bifidobacterium lactis HN019, Bacillus licheniformis M2132, Lactobacillus plantarum CICC6240, Bacillus caldolyticus M3304, Bacillus subtilis ATCC6633, Bacillus thuringiensis CICC21708, Lactobacillus acidophilus CICC6075, Lactobacillus curvatus M3001, Lactobacillus fructivorans M0462, Lactobacillus buccae M2214, Bacillus coagulans CICC25162, Bacillus coagulans CICC21736, Bacillus coagulans MFSC1626, Bacillus coagulans MFSC1947, N: ddH2O (negative control sample); M is DL2000 bp DNA Marker.

[0056] From the results of Table 1, it can be seen that, except for two Bacillus coagulans standard strains and two Bacillus coagulans isolated strains, the rest of the negative control strains have no specific amplification bands (593 bp). In Table 1, one of the Bacillus coagulans standard strains and the Bacillus coagulans isolated strains represents a typical strain of all species within the genus. Bacillus subtilis, Bacillus licheniformis, Bacillus caldolyticus and Bacillus thuringiensis, which are closely related to Bacillus coagulans, are also used as negative control strains. In addition, 11 other isolated strains are used as negative controls in this test. If these strains cannot amplify the specific fragment (593 bp) by PCR using the primer gyrB-L and the primer gyrB-R of the present application, it will be even more difficult to amplify the fragment for other strains that are less closely related to Bacillus coagulans. Therefore, through the verification of these closely related strains, the specificity of the primer gyrB-L and the primer gyrB-R described in Example 2 is ensured, and it is fully proved that the method can amplify any strain within the Bacillus coagulans species, but not any strain outside the Bacillus coagulans species level.

[0057] From the above examples, it can be seen that the test strain is cultured in TSB medium for not more than 16 h, the genomic DNA is extracted by the CTAB method for about 2 h, and the PCR detection of whether it is a Bacillus coagulans strain takes about 3 hours. Therefore, the method for detecting Bacillus coagulans of the present application needs about 24 hours, and the detection time is significantly shortened compared with the traditional method, and the detection efficiency is improved.

[0058] Example 3: Sensitivity evaluation of the method for detecting Bacillus coagulans

[0059] The genomic total DNA of Bacillus coagulans CICC21736 is extracted by the method for extracting genomic DNA described in Example 1. The obtained DNA is dissolved in sterile water, and the original solution concentration is 131.8 ng / uL. Then, sterile water is used for 10-fold gradient dilution, and a total of 8 gradients are diluted: 13.18 ng / uL, 1.318 ng / uL, 131.8 pg / uL, 13.18 pg / uL, 1.318 pg / uL, 131.8 fg / uL, 13.18 fg / uL, 1.318 fg / uL. 2 μL of the original solution concentration and each gradient are taken as a template to add to the PCR reaction system. The PCR reaction system and the PCR reaction program are carried out according to the description in Example 2, and the amplification product is detected by gel electrophoresis. The gel electrophoresis results are observed in the gel imaging instrument, as shown in Figure 2. Figure 3

[0060] Figure 3 ​This is the chromatogram of primer sensitivity verification by 2.0% agarose gel electrophoresis of the PCR products in Example 3. Lanes 1-9 represent 263.6 ng / PCR, 26.36 ng / PCR, 2.636 ng / PCR, 263.6 pg / PCR, 26.36 pg / PCR, 2.636 pg / PCR, 263.6 fg / PCR, 26.36 fg / PCR, and 2.636 fg / PCR, respectively; N: ddH2O (negative control sample); M is a DL2000 bp DNA Marker.

[0061] Depend on Figure 3 As can be seen, a clear band (593 bp) was observed in lane 6, corresponding to a DNA concentration of 2.636 pg / PCR, while no amplification bands were observed in lanes 7 and beyond. Therefore, the PCR detection sensitivity was determined to be 2.636 pg / PCR, indicating high sensitivity.

[0062] Example 4: Detection of Bacillus coagulans in food samples

[0063] Eight food samples (four feed samples and four food samples) were purchased from large supermarkets. 25 g of each sample was diluted in 225 mL of sterile saline, and *Bacillus coagulans* was isolated and identified according to the standard method GB / T 26428-2010. The final identification results were compared with those obtained using the PCR method.

[0064] (1) Take 1 mL of sample and extract genomic DNA using the CTAB method described in Example 1. Dilute the total genomic DNA to 20 ng / µL as a PCR template. Use sterile water as a negative control. Perform amplification according to the PCR reaction system and PCR reaction procedure described in Example 1. Repeat each sample twice. The results are shown in the figure. Figure 4 .like Figure 4 As shown, four samples (feed samples 1-4) tested positive for a specific fragment (593 bp), while the other four samples (food samples 1-4) did not test positive for a specific fragment (593 bp).

[0065] (2) According to the standard method GB / T 26428-2010, feed samples 1-4 were also isolated and identified as Bacillus coagulans by the national standard method, while food samples 1-4 were not identified as Bacillus coagulans.

[0066] Four samples (feed sample 1-feed sample 4) were found to contain a specific fragment (593 bp), and this sample was also isolated and identified as Bacillus coagulans by the national standard method. This shows that the method of the present invention has very high reliability.

[0067] While the application has been described and illustrated with reference to specific preferred embodiments, it is not intended that it be limited to these particulars. Various changes in form and detail can be made without departing from the spirit and scope of the application.

Claims

1. A primer pair for specifically amplifying Bacillus coagulans strains, characterized in that, The primer pair includes primer gyrB-L and primer gyrB-R; The nucleotide sequence of primer gyrB-L is shown in SEQ ID NO.1, and the nucleotide sequence of primer gyrB-R is shown in SEQ ID NO.

2.

2. A kit for specifically amplifying Bacillus coagulans strains, characterized in that, The kit contains primer pairs that specifically amplify Bacillus coagulans strains; The primer pair includes primer gyrB-L and primer gyrB-R; the nucleotide sequence of primer gyrB-L is shown in SEQ ID NO.1, and the nucleotide sequence of primer gyrB-R is shown in SEQ ID NO.

2.

3. The reagent kit according to claim 2, characterized in that, The kit also includes dNTPs, PCR buffer, and Mg. 2 + Taq DNA polymerase, ddH2O.

4. The reagent kit according to claim 2, characterized in that, The kit also includes genomic DNA extraction reagents.

5. A method for detecting Bacillus coagulans strains, characterized in that, Includes the following steps: Step (1): Extract genomic DNA from the sample to be tested, and use the extracted genomic DNA as a template for PCR amplification; the PCR amplification primers are primer gyrB-L with nucleotide sequence as shown in SEQ ID NO.1 and primer gyrB-R with nucleotide sequence as shown in SEQ ID NO.2; Step (2): Detect whether there is a single amplification product at the 593 bp position in the PCR amplification product. If there is a single amplification product at the 593 bp position, it means that the sample to be tested contains Bacillus coagulans strain. If there is no single amplification product at the 593 bp position, the sample to be tested does not contain Bacillus coagulans.

6. The method according to claim 5, characterized in that, In step (1), the sample to be tested is food; the genomic DNA is extracted using the CTAB method.

7. The method according to claim 5, characterized in that, In step (1), the PCR amplification reaction system is: 1×PCR reaction buffer, 12.5 mmol / L Mg 2+ The sample contained 0.25 mmol / L dNTPs, 0.2 μM primer gyrB-L, 0.2 μM primer gyrB-R, 0.04 U / μL Taq enzyme, and 13.18 ng / μL genomic DNA. The PCR amplification reaction program was as follows: pre-denaturation at 94 ℃ for 5 min, followed by the following cycles, each cycle consisting of: denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 30 s, and extension at 72 ℃ for 30 s; a total of 30 cycles were performed; after each cycle, extension at 72 ℃ for 10 min was performed, followed by cooling to 12 ℃ to terminate the reaction.

8. The method according to claim 5, characterized in that, In step (2), the method for detecting whether a single amplification product exists at the 593bp position in the PCR amplification product is 1.5%-2.0% agarose gel electrophoresis.

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