Method, primer pair and kit for detecting bacillus cereus

Abstract

The invention discloses a method, a primer pair and a kit for detecting bacillus cereus, 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: (1) extracting genome DNA (deoxyribonucleic acid) of a sample to be detected as a template, and performing PCR (polymerase chain reaction) amplification by taking primer pairs with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2 as primers; and (2) detecting whether a single amplification product exists at the 231 bp position in the PCR amplification product. 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 cereus in food.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection, and relates to a method, primer pairs, and reagent kit for detecting Bacillus cereus. Background Technology

[0002] Bacillus cereus is a Gram-positive bacterium. Colonies are short rod-shaped or slightly curved, approximately 1–1.5 µm × 3–5 µm in size, with smooth ends, and are often arranged in short or long chains. Some strains possess peritrichous flagella and are motile. Under stress, this bacterium can form spores, which are round or oval, usually located in the center of the cell or slightly off-center, and do not protrude from the cell body. The optimal growth temperature range is 25°C–37°C; the optimal growth pH is 6.0–9.0; this bacterium has some salt tolerance, tolerating 7.5% NaCl; it is facultatively aerobic (growing rapidly under aerobic conditions, and also capable of fermentation under anaerobic conditions). This bacterium can ferment polysaccharide compounds such as glucose, sucrose, maltose, starch, and sucrose. During fermentation, it also produces various enzymes (catalase, lecithinase, etc.), and some pathogenic strains can exhibit weak hemolysis on blood agar. This bacterium is commonly found in soil and the environment, and can also be found in many foods, such as rice, cheese, and canned goods. While some strains are used in industrial fermentation, others are also among the common pathogens in food. Food poisoning caused by Bacillus cereus is usually associated with its toxins, including heat-resistant exotoxins and acid-resistant toxins. Symptoms of food poisoning are usually vomiting and diarrhea, but in some cases can be more severe and even life-threatening. Due to its potential food safety risks, the detection and regulation of Bacillus cereus has received considerable attention. Current research trends focus on developing faster, more accurate, and more efficient detection methods to improve food safety levels. Traditional detection methods are time-consuming and labor-intensive, typically requiring 3–5 days for confirmation. Furthermore, traditional detection methods mainly refer to the requirements of the national standard GB / T 26427-2010 (Detection of Bacillus cereus in Feed), requiring a sample volume of at least 25 g or 25 mL. Therefore, the sensitivity of the detection is usually >4 CFU / g or >4 CFU / mL. As can be seen from the above, traditional methods for detecting Bacillus cereus in food have low detection efficiency and high detection costs. Summary of the Invention

[0003] The purpose of this invention is to provide a method, primer pairs, and reagent kit for detecting Bacillus cereus. This invention provides a detection method that is fast, low-cost, highly sensitive, and yields accurate and reliable results with good specificity.

[0004] In a first aspect, the present invention provides a primer pair for specifically amplifying Bacillus cereus, the primer pair comprising primer essB-L with nucleotide sequence as shown in SEQ ID NO.1 and primer essB-R with nucleotide sequence as shown in SEQ ID NO.2.

[0005] The sequences amplified by primers essB-L and essB-R described in this invention are the essB gene, which is a gene encoding a secretion system VII protein.

[0006] Secondly, the present invention provides a kit for specifically amplifying Bacillus cereus based on primers used for the specific amplification of Bacillus cereus. The target strain of the kit is Bacillus cereus.

[0007] The kit includes primer essB-L with the nucleotide sequence shown in SEQ ID NO.1 and primer essB-R with the nucleotide sequence shown in SEQ ID NO.2.

[0008] Furthermore, the kit also includes dNTPs, PCR buffer, and Mg. 2+ Taq DNA polymerase and ddH2O.

[0009] Furthermore, the kit also includes a genomic DNA extraction reagent.

[0010] Thirdly, the present invention provides a method for detecting Bacillus cereus based on the primer pair that specifically amplifies Bacillus cereus.

[0011] The method for detecting Bacillus cereus includes the following steps: Step (1): Extract genomic DNA from the sample to be tested. Using the extracted genomic DNA as a template, and essB-L (nucleotide sequence shown in SEQ ID NO.1) and essB-R (nucleotide sequence shown in SEQ ID NO.2) as primers, perform PCR amplification. Step (2) Detect whether there is a single amplification product at the 231 bp position in the PCR amplification product of step (1). If there is a single amplification product at the 231 bp position, it means that the sample to be tested contains Bacillus cereus. If there is no single amplification product at the 231 bp position, the sample to be tested does not contain Bacillus cereus.

[0012] Furthermore, in step (1), the sample to be tested is a conventional sample to be tested in the art, preferably a rice or flour product.

[0013] Furthermore, in step (1), the method for extracting genomic DNA is a conventional extraction method in the art; preferably, it is the CTAB method.

[0014] Furthermore, in step (1), the PCR amplification reaction system is a conventional reaction system in the art; preferably, it is 1×PCR reaction buffer, 12.5 mmol / L Mg 2+ The sample contained 0.25 mmol / L dNTPs, 0.2 μM primer essB-L, 0.2 μM primer essB-R, Taq enzyme 0.04 U / μL, and genomic DNA concentration of 25.6 ng / μL.

[0015] Furthermore, in step (1), the PCR amplification reaction procedure is a conventional reaction procedure in the art; preferably, it is a pre-denaturation at 94 °C for 5 min, followed by the following cycle, with each cycle consisting of: denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s; a total of 30 cycles; after the cycle, extension at 72 °C for 10 min, cooling to 12 °C, and then ending.

[0016] Furthermore, in step (2), the detection method is a conventional detection method in the art, which can be achieved by observing the amplification product, such as gel electrophoresis detection; preferably, it is 1.5%-2.0% agarose gel electrophoresis detection; most preferably, it is 2.0% agarose gel electrophoresis detection.

[0017] Beneficial effects: The detection method of this invention for Bacillus cereus can detect bacteria in as little as 24 hours, improving detection efficiency; it is simple and easy to implement, with low detection cost; the detection results are reliable, and the result interpretation is simple, allowing for specific determination of whether Bacillus cereus is present. This invention provides a simple, rapid, and sensitive method for detecting Bacillus cereus in food testing, and it has significant application value in determining whether rice and flour products contain Bacillus cereus. Attached Figure Description

[0018] Figure 1 This is a chromatogram of the primer results verified by 2.0% agarose gel electrophoresis of the PCR products in Example 1. Lanes 1-7 are, in order, Bacillus cereus CICC 21252, Bacillus cereus M3475, Bacillus cereus M3476, Bacillus cereus M3477, Bacillus cereus M15364, Bacillus cereus M15365, and Bacillus cereus M15366. N is ddH2O (negative control sample); M is DL2000 bp DNA Marker.

[0019] Figure 2The image shows the primer specificity verification experiment performed by 2.0% agarose gel electrophoresis on the PCR products in Example 2. Lanes 1-20 are, in order: Bacillus cereus CICC 21252, Bacillus cereus M3475, Bacillus cereus M15364, Staphylococcus aureus M3223, Listeria monocytogenes M3261, Enterococcus faecalis ATCC 8459, Salmonella ATCC 14028, Cronobacter sakazakii ATCC 29544, Bifidobacterium lactis HN019, Bacillus licheniformis M2132, Lactobacillus plantarum CICC 6240, Bacillus thermophilus M3304, Bacillus subtilis ATCC 6633, Bacillus thuringiensis CICC 21708, Lactobacillus acidophilus CICC 6075, Lactobacillus curvaturei M3001, Lactobacillus fructose-eating M0462, Lactobacillus brunelli M2214, Bacillus coagulans CICC 25162, and Bacillus coagulans CICC 21736. N represents ddH2O (negative control sample); M represents DL2000 bp DNA Marker.

[0020] Figure 3 This is the primer sensitivity verification diagram obtained by 2.0% agarose gel electrophoresis of the PCR products in Example 3. Lanes 1-8 are 2.56 fg / μL, 25.6 fg / μL, 256 fg / μL, 2.56 pg / μL, 25.6 pg / μL, 256 pg / μL, 2.56 ng / μL, and 25.6 ng / μL, respectively; M is the DL2000 bp DNA Marker.

[0021] Figure 4 The images show the PCR products from five actual samples in Example 4, analyzed by PCR and then electrophoretically analyzed on a 2.0% agarose gel. Lanes 1-10 are, in order: Rice Sample 1, Rice Sample 2, Rice Sample 3, Rice Sample 4, Rice Sample 5, Rice Sample 5; N is ddH2O (negative control); and M is a 2000bp DNA Marker. Detailed Implementation

[0022] The technical solution of the present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0023] The standard strains used in the embodiments of this invention were purchased from the China Industrial Microbial Culture Collection Center, and the isolated strains were deposited in the Food Science Center Laboratory of bioMérieux Detection Technologies (Shanghai) Co., Ltd.; the culture media were purchased from Beijing Luqiao Technology Co., Ltd.; and the reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0024] Example 1: Primer synthesis and PCR detection for Bacillus cereus I. Primer Synthesis Primers capable of PCR amplification of conserved sequences in the gene sequence encoding the secretion system VII protein of Bacillus cereus were synthesized (by Shanghai Sangon Biotech Co., Ltd.). The primer sequences are as follows: essB-L: 5'-CCTCCAGCTTATTATGTACGGCCT-3' (SEQ ID NO: 1); essB-R: 5'-TTGCTCCATCCGTGTTCACT-3' (SEQ ID NO: 2).

[0025] II. PCR Detection Using the primers described above, and with the genomic DNA of the Bacillus cereus standard strain CICC 21252 as a template, the PCR reaction system and procedure were established and optimized. It was found that the following reaction system and procedure could produce a single amplification product of about 231 bp.

[0026] Obtaining the genomic DNA of the Bacillus cereus standard strain CICC 21252 includes the steps of collecting bacterial cells and extracting genomic DNA using the CTAB method, as detailed below: 1. Collection of bacterial cells: Bacillus cereus CICC 21252 was inoculated into 5 mL of TSB liquid medium and enriched at 37 ℃ for 2 h. 1 mL of the bacterial suspension was then transferred to a 1.5 mL centrifuge tube and centrifuged at 10000 r / min for 10 min to collect the bacterial cells. The cells were resuspended in sterile double-distilled water, washed by centrifugation, and then 460 μL of sterile TE buffer (10 mM) was added to obtain a bacterial suspension.

[0027] 2. Genomic DNA extraction using the CTAB method: (1) Add 24 μL of lysozyme and incubate in a water bath or metal bath at 37 ℃ for 1-2 h; then add 53 μL of 10% SDS (sodium dodecyl sulfate) and incubate at 68 ℃ for 15 min; then add 87 μL of 5 M NaCl and 69 μL of 1% CTAB (hexadecyltrimethylammonium bromide) and incubate at 68 ℃ for 15 min. (2) Add 250 μL of phenol and 250 μL of chloroform-isoamyl alcohol (24:1, v / v), let stand, and centrifuge at 12,000 r / min for 10 min. (3) Take 400-500 μL of supernatant, add an equal volume (400-500 μL) of chloroform-isoamyl alcohol (24:1, v / v), centrifuge at 12,000 r / min for 5 min, take 300 μL of supernatant, add 600 μL of anhydrous ethanol, and place at -20 ℃ for 30 min-120 min. (4) Centrifuge at 10,000 r / min for 2 min, discard the supernatant, wash with 70% ethanol, centrifuge at 12,000 r / min for 2 min, discard the supernatant and air dry at room temperature. Add 50-100 μL of sterile deionized water or sterile TE (10 mM) solution to dissolve the DNA, store at -20 ℃ for later use, and it can be used as a template for subsequent PCR reaction systems.

[0028] The PCR reaction system consisted of: 1×PCR reaction buffer, 10⁻¹⁵ mmol / L Mg 2+ 0.2-0.3 mmol / L dNTPs, 0.1-0.3 μM primer essB-L, 0.1-0.3 μM primer essB-R, Taq enzyme 0.01-0.1 U / μL, DNA template 10-100 ng / μL.

[0029] The PCR amplification program is as follows: pre-denaturation at 92-95 ℃ for 3-6 min, followed by the following cycles, each cycle consisting of: denaturation at 92-95 ℃ for 20-40 s, annealing at 58-63 ℃ for 20-40 s, and extension at 68-74 ℃ for 20-40 s; for a total of 30-35 cycles; after each cycle, extension at 70-74 ℃ for 8-10 min, followed by cooling to 4-15 ℃ to terminate the process.

[0030] Preliminary experiments revealed that the PCR reaction system with the highest yield of amplified products and the clearest electrophoretic bands when the annealing temperature was 55-60℃ was: 1×PCR reaction buffer, 12.5 mmol / L MgSO4. 2+ The PCR amplification program consisted of 0.25 mmol / L dNTPs, 0.2 μM primer essB-L, 0.2 μM primer essB-R, 0.04 U / μL Taq enzyme, and 25.6 ng / μL DNA template. The highest yield and clearest electrophoretic bands were observed in amplified products around 231 bp. The PCR amplification program was as follows: pre-denaturation at 94 ℃ for 5 min, followed by the following cycles: 94 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s, and 72 ℃ extension for 30 s; a total of 30 cycles were performed. After each cycle, the product was extended at 72 ℃ for 10 min, then cooled to 12 ℃ to terminate the amplification.

[0031] PCR amplification products were detected by agarose gel electrophoresis. The electrophoresis results are shown in [Figure number missing]. Figure 1 ,Figure 1 Lanes 1-7 in the middle swimming pool contain Bacillus cereus CICC 21252, Bacillus cereus M3475, Bacillus cereus M3476, Bacillus cereus M3477, Bacillus cereus M15364, Bacillus cereus M15365, Bacillus cereus M15366, N is ddH2O (negative control sample), and M is DL2000 bp DNA Marker.

[0032] It can be seen that the PCR amplification products of these seven strains, namely Bacillus cereus CICC 21252, Bacillus cereus M3475, Bacillus cereus M3476, Bacillus cereus M3477, Bacillus cereus M15364, Bacillus cereus M15365, and Bacillus cereus M15366, all showed a single amplified band at the 231 bp position.

[0033] Example 2: Specificity evaluation for detecting Bacillus cereus (1) Obtaining the genomic DNA template of the strain The standard strain CICC 21252 of Bacillus cereus, Bacillus cereus M3475, Bacillus cereus M15364 and 17 other negative control strains (as shown in Table 1) were collected according to the steps in Example 1. Genomic DNA was extracted by CTAB method and used as PCR template for detecting Bacillus cereus.

[0034] (2) PCR detection to determine if it is Bacillus cereus Take 2 μL of the DNA solution (DNA concentration 11.6 ng / μL) of each strain obtained in step (1) as a PCR reaction template and add it to the PCR reaction system for amplification.

[0035] The PCR reaction system consisted of: 1×PCR reaction buffer, 12.5 mmol / L Mg... 2+ The reaction mixture consisted of 0.25 mmol / L dNTPs, 0.2 μM primer essB-L, 0.2 μM primer essB-R, 0.04 U / μL Taq enzyme, and 11.6 ng / μL DNA template, with sterile deionized water used as a negative control.

[0036] The PCR reaction program was as follows: pre-denaturation at 94 ℃ for 5 min, followed by the following cycles. Each cycle consisted of denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 30 s, extension at 72 ℃ for 30 s, for a total of 30 cycles. After each cycle, the extension was performed at 72 ℃ for 10 min, followed by cooling to 12 ℃ to finish the reaction.

[0037] The PCR amplification products were detected by agarose gel electrophoresis to determine whether a single amplified band was present at the 231 bp position. If a single amplified band was present at the 231 bp position, it was marked as "+", indicating that it was identified as *Bacillus cereus*; otherwise, it was marked as "-". The results are shown in Table 1, and the electrophoresis results are as follows: Figure 2 .

[0038] Table 1. Strains used for specificity evaluation and test results Strain name No. Bacterial count Result Bacillus cereus CICC 21252 1 + Bacillus cereus M3475 1 + Bacillus cereus M15364 1 + Staphylococcus aureus M3223 1 - Listeria monocytogenes M3261 1 - Enterococcus faecalis ATCC 8459 1 - Salmonella ATCC 14028 1 - Cronobacter sakazakii ATCC 29544 1 - Bifidobacterium lactis HN019 1 - Bacillus licheniformis M2132 1 - Lactobacillus plantarum CICC 6240 1 - Bacillus caldolyticus M3304 1 - Bacillus subtilis ATCC 6633 1 - Bacillus thuringiensis CICC 21708 1 - Lactobacillus acidophilus CICC 6075 1 - Lactobacillus curvatus M3001 1 - Lactobacillus fructivorans M0462 1 - Lactobacillus buchneri M2214 1 - Bacillus coagulans CICC 25162 1· - Bacillus coagulans CICC 21736 1 - Figure 2 This is the chromatogram of primers used to verify the specificity of the PCR products in Example 2 via 2.0% agarose gel electrophoresis. Lanes 1-20 are, in order: Bacillus cereus CICC 21252, Bacillus cereus M3475, Bacillus cereus M15364, Staphylococcus aureus M3223, Listeria monocytogenes M3261, Enterococcus faecalis ATCC 8459, Salmonella ATCC 14028, Cronobacter sakazakii ATCC 29544, Bifidobacterium lactis HN019, Bacillus licheniformis M2132, Lactobacillus plantarum CICC 6240, and heat-consuming amyloliquefaciens. Bacillus M3304, Bacillus subtilis ATCC6633, Bacillus thuringiensis CICC21708, Lactobacillus acidophilus CICC6075, Lactobacillus curvaturei M3001, Lactobacillus fructosemide M0462, Lactobacillus brunelli M2214, Bacillus coagulans CICC25162, Bacillus coagulans CICC21736, N is ddH2O (negative control sample); M is DL2000 bp DNA Marker.

[0039] As shown in Table 1, apart from one Bacillus cereus standard strain and two Bacillus cereus isolates, the remaining 17 negative control strains did not exhibit specific amplification bands (231 bp). The one Bacillus cereus standard strain and two Bacillus cereus isolates in Table 1 represent typical strains of all species within this genus. Bacillus thuringiensis and Bacillus subtilis, which are closely related to Bacillus cereus, were also used as negative control strains. In addition, 13 other isolates were used as negative controls in this experiment. If these strains fail to amplify the specific fragment (231bp) using the primers essB-L and essB-R of this invention via PCR, then other strains more distantly related to Bacillus cereus will find it even more difficult to amplify this fragment. Therefore, the verification by these closely related strains ensures the specificity of the primers essB-L and essB-R described in Example 2, and also fully demonstrates that this method can amplify any strain within the Bacillus cereus species, without amplifying any strains other than those at the Bacillus cereus species level.

[0040] As can be seen from the above examples, the strain to be tested is enriched in TSB medium for no more than 18 hours, the genomic DNA is extracted by CTAB method for about 2 hours, and the PCR detection of whether it is Bacillus cereus takes about 2 hours. Therefore, the method for detecting Bacillus cereus of the present invention takes about 24 hours in total, which is significantly shorter than the traditional method and improves the detection efficiency.

[0041] Example 3: Sensitivity evaluation of the method for detecting Bacillus cereus Genomic DNA was extracted from Bacillus cereus BD452 using the CTAB method described in Example 1. The obtained DNA was dissolved in sterile water to a concentration of 256 ng / μL, and then serially diluted 10-fold with sterile water to a total of eight gradients: 2.56 fg / μL, 25.6 fg / μL, 256 fg / μL, 2.56 pg / μL, 25.6 pg / μL, 256 pg / μL, 2.56 ng / μL, and 25.6 ng / μL. 2 μL of each gradient was used as template and added to the PCR reaction system. Amplification was performed using essB-L and essB-R primers according to the PCR reaction system and procedure described in Example 2. The amplification products were detected by gel electrophoresis, and the gel electrophoresis results were observed in a gel imaging system. Figure 3 As shown.

[0042] Figure 3 Lanes 1-8 of the middle swimming pool contained 5.12 fg / PCR, 51.2 fg / PCR, 512 fg / PCR, 5.12 pg / PCR, 51.2 pg / PCR, 512 pg / PCR, 5.12 ng / PCR, and 51.2 ng / PCR, respectively; M was a DL 2000 bp DNA Marker, and N was ddH2O (negative control sample); Figure 3 As can be seen, a clear band (231 bp) was observed in lane 5, corresponding to a DNA concentration of 51.2 pg / PCR. Therefore, the PCR detection sensitivity was determined to be 51.2 pg / PCR, indicating high sensitivity.

[0043] Example 4: Detection of Bacillus cereus in food samples Five rice samples were prepared, and 25 g of each sample was diluted in 225 mL of sterile physiological saline. Bacillus cereus was isolated and identified according to the recommended national standard method GB / T 26427-2010 (Table 2). Table 2 shows the physiological and biochemical identification results of the isolated Bacillus cereus positive strains, and the final identification results were compared with those of the PCR method.

[0044] Table 2. Detection results of Bacillus cereus in rice samples

[0045] Simultaneously, genomic DNA was extracted from 1 g of sample using the CTAB method described in Example 1, and the total genomic DNA was diluted to 20 ng / uL as a PCR template. Sterile water was used as a negative control. Amplification was performed according to the PCR reaction system and procedure described in Example 1. Each sample was repeated twice. The results are shown in […]. Figure 4 .

[0046] Figure 4 The images show the PCR products from five actual samples in Example 4, analyzed by PCR and then electrophoretically analyzed on a 2.0% agarose gel. Lanes 1-10 are, in order: Rice Sample 1, Rice Sample 1, Rice Sample 2, Rice Sample 2, Rice Sample 3, Rice Sample 3, Rice Sample 4, Rice Sample 4, Rice Sample 5, Rice Sample 5. N represents ddH2O (negative control), and M represents a 2000 bp DNA Marker.

[0047] like Figure 4 As shown, four samples (lanes 3-10) were found to contain a specific fragment (231 bp), and this sample was also isolated and identified as Bacillus cereus by the national standard method. Its physiological and biochemical characteristics are consistent with Table 2. This shows that the method of the present invention has very high reliability.

[0048] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention.

Claims

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

2.

2. A kit for specific amplification of Bacillus cereus prepared based on the primer pair described in claim 1, characterized in that, The kit contains the primer pair as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also includes dNTPs, PCR buffer, and Mg. 2+ Taq DNA polymerase and 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 cereus based on the primer pair described in claim 1.

6. The method according to claim 5, 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 and the primer pair described in claim 1 as primers to perform PCR amplification; Step (2): Detect whether there is a single amplification product at the 231 bp position in the PCR amplification product. If there is a single amplification product at the 231 bp position, it means that the sample to be tested contains Bacillus cereus. If there is no single amplification product at the 231 bp position, the sample to be tested does not contain Bacillus cereus.

7. The method according to claim 6, characterized in that, In step (1), the sample to be tested is a rice or flour product.

8. The method according to claim 6, characterized in that, In step (1), the genomic DNA is extracted using the CTAB method.

9. The method according to claim 6, characterized in that, In step (1), the PCR amplification reaction system is: 1×PCR reaction buffer, 12.5 mmol / L Mg 2+ 0.25 mmol / L dNTP, 0.2 μM primer essB-L, 0.2 μM primer essB-R, Taq enzyme 0.04 U / μL, the genomic DNA concentration of the sample to be tested was 25.6 ng / μL; In step (1), the PCR amplification reaction procedure is as follows: Pre-denaturation at 94 ℃ for 5 min, followed by the following cycle, with 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; after each cycle, extension at 72 ℃ for 10 min, followed by cooling to 12 ℃, and then the cycle ends.

10. The method according to claim 6, characterized in that, In step (2), the method for detecting whether a single amplification product exists at the 231 bp position in the PCR amplification product is 1.5%-2.0% agarose gel electrophoresis.

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