A fluorescent quantitative PCR primer probe set for detecting pathogen of porcine bacterial diarrhea

Abstract

The application discloses a multiplex real-time fluorescent quantitative PCR primer probe set, which is composed of three groups of primers and probes respectively aiming at three kinds of pig bacterial diarrhea pathogens, and the sequences of the primers and the probes are shown as SEQ ID NO:1-9. The primers are designed aiming at the aspA gene sequence of Lawsonia intracellularis (LI), the beta toxin gene sequence of C. perfringens and the Hypothetical Protein gene sequence of B. hyo, so that more primer probe options can be provided for clinical detection and diagnosis of the pig bacterial diarrhea pathogens, and the accuracy and sensitivity of detection are improved.

Description

Technical Field

[0001] This invention belongs to the field of pathogen detection technology, specifically relating to a multiplex real-time quantitative PCR primer and probe set for detecting porcine bacterial diarrhea pathogens. Specifically, the porcine bacterial diarrhea pathogens are *Lowsonia intracellularis* (LI), *Clostridium perfringens* Type C (C. perfringen), and *Brachyspira hyodysenteriae* (B. hyo). This invention also relates to the application of this primer and probe set in the preparation of a porcine diarrhea pathogen detection kit. Background Technology

[0002] Porcine proliferative enteritis, swine clostridial enteritis, and swine spirochetal dysentery are all bacterial diarrheal diseases caused by *Lowsonia intracellularis* (LI), *Clostridium perfringens type C* (C. perfringen), and *Brachyspira hyodysenteriae* (B. hyo), respectively. Porcine proliferative enteritis is a contagious disease transmitted through contact; acute infection can lead to hemorrhagic diarrhea and death in pigs. *Clostridium perfringens type C* infection causes bloody diarrhea and persistent diarrhea in pigs. Swine spirochetal dysentery is a serious intestinal infectious disease; infected pigs may exhibit diarrhea, mucoid hemorrhagic diarrhea, and emaciation; in severe cases, it can lead to hemorrhagic, catarrhal, or necrotic colitis and cecal inflammation. All three bacteria can cause bloody diarrhea, acute onset, and death in infected pigs, making direct differential diagnosis difficult based on clinical symptoms alone. Furthermore, mixed infections are common, further complicating the differential diagnosis process. Currently, using microbiological and serological methods for bacterial pathogen detection is time-consuming and labor-intensive. Therefore, there is an urgent need to establish a rapid and efficient detection method that can directly differentiate and diagnose these three types of bacteria, which would play a very important role in disease prevention and control.

[0003] To date, only two Lawsonia intracellularis isolates have had their complete genome sequences published on NCBI: PHE / MN1-00 and N343. These two genomes contain eight single nucleotide polymorphisms (SNPs) and 70 insertions or deletions within their intragenetic regions. Additionally, 16 SNPs and 20 insertions or deletions were identified in the intergenetic regions. Currently, commonly used detection genes include aspA, ubiE, 16S rDNA, or dnaA. Although 16S rDNA is highly conserved, the homology of 16S rDNA among different bacteria is high, leading to false positives. Studies have found that aspA in Lawsonia intracellularis isolated from pig feces has a high detection rate and is highly conserved, exhibiting a high copy number within the strain.

[0004] Clostridium perfringens has four main toxins: α, β, ε, and ι, which possess lethal, necrotic, and cytotoxic activities. Based on the type of toxin secreted, the bacterium can be classified into five types: A, B, C, D, and E. Type C Clostridium perfringens produces both α and β toxins; β toxin is considered the cause of necrotizing enterocolitis in type C strains. Although the genome of type C strains also carries genes for transcribing other toxins, such as Clostridium perfringens α toxin and Clostridium polycystic lysin, research evidence indicates that β toxin is the main lethal toxin of type C strains. While type B Clostridium perfringens also possesses β toxin, it is primarily found in the intestines of animals, with extremely low detection rates in feces.

[0005] The *B. hyo* hyrospira gene is carried on a highly virulent plasmid of approximately 65-110 kb, which may contain additional toxin genes encoding CPE or TpeL. *B. hyo* differs from other spirochetes in signal transduction, amino acid transport, and metabolic systems, and 15 potential virulence genes and 6 hemolysin genes have been identified. Later studies have found that hemolysin, flagella, bacterial chemotaxis and motility, outer membrane proteins, NADH oxidase, and iron metabolism proteins all play a role in pathogenicity. The *Hypothetical Protein* gene plays a crucial role in the pathogenesis of disease caused by *B. hyo* hyrospira, and this gene is highly conserved.

[0006] Currently, no methods have been reported for designing multiplex real-time quantitative PCR primers and probes using the aspA gene, β-toxin gene, and Hypothetical Protein gene to simultaneously detect porcine bacterial diarrhea pathogens. CN115058528A discloses a primer and probe set for detecting porcine diarrhea pathogens, which can be used to detect six pathogens, including Lawsonia intracellularis, Clostridium perfringens type C, and Treponema swine dysenteryum. The applicant matched the primers reported therein and confirmed that the target genes for detecting the three pathogens are 16S rDNA, GQX59-13090, and plc gene, respectively. Furthermore, the applicant used the primer and probe set for these three pathogens to detect Lawsonia intracellularis, Clostridium perfringens type C, and Treponema swine dysenteryum, and found that it had defects such as low detection sensitivity.

[0007] Based on this, we designed a triple real-time quantitative PCR primer and TaqMan probe set, which can rapidly and accurately detect three pathogens: LI, C. perfringens, and B. hyo. We selected the aspA gene of Lawsonia intracellularis, the β-toxin gene of Clostridium perfringens type C, and the Hypothetical Protein gene of Treponema swine dysenteryi as targets. The designed primers have the advantages of high specificity and good sensitivity. Summary of the Invention

[0008] The primary objective of this invention is to provide a set of multiplex real-time quantitative PCR primers and TaqMan probes capable of simultaneously detecting three porcine bacterial diarrhea pathogens: *Livistona spp.*, *C. perfringens*, and *B. hyo*. On one hand, this invention provides more multiplex PCR primer and probe options for the clinical detection and diagnosis of porcine bacterial diarrhea pathogens; on the other hand, it also helps to improve the accuracy and sensitivity of detection.

[0009] To achieve the above objectives, the applicant designed and validated multiple sets of primers and probes targeting the aspA gene sequence of *Lawsonia intracellularis* (LI), the β-toxin gene sequence of *C. perfringens*, and the hydrothetical protein gene sequence of *B. hyo*, based on the principles of multiplex real-time quantitative PCR primer and probe design. The designed sets of primers and probes were screened based on the accuracy, specificity, and sensitivity of quantitative PCR amplification, ultimately resulting in a real-time quantitative PCR primer and probe set consisting of three sets of primers and probes targeting the three aforementioned porcine bacterial diarrhea pathogens. The specific primer and probe sequences are as follows:

[0010] The first group consists of primers and probes designed for the aspA gene of Lawsonia intracellularis. The upstream primer (LI-qF) sequence is shown in SEDID NO:1, the downstream primer (LI-qR) sequence is shown in SEQ ID NO:2, and the probe (LI-P) sequence is shown in SEQ ID NO:3.

[0011] The second group consists of primers and probes designed for the Clostridium perfringens β-toxin gene. The upstream primer (CqF) sequence is shown in SED ID NO:4, the downstream primer (CqR) sequence is shown in SEQ ID NO:5, and the probe (CP) sequence is shown in SEQ ID NO:6.

[0012] The third group consists of primers and probes designed for the Hypothetical Protein gene of Porcine dysentery short spirochetes. The sequence of the upstream primer (B.hyo-qF) is shown in SED ID NO:7, the sequence of the downstream primer (B.hyo-qR) is shown in SEQ ID NO:8, and the sequence of the probe (B.hyo-P) is shown in SEQ ID NO:9.

[0013] When using these primers and probes, they are not independent but are mixed together in a certain proportion to form a system.

[0014] Furthermore, the probe has a fluorescent group attached to its 5' end and a fluorescence quencher attached to its 3' end. Specifically, the first group of probes (LI-P) has a FAM fluorescent group and a BHQ1 fluorescence quencher; the second group of probes (CP) has a Cy3 fluorescent group and a BHQ1 fluorescence quencher; and the third group of probes (B.hyo-P) has a Hex fluorescent group and a BHQ1 fluorescence quencher.

[0015] A second objective of this invention is to provide the application of the aforementioned multiplex real-time quantitative PCR primer-probe set in the detection of pathogens causing porcine bacterial diarrhea. Specifically, it involves using the primer-probe set for disease diagnosis of porcine bacterial diarrhea, or for laboratory screening and identification of pathogens causing porcine bacterial diarrhea.

[0016] A third objective of this invention is to provide the application of the aforementioned multiplex real-time quantitative PCR primer and probe set in the preparation of a kit for detecting porcine bacterial diarrhea pathogens.

[0017] A fourth objective of this invention is to provide a kit for detecting porcine bacterial diarrhea pathogens, the kit comprising the multiplex real-time quantitative PCR primer and probe set described above.

[0018] The fifth objective of this invention is to provide a method for detecting porcine bacterial diarrhea pathogens, the method comprising the steps of adding the aforementioned primer and probe set to a reaction system and performing multiplex real-time quantitative PCR amplification.

[0019] Furthermore, the reaction system is as follows:

[0020]

[0021] Furthermore, the amplification reaction procedure is as follows: reaction at 25℃ for 10 min, pre-denaturation at 95℃ for 30 s; denaturation at 95℃ for 5 s, annealing and extension at 54℃ for 30 s, cycled 45 times.

[0022] The beneficial effects of this invention are:

[0023] (1) The main clinical symptoms of porcine proliferative enteritis, porcine clostridial enteritis, and porcine spirochetal dysentery are hemorrhagic diarrhea and acute death, and mixed infections are common. Various methods have been developed for detecting these pathogens, but microbiological and serological methods are time-consuming, labor-intensive, and lack sensitivity and specificity. Conventional PCR methods can only detect one bacterium at a time, resulting in low detection efficiency. Therefore, rapid diagnosis of these pathogens is crucial for providing timely and effective prevention and control strategies. To this end, this invention develops a multiplex real-time quantitative PCR method for the simultaneous detection of three pathogens, providing a high-value tool for large-scale bacterial surveillance, epidemiological investigations, and prevention and control of these diseases, including Lawsonia intracellularis, Clostridium perfringens type C, and Spirochetes swine dysentery. This invention enriches the types of primers and probes for detecting these pathogens, providing more options for clinical detection and diagnosis.

[0024] (2) Currently, most detection methods for the aspA gene, β-toxin gene, and Hypothetical Protein gene involve singleton PCR. However, the applicant, based on the design principles of multiplex PCR primers and probes and after extensive screening, has developed a primer and probe set that exhibits advantages such as high amplification efficiency, strong fluorescence signal, and no interference. This invention not only enables the detection of these three pathogens but also improves the accuracy and sensitivity of the detection.

[0025] In terms of specificity, the method established in this invention only shows amplification signals for three bacteria: L. LI, C. perfringens, and B. hyo, while no amplification signals are found for DNA from Escherichia coli, Salmonella, Staphylococcus aureus, epidemic diarrhea virus, infectious gastroenteritis virus, rotavirus, and coccidia, indicating that the method has good specificity.

[0026] In terms of sensitivity, the method established in this invention has a minimum detection limit of 10 for LI plasmids. 1The minimum detection limit for C. perfringens plasmid and B. hyo plasmid is 10 copies / μL. 0 The detection limit is 300 pg / μL for nucleic acid in *Li* bacterial culture and 30 pg / μL for nucleic acid in *C. perfringens* and *B. hyo* bacterial cultures. The detection sensitivity of this invention is significantly higher than that of similar methods reported in the literature.

[0027] (3) Real-time quantitative PCR does not require observation of PCR products by gel electrophoresis, which reduces time and risk of cross-contamination, and has the advantages of being simple and fast. Attached Figure Description

[0028] Figure 1 These are the results of a screening experiment using a triple real-time quantitative PCR primer and probe set.

[0029] Figure 2 This indicates the detection sensitivity of the present invention for LI plasmids; 1-9 in the figure represent 10. 8 -10 0 A mixed plasmid template of 10 copies / μL, with 10 serving as a negative control.

[0030] Figure 3 This invention demonstrates the detection sensitivity of C. perfringens plasmids; figures 1-9 represent 10. 8 -10 0 A mixed plasmid template of 10 copies / μL, with 10 serving as a negative control.

[0031] Figure 4 This indicates the detection sensitivity of the present invention for B. hyo plasmids; 1-9 in the figure represent 10. 8 -10 0 A mixed plasmid template of 10 copies / μL, with 10 serving as a negative control.

[0032] Figure 5 This invention relates to the detection sensitivity of LI bacterial culture nucleic acid.

[0033] Figure 6 This invention relates to the detection sensitivity of nucleic acid in C. perfringens bacterial culture.

[0034] Figure 7 This invention relates to the detection sensitivity of B. hyo bacterial culture nucleic acid.

[0035] Figure 8 The figure shows the detection sensitivity of nucleic acid in the culture of three pathogens after the improvement of the method in the literature (CN 115058528A). In the figure, A is LI, B is C. perfringens, and C is B. hyo. Detailed Implementation

[0036] The present invention will now be described in detail with reference to specific embodiments. It should be understood that the specific embodiments described are merely illustrative of the invention and are not intended to limit the invention.

[0037] The experimental procedure of this invention mainly includes the following steps:

[0038] 1) Screening of primer and probe combinations for triple real-time quantitative PCR of LI, C. perfringens and B. hyo: Multiple sets of PCR primers and probes were designed and screened according to the design principles of multiplex PCR primers and probes to obtain the optimal combination;

[0039] 2) Determination of triplet real-time quantitative PCR reaction conditions: Optimize the reaction system and conditions for multiplex real-time quantitative PCR amplification;

[0040] 3) Validate the determined detection method, including sensitivity, specificity, and repeatability.

[0041] Example 1: Screening of triple real-time quantitative PCR primer and probe combinations

[0042] 1. First, the complete aspA gene sequence of *Lawsonia intracellularis*, the complete β-toxin gene sequence of *Clostridium perfringens* type C, and the complete Hypothetical Protein gene sequence of *Spirometra hygroscopicus* were retrieved from NCBI. Then, all sequences were downloaded and imported into CLC Genomics Workbench for comparison. The conserved sequences of each gene were analyzed, and primers were designed from the highly conserved sequences. Each primer was allowed a maximum of one base mismatch. According to the design principles of triple real-time quantitative PCR primers and probes, three sets of primers and probes were designed, totaling 9 pairs (numbered 1-9, with 1-3 as the first set, 4-6 as the second set, and 7-9 as the third set). Each pair consists of one upstream and one downstream primer and one TaqMan probe, as shown in Table 1.

[0043] Table 1: Primers and probes for triple real-time quantitative PCR

[0044]

[0045]

[0046] 2. Primer screening for triple real-time quantitative PCR

[0047] (1) Preparation of three gene recombination expression plasmids

[0048] After retrieving the complete aspA gene sequence of Lawsonia intracellularis, the complete β-toxin gene sequence of Clostridium perfringens type C, and the complete Hypothetical Protein gene sequence of Spirochetes swine dysenteriae from NCBI, primers were designed from both ends of each sequence to amplify the complete sequence. The sequence was then ligated into the pMD19-T vector and introduced into Escherichia coli. Positive colonies were selected for amplification culture and plasmid extraction was performed.

[0049] (2) Mixing of expression plasmids (preparation of templates with different concentrations)

[0050] The concentration of the extracted standard plasmid was determined using a K5800 / C / H / T ultra-micro spectrophotometer, and the copy number of DNA per unit volume of plasmid was calculated. Then, the three plasmids were mixed and diluted to a concentration of 10 for each plasmid. 8 -10 0 Copy the mixing template by / μL.

[0051] (3) Triple real-time quantitative PCR amplification

[0052] Reaction system: 2 μL template, 0.25 μL LI-qF (10 μmol / L), 0.25 μL LI-qR (10 μmol / L), 0.5 μL LI-P (10 μmol / L), 0.25 μL CqF (10 μmol / L), 0.25 μL CqR (10 μmol / L), 1.5 μL CP (10 μmol / L), 0.5 μL B.hyo-qF (10 μmol / L), 0.5 μL B.hyo-qR (10 μmol / L), 0.5 μL B.hyo-P (10 μmol / L), 12.5 μL 2×Probe qPCR Mix (with UNG), 0.4 μL ROX, 5.6 μL ddH2O, total 25 μL.

[0053] Reaction program: 25℃ for 10 min, 95℃ for 30 s, 95℃ for 5 s, 54℃ for 30 s, with each of the 95℃ for 5 s and 54℃ for 30 s cycles repeated 45 times.

[0054] 3. Test Results

[0055] Figure 1 The three sets of designed primers and probes were used to amplify the concentration of 10. 8 Amplification curves of a mixed template containing copies / μL of LI, C. perfringens, and B. hyo plasmids. Figure 1It can be seen that when using the first set of primers and probes, primers 1, 2, and 3 can amplify the LI, C. perfringens, and B. hyo genes respectively, and the amplification curves are good. When using the second set of primers and probes, although primer 4 can amplify the aspA gene, its amplification curve is not as good as that of primer 1, and the fluorescence signals of primers 5 and 6 are not strong. When using the third set of primers and probes, primer 7 can amplify the aspA gene, but it interferes with primers 8 and 9, resulting in no amplification curves for the β-toxin gene and the Hypothetical Protein gene.

[0056] Therefore, based on a comprehensive comparison of accuracy, specificity, and sensitivity, the first set of primers and probes was determined to be the optimal primer and probe set for triple real-time quantitative PCR, and the reaction system and conditions were optimized accordingly.

[0057] Example 2: Optimization of Triple Real-Time Quantitative PCR Reaction System and Conditions

[0058] The reaction system and conditions of triplet real-time quantitative PCR affect dimer amplification, non-specific amplification, and the amplification curve. Therefore, the PCR reaction system and conditions also have a certain impact on the amplification effect. Based on this, we further optimized the reaction system and conditions of triplet real-time quantitative PCR, selecting the system with high amplification efficiency, strong fluorescence signal intensity, and low Ct value as the optimal system. The specific method is as follows:

[0059] (1) First, determine the optimal annealing temperature range. Divide the sum of the Tm values ​​of the upstream and downstream primers by two to obtain an intermediate value. Using this as a reference, set the temperature gradients to 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, and 57℃, respectively. In the experiment, 10 5 A plasmid mixture template of copies / μL was added to the reaction system, and gradient quantitative PCR was performed using the matrix method to determine the optimal annealing temperature. The results showed that at 54℃, the Ct value for LI was 20.35, for C. perfringens it was 21.17, and for B. hyo it was 19.9, indicating strong fluorescence signal intensity and low Ct values. Therefore, 54℃ was determined to be the optimal annealing temperature.

[0060] (2) Next, the concentrations of upstream and downstream primers and probes were determined. Four gradients were set at 200 nmol / L intervals, with a final concentration ranging from 200 to 1000 nmol / L, for matrix optimization. Based on the optimal system for single-phase real-time quantitative PCR, the reaction systems of *B. hyo* and *C. perfringens* were first fixed. The amount of primer (10 μmol / L) added to *B. hyo* was 1 μL, and the amount of probe (10 μmol / L) added was 1 μL. The amount of primer (10 μmol / L) added to *C. perfringens* was 0.8 μL, and the amount of probe (10 μmol / L) added was 0.8 μL. The LI primers and probes were cross-reacted in amounts of 0.25 μL, 0.5 μL, 1 μL, and 1.5 μL to find the optimal reaction system for LI (see Table 2). The results showed that the optimal amount of primer added to LI was 0.25 μL, and the optimal amount of probe added was 0.5 μL.

[0061] Table 2: Ct values ​​of LI, C. perfringens, and B. hyo for different primer and probe amounts of LI

[0062]

[0063]

[0064] The reaction systems of LI and B. hyo were then fixed using the same method. The amount of primer (10 μmol / L) added to LI was 0.25 μL, and the amount of probe (10 μmol / L) added was 0.5 μL. For B. hyo, the amount of primer (10 μmol / L) added was 0.4 μL, and the amount of probe (10 μmol / L) added was 0.8 μL. The optimal reaction system for C. perfringens was explored by cross-reacting with 0.25 μL, 0.5 μL, 1 μL, and 1.5 μL of primer and probe, as shown in Table 3. The results showed that the optimal amount of primer and probe for C. perfringens was 0.25 μL and 1.5 μL, respectively.

[0065] Table 3: Ct values ​​of LI, C. perfringens, and B. hyo for different primer and probe amounts in C. perfringens

[0066]

[0067]

[0068] Finally, the optimal primer-probe ratio for *B. hyo* was determined. The optimal primer (10 μmol / L) and probe (10 μmol / L) concentrations for *Li* were 0.25 μL and 0.5 μL, respectively. Similarly, the optimal primer (10 μmol / L) and probe (10 μmol / L) concentrations for *C. perfringens* were 0.25 μL and 1.5 μL, respectively. Cross-reactions with 0.25 μL, 0.5 μL, 1 μL, and 1.5 μL of *B. hyo* primers and probes were explored, as shown in Table 4. The results showed that the optimal primer and probe concentrations for *B. hyo* were both 0.5 μL and 0.5 μL, respectively.

[0069] Table 4: Ct values ​​of LI, C. perfringens, and B. hyo for different primer and probe amounts of B. hyo

[0070]

[0071] The optimal triplet real-time quantitative PCR reaction system and conditions were finally determined as follows:

[0072] Table 5 Optimal Reaction System for Triple Real-Time Quantitative PCR

[0073]

[0074]

[0075] The triple real-time quantitative PCR amplification reaction program is as follows: 25℃ for 10 min, 95℃ for 30 s, 95℃ for 5 s, and 54℃ for 30 s, with each of the 95℃ for 5 s and 54℃ for 30 s cycles repeated 45 times.

[0076] Standard curves for triple real-time quantitative PCR of LI, C. perfringens, and B. hyo were obtained. The amplification efficiency of LI was 100.8%, that of C. perfringens was 110.074%, and that of B. hyo was 100.788%. The linear equations were Y(LI) = -3.303X(LI) + 40.846, Y(C. perfringens) = -3.102X(C. perfringens) + 40.462, and Y(B. hyo) = -3.303X(B. hyo) + 42.625, respectively.

[0077] Example 3: Verification Experiment of Detection Effect

[0078] 1. Specificity test

[0079] The specificity of triplet real-time quantitative PCR was studied using the optimal reaction system and conditions. Amplification was performed using LI nucleic acid, C. perfringens nucleic acid, B. hyo nucleic acid, Escherichia coli nucleic acid, Salmonella nucleic acid, Staphylococcus aureus nucleic acid, epidemic diarrhea virus nucleic acid, infectious gastroenteritis virus nucleic acid, rotavirus nucleic acid, coccidia nucleic acid, and double-distilled water as templates to verify the specificity of triplet real-time quantitative PCR.

[0080] The experimental results showed that the established triplet real-time quantitative PCR method only showed positive amplification signals for the genes of three bacteria: LI, C. perfringens, and B. hyo. No fluorescence signals were detected for Escherichia coli nucleic acid, Salmonella nucleic acid, Staphylococcus aureus nucleic acid, epidemic diarrhea virus nucleic acid, infectious gastroenteritis virus nucleic acid, rotavirus nucleic acid, coccidia nucleic acid, and double-distilled water, indicating that the established triplet real-time quantitative PCR method has good specificity.

[0081] 2. Sensitivity test

[0082] (1) Sensitivity to plasmid detection

[0083] Using the optimal reaction system and conditions for triple real-time quantitative PCR, amplification was performed on prepared copies of different numbers (10⁻⁶). 8 -10 0 Sensitivity tests were performed on a mixed plasmid template containing copies / μL, and the results are shown in […]. Figure 2-4 The minimum detection limit for plasmids is LI: 10. 1 copy / μL ( Figure 2 ), C. perfringens: 10 0 copy / μL ( Figure 3 ), B.hyo: 10 0 copy / μL ( Figure 4 ).

[0084] (2) Sensitivity to bacterial suspension detection

[0085] First, nucleic acid templates of different concentrations of bacterial suspensions were prepared. Nucleic acid was extracted from three types of bacteria separately, and the concentration of each bacterial nucleic acid was determined. The nucleic acid concentrations of the three bacteria were all diluted to 90 ng / μL. Then, the nucleic acids from the three bacteria were mixed in a 1:1:1 ratio to obtain templates with a nucleic acid concentration of 30 ng / μL for each bacterium. These templates were then serially diluted 10-fold to obtain mixed templates with concentrations of 30 ng / μL, 3 ng / μL, 300 pg / μL, 30 pg / μL, 3 pg / μL, and 0.3 pg / μL. The prepared mixed nucleic acid templates of different bacterial suspension concentrations were amplified using the optimal reaction system and conditions. Results are shown below. Figure 5-7The detection limit of the triple real-time quantitative PCR of the present invention for nucleic acid in LI bacterial culture is 300 pg / μL. Figure 5 The detection limit for nucleic acids in *C. perfringens* and *B. hyo* bacterial cultures was 30 pg / μL. Figure 6 ,7).

[0086] In addition, using the primers disclosed in CN 115058528A and designing corresponding probes, nucleic acid templates of different concentrations of bacterial cultures were prepared. Nucleic acid was extracted from three types of bacteria, and the nucleic acid concentration of each bacterium was determined. The nucleic acid concentrations of the three bacteria were all diluted to 30 ng / μL, and then mixed in a 1:1:1 ratio to obtain templates with a nucleic acid concentration of 10 ng / μL for each bacterium. These templates were then serially diluted 10-fold to obtain mixed templates with concentrations of 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, and 1 pg / μL. Detection was performed under the following conditions, and the results are shown below. Figure 8 The detection limit of this method for three nucleic acids, LI, C. perfringens, and B. hyo, is 1 ng / μL.

[0087] LI primers and probes:

[0088] LI-F: 5'-CCGTGAGGTGTTGGGTTAAGTC-3'

[0089] LI-R: 5'-TCTAGAGTGCCCAACTTTACTTGATG-3'

[0090] LI-P:ROX-CGCAACGAGCGCAACCCTTATCTTTAG-BHQ2

[0091] C. perfringens primers and probes:

[0092] Cp-F: 5'-GCAGAGGAAAGAAAAGAACAGTA-3'

[0093] Cp-R: 5'-ACCTCTTGCATATTCTTTTGACC-3'

[0094] Cp-P:HEX-AAAATAAACACAGTAGGTTGC-BHQ1

[0095] B.hyo primers and probes:

[0096] BH-F: 5'-GGAATGTGGGAGAGATTGATCAA-3'

[0097] BH-R: 5'-TCGGCAGATCTTTGGTATAAATGA-3'

[0098] BH-P; CY5-ATCTTCCTCCATGCCATATGATGTGCCA-BHQ3

[0099] Reaction system:

[0100]

[0101]

[0102] Reaction procedure:

[0103] The temperature was set at 50℃ for 2 minutes, 93℃ for 2 minutes, 93℃ for 15 seconds, and 56℃ for 30 seconds, with each of the 93℃ for 15 seconds and 56℃ for 30 seconds cycles repeated 40 times.

[0104] 3. Repeatability test

[0105] Nucleic acid was extracted from three different batches of cultured bacteria, then mixed, and detected using the optimal reaction system and conditions for triple real-time quantitative PCR. 6 copies / μL, 10 5 copies / μL, 10 4 For the mixed plasmids at a concentration of copies / μL, the coefficients of variation within and between groups were calculated. Repeatability test results showed that the coefficients of variation (Ct) for plasmid standards at different dilution gradients were below 4, indicating good repeatability (see Table 6).

[0106] Table 6 Results of Triple Real-Time Quantitative PCR Repeatability Tests

[0107]

[0108] 4. Clinical sample testing

[0109] (1) Results of clinical sample detection by single-phase quantitative PCR

[0110] Single-color quantitative PCR was used to detect 226 clinical stool samples. 73 samples were positive for Lilium oryzae (32.30%), 41 samples were positive for C. perfringens (18.14%), and 29 samples were positive for B. hyo (12.83%) (see Table 7).

[0111] Table 7 Results of clinical sample testing using singlet quantitative PCR method

[0112]

[0113] (2) Results of Triple Real-Time PCR Detection of Clinical Samples

[0114] Triple quantitative PCR was used to detect 226 clinical stool samples. 69 samples were positive for *Ligusticum striatum* (LI) (detection rate 30.53%), 41 samples were positive for *C. perfringens* (detection rate 18.14%), and 29 samples were positive for *B. hyo* (detection rate 12.83%). Among these, 4 samples showed co-detection of LI and *C. perfringens*, 20 samples showed co-detection of LI and *B. hyo*, 4 samples showed co-detection of *C. perfringens* and *B. hyo*, and 10 samples showed co-detection of LI, *C. perfringens*, and *B. hyo* (see Table 8).

[0115] Table 8 Results of clinical sample testing using triple fluorescence quantitative PCR method

[0116]

[0117] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A multiplex real-time fluorescent quantitative PCR primer probe set, characterized in that It consists of three sets of primers and probes targeting three pathogens causing porcine bacterial diarrhea: Lawsonia intracellularis, Clostridium perfringens type C, and Treponema swine dysenteryi. The sequences of the primers and probes are as follows: The first group consists of primers and probes designed for the aspA gene of Lawsonia intracellularis. The upstream primer sequence is shown in SED ID NO:1, the downstream primer sequence is shown in SEQ ID NO:2, and the probe sequence is shown in SEQ ID NO:

3. The second group consists of primers and probes designed for the Clostridium perfringens β-toxin gene. The upstream primer sequence is shown in SED ID NO:4, the downstream primer sequence is shown in SEQ ID NO:5, and the probe sequence is shown in SEQ ID NO:

6. The third group consists of primers and probes designed for the Hypothetical Protein gene of Porcine dysentery short spirochetes. The upstream primer sequence is shown in SED ID NO:7, the downstream primer sequence is shown in SEQ ID NO:8, and the probe sequence is shown in SEQ ID NO:

9.

2. The multiplex real-time fluorescent quantitative PCR primer probe set of claim 1, wherein: The probe has a fluorescent group attached to its 5' end and a fluorescence quenching group attached to its 3' end.

3. The multiplex real-time fluorescent quantitative PCR primer probe set of claim 2, wherein: The first group of probes has a fluorescent group of FAM and a fluorescence quenching group of BHQ1; the second group of probes has a fluorescent group of Cy3 and a fluorescence quenching group of BHQ1; the third group of probes has a fluorescent group of Hex and a fluorescence quenching group of BHQ1.

4. The application of the multiplex real-time quantitative PCR primer and probe set according to any one of claims 1-3 in the detection of porcine bacterial diarrhea pathogens for non-diagnostic purposes, wherein the porcine bacterial diarrhea pathogens are Lawsonia intracellularis, Clostridium perfringens type C, and Treponema swine dysenteryis.

5. The application of the multiplex real-time quantitative PCR primer and probe set according to any one of claims 1-3 in the preparation of a kit for detecting porcine bacterial diarrhea pathogens, wherein the porcine bacterial diarrhea pathogens are Lawsonia intracellularis, Clostridium perfringens type C, and Treponema swine dysenteryi.

6. A kit for detecting pathogens of porcine bacterial diarrhea, the kit comprising the multiplex real-time quantitative PCR primer and probe set as described in any one of claims 1-3, wherein the pathogens of porcine bacterial diarrhea are Lawsonia intracellularis, Clostridium perfringens type C, and Treponema swine dysenteryi.

7. A method of detecting a pathogen of porcine bacterial diarrhoea for non-diagnostic purposes, characterised in that: The method includes the step of adding the primer and probe set according to any one of claims 1-3 to the reaction system and performing multiplex real-time quantitative PCR amplification, wherein the pathogen of swine bacterial diarrhea is Lawsonia intracellularis, Clostridium perfringens type C and Treponema swine dysenteryis.

8. The detection method as described in claim 7, characterized in that, The reaction system is as follows:

9. The detection method as described in claim 7, characterized in that, The amplification reaction procedure is as follows: reaction at 25℃ for 10 min, pre-denaturation at 95℃ for 30 s; denaturation at 95℃ for 5 s, annealing and extension at 54℃ for 30 s, cycled 45 times.

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