A primer set, kit and application for simultaneously detecting clostridium perfringens, clostridium novyi, clostridium septicum and clostridium chauvoei

Abstract

The application discloses a primer group, a kit and application for simultaneously detecting Clostridium perfringens, Clostridium novyi, Clostridium putrefaciens and Clostridium chauvoei, and belongs to the technical field of molecular biology detection. The primer comprises PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3, PrimerR3, PrimerF4 and PrimerR4, and the nucleotide sequences of the primers are shown in SEQ ID NO. 1 to SEQ ID NO. 8. The primer and the kit have the advantages of specificity, sensitivity, rapidness, good repeatability and the like.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, and in particular to a primer set, kit, and application for the simultaneous detection of Clostridium perfringens, Clostridium novie, Clostridium putrefactiveum, and Clostridium emphysematous. Background Technology

[0002] Clostridium diseases are a general term for diseases caused by pathogenic bacteria of the genus *Clostridium*. They are characterized by rapid onset, short course, and high mortality, and are distributed worldwide. *Clostridium perfringens*, *Clostridium novyi*, *Clostridium septicum*, and *Clostridium chauvoei* all belong to the family Clostridiaceae and are all Gram-positive bacteria. Their spores are widely present in soil, feces, and animal intestines, and can invade the body through environmental contamination or through wounds and the digestive tract. The spores are extremely resistant and can survive for long periods in harsh environments. The exotoxins produced after germination can cause serious diseases such as gas gangrene and necrotizing enteritis, causing enormous economic losses to global livestock farming, especially large-scale cattle and sheep farming.

[0003] The clinical symptoms and necropsy findings caused by the four Clostridium species mentioned above are highly similar, and mixed infections are common in clinical practice, increasing the difficulty of differential diagnosis. Traditional diagnostic methods often rely on bacterial isolation and culture, morphological staining, biochemical identification, and animal inoculation experiments. However, Clostridium is a strict anaerobe with demanding culture conditions, and it is easily inactivated during sampling, transportation, and handling. The detection cycle can take several days, making it difficult to meet the rapid diagnostic needs of acute cases. Furthermore, since all four bacteria belong to the Clostridium genus, identification is easily confused, and mutual interference is likely. Therefore, establishing efficient, specific molecular diagnostic techniques capable of simultaneously detecting multiple pathogenic Clostridium species is of great significance for early disease warning, epidemic tracing, and scientific prevention and control.

[0004] Currently, there are various PCR detection methods for Clostridium perfringens, Clostridium noviformis, Clostridium putrefactivee, and Clostridium emphysematous, including multiplex PCR methods for detecting single genotypes and multiple genotypes simultaneously. However, multiplex PCR methods for simultaneously detecting Clostridium perfringens, Clostridium noviformis, Clostridium putrefactivee, and Clostridium emphysematous have not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to provide a primer set, kit, and application for the simultaneous detection of Clostridium perfringens, Clostridium novie, Clostridium putrefactivee, and Clostridium emphysematous. The primer set and kit can simultaneously detect Clostridium perfringens, Clostridium novie, Clostridium putrefactivee, and Clostridium emphysematous, and have the advantages of high detection sensitivity and strong specificity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a primer set for the simultaneous detection of *Clostridium perfringens*, *Clostridium novesiculosus*, *Clostridium putrefactive*, and *Clostridium emphysematous*. The primer set includes primers PrimerF1 and PrimerR1 designed using the phospholipase C gene of *Clostridium perfringens* as a template; primers PrimerF2 and PrimerR2 designed using the flagellin C gene of *Clostridium novesiculosus* as a template; primers PrimerF3 and PrimerR3 designed using the α-toxin gene of *Clostridium putrefactive* as a template; and primers PrimerF4 and PrimerR4 designed using the cytotoxin A gene of *Clostridium emphysematous* as a template. The sequences of the primers are: PrimerF1: SEQ ID NO.1, PrimerR1: SEQ ID NO.2, PrimerF2: SEQ ID NO.3, PrimerR2: SEQ ID NO.4, PrimerF3: SEQ ID NO.5, PrimerR3: SEQ ID NO.6, PrimerF4: SEQ ID NO.7, PrimerR4: SEQ ID NO.8.

[0008] This invention provides a kit for the simultaneous detection of Clostridium perfringens, Clostridium novie, Clostridium putrefactiveum, and Clostridium emphysematous, the kit comprising the primer set described in the above scheme.

[0009] Preferably, the kit also includes 2×Rapid Taq premix and ultrapure water.

[0010] Preferably, the kit also includes a positive control and a negative control.

[0011] Preferably, the positive control includes Clostridium perfringens genomic DNA, Clostridium noviformis genomic DNA, Clostridium putrefactivee genomic DNA, and Clostridium emphysematous genomic DNA; the negative control is ultrapure water.

[0012] This invention also provides the application of the primer set or kit described above in the multiplex PCR detection of Clostridium perfringens, Clostridium novie, Clostridium putrefactiveum, and Clostridium perfringens for non-diagnostic purposes, the application comprising the following steps:

[0013] (1) Extract genomic DNA from the sample to be tested;

[0014] (2) Using the genomic DNA extracted from the sample to be tested as a template, a multiplex PCR amplification reaction was performed using the primer set described in the above scheme to obtain the multiplex PCR amplification product;

[0015] (3) Multiplex PCR amplification products were detected by agarose gel electrophoresis;

[0016] (4) If a specific fragment of 731 bp is amplified, the test result is Clostridium perfringens; if no specific fragment of 731 bp is amplified, the test result is negative for Clostridium perfringens.

[0017] If a specific fragment of 529 bp is amplified, the test result is positive for Clostridium novipella; if no specific fragment of 529 bp is amplified, the test result is negative for Clostridium novipella.

[0018] If a specific fragment of 230 bp is amplified, the test result is positive for Clostridium putrefactivee; if no specific fragment of 230 bp is amplified, the test result is negative for Clostridium putrefactivee.

[0019] If a specific fragment of 117 bp is amplified, the test result is positive for Clostridium emphysematous; if no specific fragment of 117 bp is amplified, the test result is negative for Clostridium emphysematous.

[0020] Preferably, the reaction system used in step 2) for the multiplex PCR amplification reaction comprises, in 25 μL increments: 12.5 μL of 2×RapidTaq premix, 3.5 μL of ultrapure water, 0.5 μL each of PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3 and PrimerR3, 1 μL each of PrimerF4 and PrimerR4, and 4 μL of DNA template.

[0021] The primer set consists of the following components at final concentrations: PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3, PrimerR3, PrimerF4, and PrimerR4, each with a concentration of 10 μmol / L.

[0022] The procedure for the multiplex PCR amplification reaction was as follows: 95 ℃ for 3 min; 95 ℃ for 15 s, 55 ℃ for 30 s, 72 ℃ for 5 s, 30 cycles; 72 ℃ for 5 min.

[0023] Beneficial effects of the invention: This invention provides a primer set for the simultaneous detection of *Clostridium perfringens*, *Clostridium novie*, *Clostridium putrefactive*, and *Clostridium emeticae*. The primer set includes Primer F1, Primer R1, Primer F2, Primer R2, Primer F3, Primer R3, Primer F4, and Primer R4. This primer set can simultaneously amplify four specific fragments: 731 bp, 529 bp, 230 bp, and 117 bp. The specificity, sensitivity, and repeatability of this invention were evaluated, and the method was applied to detect clinically collected bovine and ovine tissue and fecal samples. The results showed high specificity, amplifying the expected bands only for the four target pathogens, while showing negative results for non-target pathogens such as *Clostridium tetani*, *Escherichia coli*, *Salmonella*, and *Staphylococcus aureus*. High sensitivity was also demonstrated, with the limits of detection for the recombinant plasmid standards for the four pathogens reaching 10 μL. 2 copies / μL, 10 3 copies / μL, 10 4 copies / μL and 10 3 The sample size is [copies / μL]; it exhibits good repeatability, with consistent results between intra- and inter-batch tests. In a study of 206 clinical samples, the positive rates for *Clostridium perfringens* were 0%, *Clostridium novesiculum* 2%, *Clostridium putrefactiveum* 12%, and *Clostridium emphysematous* 9%, with a 100% concordance rate verified by singleton PCR and sequencing. The primer set of this invention uses four primer pairs within a single PCR reaction system without interference, offering advantages such as high efficiency, systematic approach, and cost-effectiveness. It is suitable for rapid and accurate pathogen identification in clinical and laboratory settings, providing technical support for non-diagnostic work such as epidemiological investigations of bovine and ovine clostridium diseases, vaccine quality inspection, and vaccine development. Attached Figure Description

[0024] Figure 1 Amplification results of *Clostridium perfringens*, *Clostridium novesiculum*, *Clostridium putrefactivee*, and *Clostridium emphysematous*; where M: DNA relative molecular mass standard DL 2000 Plus; 1: *Clostridium perfringens*; 2: *Clostridium perfringens* negative control; 3: *Clostridium novesiculum*; 4: *Clostridium novesiculum* negative control; 5: *Clostridium putrefactivee*; 6: *Clostridium putrefactivee* negative control; 7: *Clostridium emphysematous*; 8: *Clostridium emphysematous* negative control.

[0025] Figure 2 Singleton PCR specific detection results; where AD: detection results of Clostridium perfringens, Clostridium novie, Clostridium putrefactivee, and Clostridium emphysematous, respectively; M: DNA relative molecular mass standard DL 2000 Plus; 1: negative control; 2-9: Clostridium perfringens, Clostridium novie, Clostridium putrefactivee, Clostridium emphysematous, Clostridium tetani, Escherichia coli, Salmonella, and Staphylococcus aureus; 10 template-free blank control.

[0026] Figure 3 Multiplex PCR specificity test; where M: DNA relative molecular mass standard DL 2000 Plus; 1: positive control; 2: negative control; 3-6 are respectively: Clostridium perfringens CVCC 37, Clostridium perfringens CVCC60081, Clostridium perfringens CVCC60102, Clostridium perfringens CVCC60201; 7 and 8 are respectively: Clostridium novie CVCC 60281, Clostridium novie CVCC 60282; 9: Clostridium putrefactiveum CVCC 60022; 10: Clostridium emphysematous CVCC 60002; 11: Clostridium tetani; 12: Escherichia coli; 13: Staphylococcus aureus.

[0027] Figure 4 Multiplex PCR sensitivity assay; where M: DNA molecular weight standard DL 2000 Plus; 1-11: 10 9 -10 -1 ;12: Negative control.

[0028] Figure 5 Multiplex PCR repeatability test; where M: DNA relative molecular mass standard DL 2000 Plus; 1: negative control; 2-4: three replicate experiments.

[0029] Figure 6 Clinical sample test results; where A: distribution of positive samples; B: mixed infection status of positive samples.

[0030] Figure 7 Multiplex PCR primer screening and verification results: AF correspond to multiplex PCR amplification results of primer groups 1-6, respectively; where M: DNA relative molecular mass standard (D2000); 1: 4-way multiplex positive control mixture; 2: negative control; 3-4: Clostridium novie (CVCC 60281, CVCC 60282); 5-8: Clostridium perfringens (CVCC 37, CVCC60081, CVCC60102, CVCC60201); 9: Clostridium putrefactive (CVCC 60022); 10: Clostridium perfringens (CVCC 60002).

[0031] Figure 8Multiplex PCR primer screening and verification results 2: AB: Multiplex PCR amplification results of primer groups 7-8. M: DNA relative molecular mass standard (D2000); 1: Quadruple positive control mixture; 2: Negative control; Clostridium novii (CVCC 60281, CVCC60282); 5-8: Clostridium perfringens (CVCC 37, CVCC60081, CVCC60102, CVCC60201); 9: Clostridium putrefactive (CVCC 60022); 10: Clostridium perfringens (CVCC 60002).

[0032] Figure 9 Multiplex PCR primer screening and verification results 3: M: DNA relative molecular mass standard (D2000); 1-2: Group 9 multiple positive control mixture; 3-4: Group 9 negative control; 5-6: Group 10 multiple positive control mixture; 7-8: Group 10 negative control.

[0033] Figure 10 Optimized annealing temperature results for multiplex PCR: M: DNA relative molecular mass standard (DL 2000 Plus); 1: 60℃; 2: negative control; 3: 59℃; 4: negative control; 5: 58℃; 6: negative control; 7: 56.7℃; 8: negative control; 9: 55℃; 10: negative control; 11: 53℃; 12: negative control; 13: 52℃; 14: negative control; 15: 51℃; 16: negative control.

[0034] Figure 11 Multiplex PCR primer screening and verification results: M: DNA relative molecular mass standard (DL 2000 Plus); 1: Multiplex positive control mixture; 2: Negative control; C. novyi (CVCC 60281, CVCC 60282); 5-8: Clostridium perfringens (CVCC 37, CVCC60081, CVCC60102, CVCC60201); 9: Clostridium putrefactive (CVCC 60022); 10: Clostridium perfringens (CVCC 60002).

[0035] Figure 12Multiplex PCR primer concentration optimization results, where M: DNA relative molecular mass standard (DL 2000 Plus); 1: Clostridium perfringens, Clostridium novie, Clostridium putrefactivee, and Clostridium emphysematous ratio: 0.5 μL: 0.5 μL: 0.5 μL: 0.5 μL; 2: Negative control; 3: Clostridium perfringens, Clostridium novie, Clostridium putrefactivee, and Clostridium emphysematous ratio: 0.5 μL: 0.5 μL: 0.5 μL: 1 μL; 4: Negative control; 5: Clostridium perfringens, Clostridium novie, Clostridium putrefactivee, and Clostridium emphysematous ratio: 0.5 μL: 0.5 μL: 0.5 μL: 1.5 μL; 6: Negative control; 7: Clostridium perfringens, Clostridium novie, The ratio of Clostridium putrefactivee and Clostridium emphysematous was 0.5 μL: 0.5 μL: 0.5 μL: 2 μL; 6: negative control. Detailed Implementation

[0036] This invention provides a primer set for the simultaneous detection of Clostridium perfringens, Clostridium noviformis, Clostridium putrefactivee, and Clostridium perfringens, wherein the primer set includes PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3, PrimerR3, PrimerF4, and PrimerR4.

[0037] The nucleotide sequence of the PrimerF1 is shown in SEQ ID NO: 1, specifically: 5'-CCCGCTGTTCCTTTTTGAGAG -3';

[0038] The nucleotide sequence of PrimerR1 is shown in SEQ ID NO: 2, specifically: 5'-TGGGCTGGGACATCAACTAA-3';

[0039] The nucleotide sequence of the PrimerF2 is shown in SEQ ID NO: 3, specifically: 5'-GCTTTAAACGAAACACACGC-3';

[0040] The nucleotide sequence of PrimerR2 is shown in SEQ ID NO: 4, specifically: 5'-CTTGAGCTGCTTGTTGAAGT-3';

[0041] The nucleotide sequence of the PrimerF3 is shown in SEQ ID NO: 5, specifically: 5'-AATTCAGTGTGCGGCAGTAGT-3';

[0042] The nucleotide sequence of PrimerR3 is shown in SEQ ID NO: 6, specifically: 5'-CGTTTGGATTAGCCGTTCCAC-3';

[0043] The nucleotide sequence of the PrimerF4 is shown in SEQ ID NO: 7, specifically: 5'-GCAGGGGGAGGAATAAATGGA-3';

[0044] The nucleotide sequence of PrimerR4 is shown in SEQ ID NO: 8, specifically: 5'-GCAACTTCCCAAGATGCACTAT-3'.

[0045] In this invention, PrimerF1 and PrimerR1 are a pair of primers; PrimerF1 and PrimerR1 are designed for the Clostridium perfringens phospholipase C gene (Plc); the nucleotide sequence of the Clostridium perfringens phospholipase C gene is shown in SEQ ID NO: 9.

[0046] In this invention, PrimerF2 and PrimerR2 are a pair of primers; PrimerF2 and PrimerR2 are designed for the Clostridium novie flagellin C gene (Flic); the nucleotide sequence of the Clostridium novie flagellin C gene is shown in SEQ ID NO: 10.

[0047] In this invention, PrimerF3 and PrimerR3 are a pair of primers; PrimerF3 and PrimerR3 are designed to target the alpha-toxin gene of Clostridium putrefactivee; the nucleotide sequence of the Clostridium putrefactivee alpha-toxin gene is shown in SEQ ID NO: 11.

[0048] In this invention, PrimerF4 and PrimerR4 are a pair of primers; PrimerF4 and PrimerR4 are designed for Clostridium emphysematous cytotoxin A (CctA); the nucleotide sequence of Clostridium emphysematous cytotoxin A is shown in SEQ ID NO: 12.

[0049] In the specific implementation of this invention, the primer set was synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd.

[0050] A kit for the simultaneous detection of Clostridium perfringens, Clostridium novesiculosum, Clostridium putrefactiveum, and Clostridium emphysematous, the kit comprising the primer set described in the above-mentioned scheme; the concentrations of primers PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3, PrimerR3, PrimerF4, and PrimerR4 are independently 10 μmol / L; the kit further comprises 2×Rapid Taq premix and ultrapure water.

[0051] In this invention, the kit further includes positive and negative controls. The positive controls include Clostridium perfringens genomic DNA, Clostridium novesiculum genomic DNA, Clostridium putrefactivee genomic DNA, and Clostridium emphysematous genomic DNA. The negative control is ultrapure water.

[0052] This invention also provides the application of the primer set or kit described above in the multiplex PCR detection of Clostridium perfringens, Clostridium novie, Clostridium putrefactiveum, and Clostridium perfringens for non-diagnostic purposes, the application comprising the following steps:

[0053] (1) Extract genomic DNA from the sample to be tested;

[0054] (2) Using the genomic DNA extracted from the sample to be tested as a template, a multiplex PCR amplification reaction was performed using the primer set described in the above scheme to obtain the multiplex PCR amplification product;

[0055] (3) Multiplex PCR amplification products were detected by agarose gel electrophoresis;

[0056] (4) If a specific fragment of 731 bp is amplified, the test result is Clostridium perfringens; if no specific fragment of 731 bp is amplified, the test result is negative for Clostridium perfringens.

[0057] If a specific fragment of 529 bp is amplified, the test result is positive for Clostridium novipella; if no specific fragment of 529 bp is amplified, the test result is negative for Clostridium novipella.

[0058] If a specific fragment of 230 bp is amplified, the test result is positive for Clostridium putrefactivee; if no specific fragment of 230 bp is amplified, the test result is negative for Clostridium putrefactivee.

[0059] If a specific fragment of 117 bp is amplified, the test result is positive for Clostridium emphysematous; if no specific fragment of 117 bp is amplified, the test result is negative for Clostridium emphysematous.

[0060] The present invention first extracts genomic DNA from the sample to be tested; the sample to be tested includes animal tissue, secretions and excrement; the present invention does not have any particular limitation on the method of extracting genomic DNA from the sample to be tested, and conventional genomic DNA extraction methods in the art can be used.

[0061] In this invention, after extracting genomic DNA from the sample to be tested, the genomic DNA is used as a template, and multiplex PCR amplification reaction is performed using the primer set described in the above scheme to obtain multiplex PCR amplification products.

[0062] In this invention, the reaction system used for the multiplex PCR amplification reaction, in 25 μL increments, includes: 12.5 μL of 2×Rapid Taq premix, 3.5 μL of ultrapure water, 0.5 μL each of PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3 and PrimerR3, 1 μL each of PrimerF4 and PrimerR4, and 4 μL of DNA template.

[0063] The primer set consists of the following components at final concentrations: PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3, PrimerR3, PrimerF4, and PrimerR4, each with a concentration of 10 μmol / L.

[0064] The procedure for the multiplex PCR amplification reaction was as follows: 95 ℃ for 3 min; 95 ℃ for 15 s, 55 ℃ for 30 s, 72 ℃ for 5 s, 30 cycles; 72 ℃ for 5 min.

[0065] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Specifying a manufacturer only indicates that the present invention uses that product, and does not limit the use of only that manufacturer's products.

[0066] Example 1

[0067] I. Strains and Sources

[0068] Clostridium perfringens strains CVCC 37, CVCC60081, CVCC60102, and CVCC60201; Clostridium novyi strains CVCC 60281 and CVCC 60282; Clostridium septicum strain CVCC60022; and Clostridium chauvoei strain CVCC 60002, etc., were all isolated, identified, and preserved by the China Institute of Veterinary Drug Control.

[0069] II. Bacterial Culture

[0070] Clostridium emphysematous, Clostridium putrefactive, and Clostridium perfringens were inoculated into enhanced Clostridium medium, and Clostridium botulinum was inoculated into GAM medium. All were then placed in a sealed box and incubated at 37°C for 24-48 h.

[0071] III. Nucleic Acid Extraction

[0072] DNA was extracted using a commercially available bacterial DNA extraction kit.

[0073] IV. Preparation of Recombinant Plasmid Standards

[0074] Using the nucleic acids corresponding to *Clostridium perfringens* (CVCC 37, CVCC60081, CVCC60102, CVCC60201), *Clostridium noviformis* (CVCC60281, CVCC 60282), *Clostridium putrefactiveum* (CVCC 60022), and *Clostridium emphysematous* (CVCC 60002) as templates, PCR amplification was performed separately. The PCR reaction system consisted of 12.5 μL of 2×Taq PCR Master Mix, 1 μL of template, 1 μL each of forward and reverse primers (10 μmol / L), and ddH2O to a final volume of 25 μL. The PCR reaction conditions were: 95 ℃ for 3 min; 95 ℃ for 15 s, 55 ℃ for 30 s, 72 ℃ for 25 s, for 35 cycles; 72 ℃ for 5 min. The amplification products were detected by 2.0% agarose gel electrophoresis, purified using a gel extraction kit, ligated into the pUCm-t vector, and transformed into DH5α competent cells. After identification by bacterial culture PCR (PCR reaction system and conditions as above) and sequencing, correctly identified positive clones were selected for amplification culture. Plasmids were extracted using a plasmid extraction kit, and OD values ​​were measured using a NanoDrop nucleic acid and protein analyzer. 260 / OD 280 The plasmid copy number was calculated, and then diluted to 1×10⁻⁶ with nucleic acid-free water. 9 Copies / μL, used as a positive standard for PCR reactions, should be stored at −80 ℃ for later use. [X: Plasmid concentration (ng / μL), A: Target fragment length (bp)]

[0075]

[0076] Vector size: 2773bp.

[0077] Based on conserved gene sequences, this invention designs and screens one pair of primers each for plc, fliC, alpha-toxin and CctA genes. The specific primers obtained are shown in Table 1. The primer screening process is described in Example 7.

[0078] Table 1 Primers used in this invention

[0079]

[0080] PCR validation showed that all four primer pairs could amplify specific bands of the expected size, such as... Figure 1 As shown, the fragment lengths of *Clostridium perfringens*, *Clostridium novie*, *Clostridium putrefactive*, and *Clostridium emphysematous* are 731 bp, 529 bp, 230 bp, and 117 bp, respectively. After cloning the target fragment into the pUCm-t vector, positive clones were screened by colony PCR and sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. Plasmids with correct sequencing results were stored as standard positive plasmid templates.

[0081] Example 2 Primer-specific detection

[0082] Using the specific primers of this invention (Primer F1, Primer R1, Primer F2, Primer R2, Primer F3, Primer R3, Primer F4, Primer R4), PCR detection was performed on DNA from *Clostridium perfringens*, *Clostridium novie*, *Clostridium putrefactiveum*, *Clostridium emeticae*, *Clostridium tetani*, *Escherichia coli*, and *Staphylococcus aureus*, respectively, as templates. The PCR reaction system consisted of 12.5 μL of 2×Taq PCR Master Mix, 1 μL of template, 1 μL each of forward and reverse primers (10 μmol / L), and ddH2O to a final volume of 25 μL. The PCR reaction conditions were: 95 ℃ for 3 min; 95 ℃ for 15 s, 55 ℃ for 30 s, 72 ℃ for 25 s, for 35 cycles; 72 ℃ for 5 min. After the amplification was completed, the amplified products were detected by electrophoresis on a 2.0% agarose gel (2.0 g of agarose was weighed, dissolved in 100 mL of TAE buffer, and heated to dissolve, thus preparing a 2.0% agarose gel).

[0083] See test results Figure 2 The following are the electrophoresis results: Lane 1 is the PCR result electrophoresis image of the negative control; Lane 2 is the PCR result electrophoresis image of Clostridium perfringens amplified by PrimerF1 and PrimerR1; Lane 3 is the PCR result electrophoresis image of Clostridium novesiculum amplified by PrimerF2 and PrimerR2; Lane 4 is the PCR result electrophoresis image of Clostridium putrefactivee amplified by PrimerF3 and PrimerR3; Lane 5 is the PCR result electrophoresis image of Clostridium emphysematous amplified by PrimerF4 and PrimerR4; Lanes 6, 7, 8 and 9 are the PCR result electrophoresis images of Clostridium tetani, Escherichia coli, Salmonella and Staphylococcus aureus respectively; Lane 10 is the template-free blank control; M is the DNA molecular weight standard DL 2000 Plus.

[0084] The results showed that when the individual detection primers of this invention were used to amplify the genomic DNA templates of Clostridium perfringens, Clostridium noviformis, Clostridium putrefactivee, Clostridium emeticae, Clostridium tetani, Escherichia coli, and Staphylococcus aureus, clear target bands of 731 bp, 529 bp, 230 bp, and 117 bp appeared in lanes 2, 3, 4, and 5, respectively, in the electrophoresis results. The amplification products of Clostridium tetani, Escherichia coli, Staphylococcus aureus, and the negative control did not show any amplification products. This indicates that the primers designed based on the genomes of each pathogen can efficiently perform PCR on the target genes.

[0085] Example 3: Specificity Validation of Multiplex PCR Detection Method

[0086] The reaction system used in the multiplex PCR amplification reaction of this invention, in 25 μL increments, comprises: 12.5 μL of 2×Rapid Taq premix, 3.5 μL of ultrapure water, 0.5 μL each of PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3, and PrimerR3, 1 μL each of PrimerF4 and PrimerR4, and 4 μL of DNA template. The primer set consists of the following components at the following final concentrations: PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3, PrimerR3, PrimerF4, and PrimerR4, each at 10 μmol / L. The multiplex PCR amplification reaction program is as follows: 95 ℃ for 3 min; 95 ℃ for 15 s, 55 ℃ for 30 s, 72 ℃ for 5 s, 30 cycles; 72 ℃ for 5 min.

[0087] Following the PCR reaction system and conditions described above, multiplex PCR amplification was performed using the genomes of Clostridium tetani, Escherichia coli, Salmonella, and Staphylococcus aureus as templates. A mixture of nucleic acids from Clostridium perfringens, Clostridium novie, Clostridium putrefactiveum, and Clostridium perfringens served as positive controls, and ddH2O as a negative control, to evaluate the specificity of this method.

[0088] The amplification products were then analyzed by 2.0% agarose gel electrophoresis. The results are shown in [link to results]. Figure 3The following are the electrophoresis results for each of the following: Lane 1 shows the PCR results for the positive control; Lane 2 shows the PCR results for the negative control; Lane 3 shows the PCR results for *Clostridium perfringens* C57-1; Lane 4 shows the PCR results for *Clostridium perfringens* C58-1; Lane 5 shows the PCR results for *Clostridium perfringens* C59-2; Lane 6 shows the PCR results for *Clostridium perfringens* C60-2; Lane 7 shows the PCR results for *Clostridium novie* C61-4; Lane 8 shows the PCR results for *Clostridium novie* C61-5; Lane 9 shows the PCR results for *Clostridium putrefactive*; Lane 10 shows the PCR results for *Clostridium emphysematous*; Lanes 11-12 show the PCR results for *Clostridium tetani*, *Escherichia coli*, and *Staphylococcus aureus*, respectively; M represents the DNA molecular weight standard DL 2000 Plus. The results showed that the present invention has high specificity and no cross-reaction with common non-target pathogens such as Clostridium tetani, Escherichia coli, Salmonella, and Staphylococcus aureus, indicating that the primer design has good species specificity.

[0089] Example 4: Sensitivity Validation of Multiplex PCR Detection Method

[0090] The standard plasmids of Clostridium perfringens, Clostridium novie, Clostridium putrefactiveum, and Clostridium emphysematous constructed in Example 1 were serially diluted 10-fold using ddH2O. 9 -10 -1 The DNA template was prepared by mixing equal volumes of the premixed DNA in groups, with nucleic acid-free water serving as a negative control. The PCR reaction system, in 25 μL volumes, included: 12.5 μL of 2×Rapid Taq premix, 3.5 μL of ultrapure water, 0.5 μL each of Primer F1, Primer R1, Primer F2, Primer R2, Primer F3, and Primer R3, 1 μL each of Primer F4 and Primer R4, and 4 μL of DNA template. The primer set consisted of the following components at final concentrations: Primer F1, Primer R1, Primer F2, Primer R2, Primer F3, Primer R3, Primer F4, and Primer R4, each independently at 10 μmol / L. The multiplex PCR amplification program was as follows: 95 ℃ for 3 min; 95 ℃ for 15 s, 55 ℃ for 30 s, 72 ℃ for 5 s, 30 cycles; 72 ℃ for 5 min. After a period of time, the amplification products were analyzed by 2.0% agarose gel electrophoresis. The results are as follows: Figure 4 As shown, *Clostridium perfringens*, *Clostridium novie*, *Clostridium putrefactive*, and *Clostridium emphysematous* correspond to bands at 731 bp, 529 bp, 230 bp, and 117 bp, respectively. The minimum concentration of each clearly detectable single band is 10. 2copies / μL, 10 3 copies / μL, 10 4 copies / μL, 10 3 The method has good sensitivity, with copies / μL.

[0091] Example 5: Repeatability Validation of Multiplex PCR Detection Method

[0092] Using a 5 ng / μL corresponding nucleic acid mixture as a template and ddH2O as a negative control, the PCR reaction system (25 μL) included: 12.5 μL of 2×Rapid Taq premix, 3.5 μL of ultrapure water, 0.5 μL each of PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3, and PrimerR3, 1 μL each of PrimerF4 and PrimerR4, and 4 μL of DNA template. The primer set consisted of the following components at final concentrations: PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3, PrimerR3, PrimerF4, and PrimerR4, each independently at 10 μmol / L. The multiplex PCR amplification program was: 95 ℃ for 3 min; 95 ℃ for 15 s, 55 ℃ for 30 s, 72 ℃ for 5 s, 30 cycles; 72 ℃ for 5 min. The assay was performed three times, once every month, and the repeatability and reliability of the results were determined by electrophoresis. The repeatability verification results are as follows: Figure 5 As shown, the electrophoresis images from the three tests all showed clear target bands at 731bp, 529bp, 230bp, and 117bp, demonstrating good repeatability.

[0093] Example 6: Detection of Clinical Samples

[0094] A total of 206 clinical bovine anal swab samples were collected from different cattle farms in Jiangsu Province. Nucleic acid was extracted using a fully automated nucleic acid extractor and detected using the multiplex PCR method of this invention. The PCR reaction system, in 25 μL volumes, included: 12.5 μL of 2×Rapid Taq premix, 3.5 μL of ultrapure water, 0.5 μL each of PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3, and PrimerR3, 1 μL each of PrimerF4 and PrimerR4, and 4 μL of DNA template. The primer set consisted of the following components at final concentrations: PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3, PrimerR3, PrimerF4, and PrimerR4, each independently at 10 μmol / L. The multiplex PCR amplification program was: 95 ℃ for 3 min; 95 ℃ for 15 s, 55 ℃ for 30 s, 72 ℃ for 5 s, 30 cycles; 72 ℃ for 5 min. min. Meanwhile, referring to published articles on *Clostridium perfringens*. Clostridium noviformis Clostridium putrefactivee Clostridium perfringens and emphysematous Singleton PCR was performed, and the results were compared with those of multiplex PCR for verification.

[0095] Multiplex PCR result interpretation: The experiment is considered valid when the DNA marker bands are clear, the negative control shows no amplification, and the positive control shows specific amplification products of the expected size. Based on a valid experiment, if the sample shows a band of the same size as any target pathogen amplification fragment, it is considered positive for that pathogen's nucleic acid; if multiple target bands appear simultaneously, it is considered positive for the corresponding multiple pathogen nucleic acids; if no specific amplification bands are found, it is considered negative.

[0096] The results of testing 206 clinical samples using the multiplex PCR detection method of this invention showed that 48 of the 206 stool samples were positive. Figure 6 A) Four cases (2%) were positive for *Clostridium noviductus*, 25 cases (12%) were positive for *Clostridium putrefactiveum*, and 19 cases (9%) were positive for *Clostridium emphysematous*. Mixed infections were observed in the samples. Figure 6 B), including one case of mixed infection with Clostridium novie and Clostridium putrefactivee, and nine cases of mixed infection with Clostridium putrefactivee and Clostridium emphysematous. The results of the multiplex PCR clinical sample tests were validated by single-particle PCR. The results of the multiplex PCR method were consistent with those of the single-particle PCR method, and it has advantages such as high detection efficiency, good sensitivity, time saving, and low cost.

[0097] The primer set or kit provided by this invention can be used for multiplex PCR detection of *Clostridium perfringens*, *Clostridium noviformis*, *Clostridium putrefactiveum*, and *Clostridium emphysematous* for non-diagnostic purposes. It can simultaneously detect *Clostridium perfringens* (Plc gene), *Clostridium noviformis* (FliC gene), *Clostridium putrefactiveum* (α-toxin gene), and *Clostridium emphysematous* (Ccta gene). This method exhibits high specificity, showing no cross-reactivity with common non-target pathogens such as *Clostridium tetani*, *Escherichia coli*, *Salmonella*, and *Staphylococcus aureus*, indicating good species specificity of the primer design. The method demonstrates good reproducibility, with consistent intra- and inter-batch detection results, indicating its stability and reliability. Regarding sensitivity, the limits of detection for the four pathogen recombinant plasmid standards of this invention are 10², 10³, and 10⁻⁶, respectively. 4 10³ copies / μL. Compared with other reported Clostridium detection methods, Jin Meijun et al. The established fluorescence quantitative PCR method for Clostridium perfringens has a limit of detection of 7.5 × 10³ copies / μL. This method is comparable to or slightly better than that method in terms of sensitivity for Clostridium perfringens, and it enables simultaneous detection of multiple Clostridium pathogens, which significantly improves the detection efficiency.

[0098] Using the kit provided by this invention, multiplex PCR can unify the amplification conditions of four DNA targets—Clostridium perfringens, Clostridium novie, Clostridium putrefactiveum, and Clostridium perfringens—in the same reaction system. Its advantages include simple operation and short processing time; the entire process from nucleic acid extraction to result interpretation can be completed within 3 hours. The challenge lies in the high homology among the four Clostridium species, leading to interference between primer bands. Simultaneously, it is crucial to ensure that the Tm values ​​of the four primer pairs are close to avoid primer dimer formation, while also ensuring uniform intervals in amplified fragment lengths for easy electrophoretic differentiation. The amplification products of the four primer pairs of this invention have lengths of 731 bp, 529 bp, 230 bp, and 117 bp, respectively, and can be clearly distinguished on a 2% agarose gel.

[0099] Example 7: Primer Exploration and Determination

[0100] Multiple primer sets were designed for each of the four target genes plc, C. novyi alpha-toxin, C. septicum alpha-toxin, and CctA, specifically conserved genes of *Clostridium perfringens*, *Clostridium novyi*, *Clostridium putrefactiveum*, and *Clostridium septicum*, and multiple combination validations were performed according to a certain gradient (as shown in Table 2). Oligo 7 analysis was used to determine the annealing range, and a suitable annealing temperature was selected for preliminary validation (e.g., ...). Figure 7 ).

[0101] Using the primers in Table 2, PCR detection was performed with DNA from *Clostridium perfringens*, *Clostridium novie*, *Clostridium putrefactive*, and *Clostridium emphysematous* as templates. The PCR reaction system consisted of 12.5 μL of 2×Taq PCR Master Mix, 1 μL of template, 1 μL each of forward and reverse primers (10 μmol / L), and ddH2O to a final volume of 25 μL. PCR conditions were as follows: 95 ℃ for 3 min; 95 ℃ for 15 s, 55 ℃ for 30 s, 72 ℃ for 25 s, for 35 cycles; 72 ℃ for 5 min. The amplified products were then detected by 2.0% agarose gel electrophoresis.

[0102] Table 2 Primers used in initial validation 1

[0103]

[0104]

[0105] The positive bands of primer sets 1-6 were not fully amplified, which may be due to differences in amplification ability among different primers. It is proposed to replace some primers, specifically those targeting bands with lower brightness, such as those for *Clostridium novie* and *Clostridium perfringens* (as shown in Table 2). This will be verified (e.g., ...). Figures 8-9 ).

[0106] Table 3. Problems and solutions related to primer sets 1-6

[0107]

[0108] Next step:

[0109] 1. Select primers (N-αT-4) with clear bands and good amplification effect for further screening of Clostridium noviformis primers.

[0110] 2. Select primers (Plc-2, Plc-3) with clear Clostridium perfringens bands and good amplification effect for further screening.

[0111] 3. Primer selection: Primers with clear Clostridium putrefactive bands and good amplification effect (S-αT-2) were selected for the next step of screening.

[0112] 4. Due to the poor multiple positive amplification effect of primers 1-6 for Clostridium emphysematous, primers (CctA-5, CctA-6) were redesigned targeting the CctA gene of Clostridium emphysematous.

[0113] After verification, it was determined that the above primers were used as the basis for reintegration, and primers were designed and combined according to the principle that the amplification fragments of the four Clostridium species were evenly spaced to facilitate electrophoretic differentiation.

[0114] Table 4 Primers used in initial validation 2

[0115]

[0116] The positive bands in groups 7-10 were verified to show that only the Clostridium noviformis band was absent, while the Clostridium perfringens band became brighter. The analysis suggested that this might be due to an inappropriate annealing temperature or interference caused by homology of the target fragment gene. Therefore, the annealing temperature was optimized first, and if the effect was not significant, the primers were improved. Specifically, the annealing temperature for group 9 was optimized (51-60℃).

[0117] Table 5. Problems and solutions related to primer sets 7-10.

[0118]

[0119] Next step:

[0120] Multiple primer set 9 was selected to verify the annealing temperature and observe whether the brightness of the Clostridium novie band changed. Using primer 9, PCR detection was performed with DNA from Clostridium perfringens, Clostridium novie, Clostridium putrefactiveum, and Clostridium perfringens as templates. The reaction mixture consisted of 12.5 μL of 2×Taq PCR Master Mix, 1 μL of template, 1 μL each of forward and reverse primers (10 μmol / L), and ddH2O to a final volume of 25 μL. PCR reaction conditions were: 95 ℃ for 3 min; 95 ℃ for 15 s, TM for 30 s, 72 ℃ for 25 s, 35 cycles; 72 ℃ for 5 min. The amplified products were then detected by 2.0% agarose gel electrophoresis. TM was set with eight annealing temperatures: 60 ℃, 59 ℃, 58 ℃, 56.7 ℃, 55 ℃, 53 ℃, 52 ℃, and 51 ℃. The results are shown below. Figure 10 As shown.

[0121] The target band of *Clostridium noviperi* remained dim after annealing at primer set 9. Analysis suggested this might be due to the homology between the *noviperi* alpha-toxin gene and the *Clostridium perfringens* plc gene, causing amplification interference from the *noviperi* primer (N-αT-4) and the *plc* primer. Therefore, the target gene of *noviperi* changed from Tcnα to fliC (as shown in Table 1). The annealing range was determined using the bioinformatics software Oligo 7, and a suitable annealing temperature was selected for preliminary verification. Using the primers in Table 1, PCR detection was performed using DNA from *Clostridium noviductii* (CVCC 60281, CVCC 60282), *Clostridium perfringens* (CVCC 37, CVCC60081, CVCC60102, CVCC60201), *Clostridium putrefactiveum* (CVCC 60022), and *Clostridium emphysematous* (CVCC 60002) as templates. The mixture consisted of 12.5 μL of 2×Taq PCR Master Mix, 1 μL of template, 1 μL each of forward and reverse primers (10 μmol / L), and ddH2O to a final volume of 25 μL. PCR reaction conditions were: 95 ℃ for 3 min; 95 ℃ for 15 s, 55 ℃ for 30 s, 72 ℃ for 25 s, 35 cycles; 72 ℃ for 5 min. The amplified products were then detected by 2.0% agarose gel electrophoresis. The results are shown below. Figure 11 As shown.

[0122] Next step:

[0123] 1. Due to the unclear multiple bands of Clostridium noviductum, consider changing the target gene and redesigning the primers (fliC).

[0124] 2. Select primers (Plc-2, Plc-3) with clear Clostridium perfringens bands and good amplification effect for further screening.

[0125] 3. Primer selection: Primers with clear Clostridium putrefactive bands and good amplification effect (S-αT-2) were selected for the next step of screening.

[0126] 4. Primers with clear Clostridium emphysema bands and good amplification effect (CctA-5, CctA-6) were selected for further screening.

[0127] Based on the verified and confirmed primers, primers were re-integrated according to the principle of uniform length spacing of the four Clostridium species amplification fragments to facilitate electrophoretic differentiation (e.g., Figure 11 ).

[0128] Primer concentration optimization (10 μmol / L):

[0129] The primer ratios for *Clostridium perfringens*, *Clostridium novesiculum*, *Clostridium putrefactive*, and *Clostridium emphysematous* were as follows: (1) 0.5 μL: 0.5 μL: 0.5 μL: 0.5 μL; (2) 0.5 μL: 0.5 μL: 0.5 μL: 1 μL; (3) 0.5 μL: 0.5 μL: 0.5 μL: 1.5 μL; (4) 0.5 μL: 0.5 μL: 0.5 μL: 2 μL; other reaction conditions were the same as above, and the results were as follows: Figure 12 As shown.

[0130] The primer set was ultimately selected as the primer set for this invention (Table 1), with four bright bands and no other extraneous bands. The primer concentrations were selected as (10 μmol / L) for Clostridium perfringens, Clostridium novesiculum, Clostridium putrefactiveum, and Clostridium emphysematous: 0.5 μL: 0.5 μL: 0.5 μL: 1 μL.

[0131] This invention utilizes multiplex PCR to unify the amplification conditions of four DNA targets in a single reaction system. Its advantages include simple operation and short processing time; the entire process from nucleic acid extraction to result interpretation can be completed within 3 hours. The challenge lies in the high homology among the four Clostridium species, leading to primer band interference. Simultaneously, it is crucial to ensure that the Tm values ​​of the four primer pairs are close to avoid primer dimer formation, while also ensuring uniform intervals in amplified fragment lengths for easy electrophoretic differentiation. In primer design and screening, we designed seven candidate primer pairs for each target gene. Specificity was assessed using Primer-BLAST, and the optimal pairing was screened through primer combination experiments. The final four primer pairs yielded amplification product lengths of 731 bp, 529 bp, 230 bp, and 117 bp, respectively, which were clearly distinguishable on a 2% agarose gel.

[0132] References:

[0133] [1] Method for detecting Clostridium perfringens toxin at border ports: SN / T 2709-2010 [S / OL].

[0134] [2] Meng Dezhi. Establishment of culture and identification methods for Clostridium novesiculum and preparation of monoclonal and polyclonal antibodies against its α-toxin [D]. Shandong Agricultural University, 2019.

[0135] [3] Li Tianmei, Wu Zhengshuang, Xu Dawei, et al. Establishment and preliminary application of rapid PCR method for identifying ovine clostridial disease [J / OL]. Progress in Animal Medicine, 2019, 40(7) [2026-05-19].

[0136] [4] Zhang Yongjia, Si Wei, Ren Chunyu, et al. Establishment and preliminary application of PCR detection method for Clostridium perfringens [J / OL]. Heilongjiang Journal of Animal Husbandry and Veterinary Medicine, 2016(16) [2026-05-19].

[0137] [5] Jin Meijun, Hu Yunhao, Xin Lingxiang, et al. Establishment of a one-step multiplex PCR / RT-PCR detection method for 11 bovine diarrhea pathogens [J]. Bulletin of Microbiology, 2025, 52(1): 383-396.

Claims

1. A primer set for simultaneous detection of *Clostridium perfringens*, *Clostridium novie*, *Clostridium putrefactive*, and *Clostridium emphysematous*, characterized in that... The primer set includes primers PrimerF1 and PrimerR1 designed using the phospholipase C gene of *Clostridium perfringens* as a template, primers PrimerF2 and PrimerR2 designed using the flagellin C gene of *Clostridium novesiculum* as a template, primers PrimerF3 and PrimerR3 designed using the α-toxin gene of *Clostridium putrefactivee* as a template, and primers PrimerF4 and PrimerR4 designed using the cytotoxin A gene of *Clostridium emphysematous* as a template. The sequences of the primers are as follows: PrimerF1: SEQ ID NO.1, PrimerR1: SEQ ID NO.2, PrimerF2: SEQ ID NO.3, PrimerR2: SEQ ID NO.4, PrimerF3: SEQ ID NO.5, PrimerR3: SEQ ID NO.6, PrimerF4: SEQ ID NO.7, PrimerR4: SEQ ID NO.

8.

2. A kit for simultaneous detection of Clostridium perfringens, Clostridium novie, Clostridium putrefactiveum, and Clostridium perfringens, characterized in that, The kit includes the primer set of claim 1.

3. The kit for simultaneous detection of *Clostridium perfringens*, *Clostridium novie*, *Clostridium putrefactive*, and *Clostridium emphysematous* according to claim 2, characterized in that... The kit also includes 2×Rapid Taq premix and ultrapure water.

4. The kit for simultaneous detection of Clostridium perfringens, Clostridium novie, Clostridium putrefactiveum, and Clostridium emphysematous as described in claim 2, characterized in that... The kit also includes a positive control and a negative control.

5. The kit for simultaneous detection of Clostridium perfringens, Clostridium novie, Clostridium putrefactiveum, and Clostridium perfringens according to claim 2, characterized in that... The positive control includes Clostridium perfringens genomic DNA, Clostridium noviformis genomic DNA, Clostridium putrefactiveis genomic DNA, and Clostridium emphysematous genomic DNA; the negative control is ultrapure water.

6. The application of the primer set according to claim 1 or the kit according to any one of claims 2 to 5 in the multiplex PCR detection of Clostridium perfringens, Clostridium novie, Clostridium putrefactivee, and Clostridium perfringens for non-diagnostic purposes, wherein the application includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the genomic DNA extracted from the sample to be tested as a template, a multiplex PCR amplification reaction is performed using the primer set described in claim 1 to obtain multiplex PCR amplification products; (3) Multiplex PCR amplification products were detected by agarose gel electrophoresis; (4) If a specific fragment of 731 bp is amplified, the test result is Clostridium perfringens; if no specific fragment of 731 bp is amplified, the test result is negative for Clostridium perfringens. If a specific fragment of 529 bp is amplified, the test result is positive for Clostridium novipella; If no specific fragment of 529 bp is amplified, the test result is negative for Clostridium novipella; If a specific fragment of 230 bp is amplified, the test result is positive for Clostridium putrefactivee; if no specific fragment of 230 bp is amplified, the test result is negative for Clostridium putrefactivee. If a specific fragment of 117 bp is amplified, the test result is positive for Clostridium emphysematous; if no specific fragment of 117 bp is amplified, the test result is negative for Clostridium emphysematous.

7. The application according to claim 6, characterized in that, The reaction system used in the multiplex PCR amplification reaction in step 2) comprises, in 25 μL increments: 12.5 μL of 2×Rapid Taq premix, 3.5 μL of ultrapure water, 0.5 μL each of PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3 and PrimerR3, 1 μL each of PrimerF4 and PrimerR4, and 4 μL of DNA template.

8. The application according to claim 7, characterized in that, The primer set consists of the following components at final concentrations: PrimerF1, PrimerR1, PrimerF2, PrimerR2, PrimerF3, PrimerR3, PrimerF4, and PrimerR4, each with a concentration of 10 μmol / L.

9. The application according to claim 6, characterized in that, The procedure for the multiplex PCR amplification reaction was as follows: 95 ℃ for 3 min; 95 ℃ for 15 s, 55 ℃ for 30 s, 72 ℃ for 5 s, 30 cycles; 72 ℃ for 5 min.

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