Kit and method for identifying bacillus thuringiensis

Abstract

The invention relates to the technical field of microbiological detection, particularly provides a kit and a method for identifying bacillus thuringiensis, and designs two sets of specific primer probe combinations: a target BT-657 is located in a Bt strain plasmid genome, and a target BT-InhA is located in a Bt strain chromosome immunosuppressive factor A (inhA) gene. A qPCR detection system is established by adopting a TaqMan probe method, and closed tube detection is realized within 2 hours through dual-channel verification. The method is high in specificity (only bacillus thuringiensis is used for amplification, and the Ct value is less than or equal to 30; the method has the advantages of high sensitivity (up to fg level) and high sensitivity (up to fg level), thoroughly solves the problems of false negative risk and gel electrophoresis pollution caused by the fact that traditional PCR depends on plasmid genes (such as Cry toxin genes), and is suitable for the fields of production quality control, environmental microorganism monitoring and clinical pathogen identification of micro-ecological viable bacteria products.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, specifically to a kit and method for identifying Bacillus thuringiensis. Background Technology

[0002] According to the 2025 edition of the Chinese Pharmacopoeia, the bacterial and viral strains used in production should undergo testing for biological characteristics, biochemical characteristics, serological tests, and molecular genetic characteristics. Currently, the main components of live microbial products are obtained through pure bacterial culture, cell collection, and freeze-drying. The bacterial strains used in production include 13 species such as *Bifidobacterium longum* subsp. *infantitidis*, *Enterococcus faecalis*, and *Bacillus cereus*. Among these, *Bacillus cereus* and *Bacillus thuringiensis* both belong to the *Bacillus cereus* group. Traditional biochemical identification methods have low accuracy and long testing times (7-8 days). The 16S rRNA sequences of the two are as homologous as 99%, making it difficult to distinguish between *Bacillus cereus* and *Bacillus thuringiensis* through 16S sequencing. Whole-genome sequencing is expensive and requires advanced technical expertise, making it difficult for ordinary laboratories to perform.

[0003] The genome of Bacillus thuringiensis is a circular DNA structure. In addition to the nucleoid genome, Bacillus thuringiensis strains may also contain other DNA replicons, namely plasmid genomes. Different BT subspecies can synthesize a variety of different insecticidal active proteins such as Cry protein, Cyt protein, Vip protein, and Sip protein. Among them, more than 70 major classes of genes encoding Cry protein have been discovered to date.

[0004] Patent CN118562987A discloses a specific composition and method for rapid detection of Bacillus thuringiensis in food. The primer set improves the accuracy of Bacillus thuringiensis identification and is used to identify Bacillus thuringiensis in food. Patent CN119177303A discloses a specific composition that can be used to detect Bacillus cereus, Bacillus thuringiensis, and Bacillus mycosis fungi in food, distinguishing the foodborne pathogen Bacillus cereus from three highly homologous bacteria. The primer sets and probes for Bacillus thuringiensis in the above two patents are identical. However, NCBI comparison shows that this gene segment only encodes the cry1 insecticidal protein, requiring primers and probes designed based on only one of these insecticidal proteins, which has significant limitations.

[0005] Patent CN113136443A discloses a rapid nucleic acid detection method for identifying Bacillus cereus and Bacillus thuringiensis. This method involves extracting genomic DNA from the sample, using the genomic DNA as a template, and employing primer pairs as amplification primers. A high-resolution melting curve method is used to distinguish between Bacillus cereus and Bacillus thuringiensis by observing their different characteristic melting curves. However, this high-resolution melting curve detection method requires a high degree of standardization and precision in experimental procedures. Any changes in the PCR reaction system, such as primer concentration, magnesium ion concentration, buffer composition, or even the quality and purity of the template DNA, can significantly affect the shape and Tm value of the melting curve, interfering with the identification of Bacillus cereus and Bacillus thuringiensis, and leading to false positives, false negatives, or complicated result interpretation.

[0006] Therefore, how to accurately, easily, and quickly identify Bacillus thuringiensis in probiotic products is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] To address the aforementioned problems in existing technologies, this invention provides a kit and method for identifying Bacillus thuringiensis. By analyzing structural differences in the genomic map, two sets of specific primer-probe combinations were designed: the target BT-657 is located in the Bt strain plasmid genome, and the target BT-InhA is located in the Bt strain chromosomal immunosuppressive factor A (inhA) gene. A qPCR detection system was established using the TaqMan probe method, and closed-tube detection was achieved within 2 hours through dual-channel validation. This method exhibits high specificity (amplification only of Bacillus thuringiensis, Ct value ≤30; no signal is observed in Bacillus cereus and other control bacteria) and high sensitivity (up to fg level), completely resolving the false-negative risk and gel electrophoresis contamination problems caused by traditional PCR-dependent plasmid genes (such as the Cryotoxin gene). This invention is applicable to the production quality control of live microbial products, environmental microbial monitoring, and clinical pathogen identification.

[0008] The main concept of this invention is as follows: The inventors conducted in-depth analysis of the genome maps of Bacillus cereus and Bacillus thuringiensis. Starting from the differences in genome structure, by comparing the genome coverage depth and conserved regions of the two bacteria, they precisely screened target sites located in the core chromosome genes and hypervariable regions of plasmids, namely the conserved gene (inhA) on the Bacillus thuringiensis chromosome and the plasmid-specific marker (BT-657). Chromosomal targets ensure the fundamental reliability of species identification, while plasmid targets enhance the recognition of the functional characteristics of Bacillus thuringiensis, forming a structurally complementary identification logic.

[0009] The specific solution of this invention is as follows: A kit for identifying Bacillus thuringiensis includes a primer and probe composition, a qPCR premix, ROX dye (ROX Reference Dye I / II), a positive control, and a negative control.

[0010] The primer-probe composition comprises two sets of primers and probes, as detailed below: 1) The target BT-657 (SEQ ID NO:1-3) is located in the Bt strain plasmid genome. Primer and probe sequences are as follows: 2) The target BT-InhA (SEQ ID NO:4-6) is located in the chromosomal immunosuppressive factor A (inhA) gene of Bt strains. Primer and probe sequences: .

[0011] In addition, the qPCR premix includes Taq DNA polymerase, dNTPs, and buffer, specifically purchased from Beijing Qingke Biotechnology Co., Ltd. as 2xT5 Fast qPCR Mix (probe) (TSE301); the ROX dye (ROXReference Dye I / II) is also purchased from Beijing Qingke Biotechnology Co., Ltd.; the positive control is Bt genomic DNA, specifically Bacillus thuringiensis from the China Industrial Microbial Culture Collection Center. Bacillus thuringiensis Whole-genome DNA extracted from CICC 22945; the negative control was ultrapure water.

[0012] In practical use, the amount of primers and probes added is 0.7-0.9 μL, more preferably 0.8 μL, the concentration of primers and probes is 10 μmol / L, and the concentration of DNA template is 50-150 ng / μL.

[0013] Based on the above-mentioned kit, the present invention further provides a method for identifying Bacillus thuringiensis, the specific steps of which are as follows: (1) Extract DNA template from the sample to be tested; For different types of samples containing Bacillus cereus, such as tablets / powders / raw material powders, a one-step bacterial genomic DNA extraction method is used to extract DNA templates. The specific steps for using the one-step bacterial genomic DNA extraction method as a DNA template provided in this invention are as follows: Tablets / Powders / Raw Bacterial Powder: Aseptically weigh 3.0g of finished tablets / powders / raw bacterial powder, add 27mL of 0.9wt% sterile sodium chloride solution or other suitable diluent, shake well, centrifuge at 13000rpm for 3-5min, discard the supernatant, take no less than 0.1g of precipitate, add 50-100μL of lysis buffer A, mix well, incubate in a 100℃ metal bath for 10-15min, quickly place on ice to cool for 3-5min, centrifuge at 13000rpm for 10-15min, and the supernatant is the bacterial genomic DNA.

[0014] The formulation of the lysis buffer A is as follows: In the aforementioned lysis buffer A, the main function of Triton X-100 is to disrupt cell membranes and organelle membranes, releasing intracellular DNA and proteins; the function of Tris-HCl (pH 8.0) is to provide a stable acid-base buffer environment; and the main function of EDTA is to chelate Ca2+. 2+ Mg 2+ It contains divalent metal ions and inhibits DNase activity; the solvent is ultrapure water.

[0015] (2) qPCR reaction system and amplification procedure: The DNA template extracted in step (1) was placed into the above kit for amplification; the qPCR reaction system (20 μL) is as follows: Preferably, the amount of both primer and probe added is 0.8 μL.

[0016] The amplification procedure is as follows: .

[0017] (3) Result determination: Bt positive: Ct values ​​of both BT-657 and BT-InhA channels are ≤30; Bc or other bacteria: No amplification was observed in either channel.

[0018] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: (1) The one-step bacterial genomic DNA extraction method provided by this invention improves the extraction efficiency of spore-forming genomic DNA by appropriately increasing the concentration of EDTA in the bacterial DNA extraction lysis buffer and adjusting the time and temperature during the extraction process, thereby simplifying the processing steps for bacterial genomic DNA extraction. Compared with commercially available bacterial genomic DNA extraction kits (such as the OMEGA Bacterial DNA Kit, which has 33 extraction steps), the extraction time is shortened from 2-3 hours to 30-40 minutes.

[0019] (2) A qPCR detection system was established using the TaqMan probe method. Through dual-channel verification, closed-tube detection was achieved within 2 hours. This method exhibits high specificity (only Bacillus thuringiensis amplifies, Ct value ≤ 30; Bacillus cereus and other control bacteria show no signal) and high sensitivity (the lowest DNA detection concentration can reach 10). -5 (ng / μL), completely eliminating the false negative risk and gel electrophoresis contamination problems caused by traditional PCR-dependent plasmid genes (such as the Cry toxin gene). This method is applicable to the production quality control of live microbial products, environmental microbial monitoring, and clinical pathogen identification.

[0020] (3) For the first time, a dual-target molecular identification system was constructed based on differences in genomic structure. By comparing the genomic coverage depth and conserved regions of the two bacteria, target sites located in hypervariable regions of plasmids and chromosomal core genes were accurately screened, avoiding cross-reactions caused by homologous recombination or horizontal gene transfer, thus improving specificity from the source. Compared with traditional methods that only focus on plasmid genes and cannot identify Bacillus thuringiensis strains that have lost plasmids in natural variations, this method is more effective.

[0021] This invention fills this structural detection blind spot by targeting chromosomes, significantly reducing the risk of false negatives, solving the structural detection loophole, and redefining the identification strategy for Bacillus cereus. Attached Figure Description

[0022] Figure 1 This is the genome map of Bacillus thuringiensis in Example 1. The red circle represents chromosome information, and the green circle represents plasmid information. Figure 2 This is the genome map of Bacillus cereus in Example 1. The red circle represents chromosome information, and the green circle represents plasmid information. Figure 3 The image shows the results of the BT-657 specificity test in Example 5. In the image, 1 represents Bacillus thuringiensis CICC22945, and 2 represents other non-target bacteria: Bacillus cereus CMCC 63303, Enterococcus faecalis CICC 10396, Lactobacillus acidophilus CICC 6075, Bifidobacterium longum subsp. infantis CICC 6069, and Escherichia coli CMCC 44102. Figure 4 The image shows the results of the BT-InhA specificity experiment in Example 5. 1 represents Bacillus thuringiensis CICC22945, and 2 represents other non-target bacteria: Bacillus cereus CMCC 63303, Enterococcus faecalis CICC 10396, Lactobacillus acidophilus CICC 6075, Bifidobacterium longum subsp. infantis CICC 6069, and Escherichia coli CMCC 44102. Figure 5This is the amplification standard curve of BT-657 in Example 6, where the standard curve is plotted with copy number log value on the x-axis and average CT value on the y-axis. Figure 6 The amplification pattern of BT-657 in Example 6; Figure 7 This is the amplification standard curve of BT-InhA in Example 6, where the standard curve is plotted with copy number log value on the x-axis and average CT value on the y-axis. Figure 8 The amplification pattern of BT-InhA in Example 6; Figure 9 The results are the actual sample test results of BT-657 in Example 7, where 1-7 correspond to the actual sample numbers 1-7 respectively; Figure 10 The results are the actual sample test results of BT-InhA in Example 7, where 1-7 correspond to the actual sample numbers 1-7 respectively. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to specific embodiments. These embodiments are only used to illustrate the technical solution of the present invention in more detail and should not be construed as limiting the scope of protection of the present invention.

[0024] Unless otherwise specified in the embodiments, the experimental methods are assumed to be conventional methods in the art. For specific operations, please refer to relevant technical documents, product manuals, or industry-standard practices. Unless otherwise specified, all experimental materials and reagents involved are commercially available products and can be obtained through commercial channels.

[0025] Example 1: Genomic mapping analysis of Bacillus cereus and Bacillus thuringiensis Genomic DNA was extracted from standard strains of Bacillus thuringiensis and Bacillus cereus using the one-step bacterial genomic DNA extraction method described in this invention. The complete genome sequence was obtained through whole-genome sequencing, and comparative genomic analysis was performed. Figure 1 , 2 The fundamental differences in genomic composition between the two are clearly demonstrated: Bacillus thuringiensis has a unique plasmid genome carrying a large number of functional genes (such as insecticidal crystal protein genes), while Bacillus cereus is mainly composed of a chromosomal genome. This structural difference directly affects the reliability of traditional detection methods—relying solely on plasmid genes (such as the Cry toxin gene) is prone to false negatives due to plasmid loss or horizontal transfer. Therefore, the inventors ultimately determined to simultaneously target the conserved gene (inhA) on the Bacillus thuringiensis chromosome and the plasmid-specific marker (BT-657) to avoid cross-reactions caused by homologous recombination or horizontal gene transfer, thereby improving detection specificity and accuracy.

[0026] Example 2: A kit for identifying Bacillus thuringiensis, comprising primer and probe composition, qPCR premix, ROX dye, positive control, and negative control, as detailed below: The primer-probe composition comprises two sets of primers and probes, as detailed below: 1) The target BT-657 (SEQ ID NO:1-3) is located in the Bt strain plasmid genome. Primer and probe sequences are as follows: 2) The target BT-InhA (SEQ ID NO:4-6) is located in the chromosomal immunosuppressive factor A (inhA) gene of Bt strains. Primer and probe sequences: In addition, the qPCR premix includes Taq DNA polymerase, dNTPs, and buffer, specifically purchased from Beijing Qingke Biotechnology Co., Ltd. as 2xT5 Fast qPCR Mix (probe) (TSE301); the positive control is Bt genomic DNA, specifically Bacillus thuringiensis from the China Industrial Microbial Culture Collection Center. Bacillus thuringiensis Whole-genome DNA extracted from CICC 22945; the negative control was ultrapure water.

[0027] Example 3: The specific steps for one-step bacterial genomic DNA extraction as a DNA template are as follows: Tablets / Powders / Raw Bacterial Powder: Aseptically weigh 3.0g of finished tablets / powders / raw bacterial powder, add 27mL of 0.9% sterile sodium chloride solution or other suitable diluent, shake well, centrifuge at 13000rpm for 3-5min, discard the supernatant, take no less than 0.1g of precipitate, add 50-100μL of lysis buffer A, mix well, incubate in a 100℃ metal bath for 10-15min, quickly place on ice to cool for 3-5min, centrifuge at 13000rpm for 10-15min, and the supernatant is the bacterial genomic DNA.

[0028] The formulation of the lysis buffer A (solvent being ultrapure water) is as follows: .

[0029] Example 4: Comparison of one-step bacterial genomic DNA extraction method with commercially available bacterial genomic DNA extraction kit (OMEGA's E.ZNA® Bacterial DNA Kit) Take Bacillus cereus CMCC 63303 ( Bacillus cereus Bacillus thuringiensis CICC 22945 Bacillus thuringiensisEscherichia coli CMCC 44102 ( Escherichia coli ), Enterococcus faecalis CICC10396 ( Enterococcus faecalis Lactobacillus acidophilus CICC 6075 Lactobacillus acidophilus ) and Bifidobacterium longum infantile subspecies CICC 6069 ( Bifidobacterium longum subsp. infantis Liquid cultures of standard bacterial strains were used as samples for comparison in bacterial genomic DNA extraction methods. All of the above strains were purchased from the China Medical Culture Collection Center (CMCC) and the China Industrial Culture Collection Center (CICC). 。

[0030] Genomic DNA extraction was performed according to the one-step bacterial genomic DNA extraction method described in Example 3, using the method outlined in the OMEGA EZNA® Bacterial DNA Kit manual. A comparison of the specific extraction steps is shown in Table 1, and the concentration and purity of the genomic DNA extracted by the two methods are shown in Table 2.

[0031] Table 1 Comparison of Methods and Procedures Table 2 DNA concentration detection results The results showed that, compared with the commercially available kits, the one-step bacterial genomic DNA extraction method could obtain genomic DNA of sufficient quality to meet the requirements of subsequent qPCR reactions (50-150 ng / μL). However, the one-step bacterial genomic DNA extraction method used in this application simplifies the operation steps and greatly shortens the detection time.

[0032] Example 5: Primer set specificity verification Using the DNA of the standard strain used in Example 4 as templates, amplification was performed using the kit in Example 2.

[0033] The qPCR reaction system (20 μL) is as follows:

[0034] The amplification procedure is as follows: Figure 3 The image shows the results of a single primer and probe BT-657 specificity experiment. The target strain, *Bacillus thuringiensis* CICC 22945, showed a positive amplification result; while the non-target strains, *Bacillus cereus* CMCC 63303, *Enterococcus faecalis* CICC 10396, *Lactobacillus acidophilus* CICC 22813, *Bifidobacterium longum* subsp. *infant* CICC 6069, and *Escherichia coli* CMCC 44102, showed negative amplification results.

[0035] Figure 4 The image shows the results of a single primer and probe BT-InhA specificity experiment. The target strain, *Bacillus thuringiensis* CICC 22945, showed a positive amplification result; while the non-target strains, *Bacillus cereus* CMCC 63303, *Enterococcus faecalis* CICC10396, *Lactobacillus acidophilus* CICC 22813, *Bifidobacterium longum* subsp. *infant* CICC 6069, and *Escherichia coli* CMCC 44102, showed negative amplification results.

[0036] The results showed that the specific primer-probe composition of the present invention could specifically amplify the target strain Bacillus thuringiensis CICC 22945; and could not specifically amplify non-target strains such as Bacillus cereus CMCC 63303, Enterococcus faecalis CICC10396, Lactobacillus acidophilus CICC 22813, Bifidobacterium longum subsp. infantis CICC 6069, and Escherichia coli CMCC 44102.

[0037] Example 6 Sensitivity and Detection Efficiency Bacillus thuringiensis CICC22945 was purchased from the China Industrial Microbial Culture Collection Center (CICC). Bacillus thuringiensis Standard strains, with genomic DNA extracted using the one-step bacterial genomic DNA extraction method as 10 1 Template, take 10 1 The template was gradient diluted to 10. 7 (Dilution factor) Genomic DNA at different dilution levels was used as nucleic acid templates and amplified using the kit described in Example 2. The amplification results are shown in Table 3 below. Table 3 Amplification Results It can be seen that the lowest detectable DNA concentration of the BT-657 gene can reach 10. -5 The lowest detectable DNA concentration for the BT-InhA gene is 10 ng / μl. -6 ng / μl, amplification efficiency graph is shown in Figure 5-8 .

[0038] Example 7: Comparison of Detection Results for Actual Samples Five batches of Bifidobacterium tetrad live bacteria tablets (sample numbers 1-5) of a certain brand were randomly selected from pharmacies. The main components were Bifidobacterium longum subsp. infantis, Lactobacillus acidophilus, Enterococcus faecalis, and Bacillus cereus. The identification of Bacillus thuringiensis and Bacillus cereus was carried out using traditional biochemical identification methods (refer to GB 4789.14-2014 "National Food Safety Standard for Microbiological Examination of Food - Examination of Bacillus cereus" 4.3.2 Biochemical Identification) and the method described in this application.

[0039] To ensure a high detection rate, two samples were selected for artificial contamination with Bacillus thuringiensis. The specific steps were as follows: 3.0 g of each of the two batches of samples numbered 4 and 5 were aseptically weighed, and 27 mL of 0.9 wt% sterile sodium chloride solution was added. The mixture was shaken thoroughly, centrifuged at 13000 rpm for 5 min, and the supernatant was discarded. 0.1 g of the precipitate was taken and 1 mL of 5.0 × 10⁻⁶ solution was added. 6 CFU / mL Bacillus thuringiensis CICC 22945 bacterial suspension was thoroughly shaken to obtain a homogenate of artificially contaminated Bacillus thuringiensis samples numbered 6-7.

[0040] Samples numbered 1-7 were extracted using the one-step bacterial genomic DNA extraction method: Sample Nos. 1-5: Aseptically weigh 3.0g of finished tablets, add 27mL of 0.9% sterile sodium chloride solution, shake well, centrifuge at 13000rpm for 5min, discard the supernatant, take 0.1g of precipitate, add 100μL of lysis buffer A, mix well, incubate in a 100℃ metal bath for 10-15min, quickly place on ice to cool for 5min, centrifuge at 13000rpm for 15min, and the supernatant is the bacterial genomic DNA.

[0041] Sample No. 6-7: Take 1000 μL of artificially contaminated sample homogenate, centrifuge at 13000 rpm for 5 min, discard the supernatant, add 100 μL of lysis buffer A, mix well, incubate in a 100℃ metal bath for 10-15 min, quickly place on ice to cool for 3-5 min, centrifuge at 13000 rpm for 10-15 min, and the supernatant is the bacterial genomic DNA.

[0042] Amplification was performed using the kit described in Example 2. The qPCR reaction volume (20 μL) is as follows: The amplification procedure is as follows: Amplification results are shown below Figure 9 and 10Samples numbered 6 and 7 both had Ct values ​​≤30, indicating they were Bt-positive. The results show that Bacillus thuringiensis was detected only in samples with human contamination, demonstrating that the market products selected in this embodiment do not contain Bacillus thuringiensis. The method described in this invention can effectively separate Bacillus thuringiensis from Bacillus cereus, and the results obtained by this method are consistent with those of traditional biochemical identification methods (see Table 4). Table 4 Results of Bacillus thuringiensis detection Note: + detected; - not detected.

[0043] Therefore, the detection method described in this invention has a detection rate comparable to that of traditional biochemical identification methods, demonstrating its reliability. At the same time, it can significantly shorten the detection time, especially compared with traditional biochemical identification methods, reducing the detection time from 7-8 days to 2.5 hours. Furthermore, it is simple to operate and convenient for practical detection applications.

[0044] The present invention has described the technical solutions in detail with reference to the accompanying drawings and embodiments. However, the specific embodiments described are merely illustrative and not restrictive. It should be understood that those skilled in the art, based on the technical concept of the present invention, can achieve various implementation forms by adjusting the implementation parameters, optimizing the combination method, or replacing equivalent technical features without departing from the scope of protection defined by the claims. All modifications, extensions, or improvements made based on the core innovations of the present invention fall within the scope of protection defined by the claims of the present invention.

Claims

1. A kit for identifying Bacillus thuringiensis, the kit comprising a primer-probe composition, a qPCR premix, ROX dye, a positive control, and a negative control; characterized in that, The primer-probe composition comprises two sets of primers and probes, as detailed below: Primer and probe sequences for target BT-657: Target BT-InhA primers and probe sequences: 。 2. The kit for identifying Bacillus thuringiensis according to claim 1, characterized in that, The target BT-657 is located in the plasmid genome of Bacillus thuringiensis strain; the target BT-InhA is located in the chromosomal immunosuppressive factor A gene of Bacillus thuringiensis strain.

3. The kit for identifying Bacillus thuringiensis according to claim 1, characterized in that, The qPCR premix includes Taq DNA polymerase, dNTPs, and buffer; the positive control is Bt genomic DNA; and the negative control is ultrapure water.

4. The kit for identifying Bacillus thuringiensis according to claim 1, characterized in that, When using the primers and probes, the amount added is 0.7–0.9 μL, the concentration of the primers and probes is 10 μmol / L, and the concentration of the DNA template is 50–150 ng / μL.

5. In the kit for identifying Bacillus thuringiensis according to claim 4, the amount of primer and probe added is 0.8 μL in specific use.

6. A method for identifying Bacillus thuringiensis using the kit described in claim 1, characterized in that, Includes the following steps: (1) Extracting DNA template from the sample to be tested: For different types of samples containing Bacillus cereus, such as tablets / powders / raw material bacterial powders, a one-step bacterial genomic DNA extraction method was used to extract DNA templates. (2) qPCR reaction system: The DNA template extracted in step (1) is placed into the kit for amplification. (3) Result determination: Bt positive: Ct values ​​of both BT-657 and BT-InhA channels are ≤30; Bc or other bacteria: No amplification was observed in either channel.

7. The method according to claim 6, characterized in that, The specific steps for the one-step bacterial genomic DNA extraction as a DNA template described in step (1) are as follows: Tablets / Powders / Raw Bacterial Powder: Aseptically weigh 3.0g of finished tablets / powders / raw bacterial powder, add to 27mL of 0.9wt% sterile sodium chloride solution, shake well, centrifuge at 13000rpm for 3-5min, discard the supernatant, take no less than 0.1g of precipitate, add 50-100μL of lysis buffer A, mix well, in a 100℃ metal bath for 10-15min, quickly place on ice to cool for 3-5min, centrifuge at 13000rpm for 10-15min, the supernatant is the bacterial genomic DNA.

8. The method according to claim 7, characterized in that, The lysis buffer A described in step (1) contains 2% by volume Triton X-100, 100 mmol / L Tris-HCl, and 20-40 mmol / L EDTA, and the solvent is ultrapure water.

9. The method according to claim 6, characterized in that, The qPCR reaction system described in step (2) is as follows: 10 μL of 1×qPCRMix, 0.7-0.9 μL of 10 μM upstream primer, 0.7-0.9 μL of 10 μM downstream primer, 0.7-0.9 μL of 10 μM probe, 0.4 μL of 1×ROX dye, 1 μL of DNA template with a concentration of 50-150 ng / μL, and ddH2O to make up to 20 μL.

10. The method according to claim 6 or 9, characterized in that, The reaction conditions for the amplification treatment in step (2) are: 95℃, 2 min, 1 cycle; 95℃ 15 s, 60℃ 30 s, 40 cycles.

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