Kit and detection method for detecting multiple piglet diarrhea pathogens

Abstract

The invention provides a kit for detecting multiple piglet diarrhea pathogens and a detection method, and belongs to the technical field of biological information detection. The kit comprises a reaction reagent, wherein the reaction reagent comprises a porcine epidemic diarrhea virus specific primer and a probe, a porcine delta coronavirus specific primer and a probe, a piglet pathogenic escherichia coli specific primer and a probe, a clostridium perfringens specific primer and a probe, a buffer solution, an enzyme mixed solution and a freeze-drying protective additive. The detection method adopts the kit for detecting various piglet diarrhea pathogens. According to the kit disclosed by the invention, simultaneous detection of four target genes including the porcine epidemic diarrhea virus, the porcine delta coronavirus, the piglet pathogenic escherichia coli and the clostridium perfringens can be realized only through one-time amplification, so that the detection time is greatly shortened while the detection cost is reduced; the kit has extremely high specificity, sensitivity and repeatability, and the primers and the probes do not interfere with each other; good normal-temperature storage stability is also realized.

Description

Technical Field

[0001] This invention relates to the field of bioinformatics detection technology, and in particular to a reagent kit and detection method for detecting multiple pathogens causing diarrhea in piglets. Background Technology

[0002] With the rapid and large-scale development of my country's pig farming industry, the number of diseases in pig farms has also increased, especially diarrheal diseases in piglets, which have become the leading cause of death in piglets, accounting for 50%-70% of the total mortality rate. In addition to some deaths, piglets suffering from diarrheal diseases may also experience problems such as decreased feed intake, digestive and absorptive disorders, and incomplete immune system development, leading to a significant increase in feed conversion ratio, immunosuppression, and an increase in the proportion of runts, causing significant economic losses to the pig farming industry.

[0003] Besides management factors, diarrheal diseases in piglets are mainly caused by various viral or bacterial pathogens. Common viruses causing piglet diarrhea include porcine epidemic diarrhea virus, porcine delta coronavirus, porcine transmissible gastroenteritis virus, and porcine rotavirus. Common bacteria causing piglet diarrhea include pathogenic Escherichia coli and Clostridium perfringens. Rapidly identifying common pathogens causing piglet diarrhea and implementing control measures as early as possible is crucial for improving piglet survival and health rates in the pig farming industry.

[0004] The detection of diarrheal diseases in piglets currently mainly involves pathogen isolation and culture, immunoassay, and PCR detection, among which PCR detection is the most commonly used due to its short detection time and high accuracy. PCR detection methods are divided into conventional PCR and real-time fluorescence PCR, both of which are applied to piglet diarrheal pathogens. Relatively speaking, real-time fluorescence PCR, with its shorter detection time and lower contamination, has greater advantages in clinical testing, and real-time fluorescence PCR detection of some antigens has been elevated to national, industry, and local standards. However, existing PCR detection methods for piglet diarrheal pathogens also have some problems. First, most existing detection reagents are for single-disease detection, with a maximum of three-disease detection, resulting in long detection times and high costs. Second, most existing detection reagents are liquid reagents, requiring cold chain transportation and storage at -20℃, which limits their wider application.

[0005] Based on the above situation, there is an urgent need to develop a reagent kit that can detect multiple pathogens at once and can be stored and transported at room temperature. Summary of the Invention

[0006] The technical solution adopted in this invention is to provide a reagent kit and detection method for detecting multiple pathogens causing diarrhea in piglets, so as to solve the technical problems of existing PCR detection methods for pathogens causing diarrhea in piglets, which have the problems of small number of pathogens detected per test and the need for cold chain transportation and preservation.

[0007] To address the aforementioned technical problems, this invention provides a kit for detecting multiple pathogens causing diarrhea in piglets, comprising reaction reagents including specific primers and probes for porcine epidemic diarrhea virus, specific primers and probes for porcine delta coronavirus, specific primers and probes for pathogenic Escherichia coli in piglets, specific primers and probes for Clostridium perfringens, buffer solution, enzyme mixture, and lyophilization protectant.

[0008] Preferably, the porcine epidemic diarrhea virus-specific primers, the porcine delta coronavirus-specific primers, the porcine pathogenic Escherichia coli-specific primers, and the Clostridium perfringens-specific primers all include an upstream primer and a downstream primer; the 5' end of the porcine epidemic diarrhea virus probe, the porcine delta coronavirus probe, the porcine pathogenic Escherichia coli probe, and the Clostridium perfringens probe includes any one of the FAM, HEX, ROX, or Cy5 fluorescent reporter groups, and the 3' end includes any one of the BHQ1 or BHQ2 fluorescent quencher groups; The nucleotide sequences of the upstream and downstream specific primers and probes are as follows: Upstream primer for porcine epidemic diarrhea virus: 5'-TAGTGGTACATTGCTTGTAG-3'; Porcine epidemic diarrhea virus downstream primer: 5'-GAGTAGTCGCCGTGTTTTGA-3'; Porcine epidemic diarrhea virus probe: 5'-FAM-ATGCTTCATCTGGCACTGGT-BHQ1-3'; Upstream primer for porcine delta coronavirus: 5'-TTTTGCCGTACTGACCTTCA-3'; Porcine delta coronavirus downstream primer: 5'-TTGGCCAGCTCTTGCCCATG-3'; Porcine delta coronavirus probe: 5'-HEX-ATGGATCCAATGGGTACATGGA-BHQ1-3'; Upstream primer for pathogenic Escherichia coli in piglets: 5'-TGCGGCACAACAGGCGGCGA-3'; Downstream primer for pathogenic Escherichia coli in piglets: 5'-CATAGAACGGTAATAAGAAG-3'; Pathogenic Escherichia coli probe for piglets: 5'-ROX-GCTCTTGGTATCGCTGGTAA-BHQ2-3'; upstream primer for Clostridium perfringens: 5'-AATATACAGCATTCCCAGAT-3'; Clostridium perfringens downstream primer: 5'-GTAAATACCACCAAAACCAA-3'; Clostridium perfringens probe: 5'-Cy5-ATGGAAAAGTTGTAGTAGAC-BHQ2-3'.

[0009] Preferably, the concentrations of the upstream and downstream primers for the porcine epidemic diarrhea virus and the porcine delta coronavirus are both 500 nmol / L, the concentrations of the upstream and downstream primers for the pathogenic Escherichia coli and the Clostridium perfringens are both 210 nmol / L, and the concentrations of the porcine epidemic diarrhea virus probe, the porcine delta coronavirus probe, the pathogenic Escherichia coli probe, and the Clostridium perfringens probe are all 100 nmol / L.

[0010] Preferably, the buffer solution comprises 30 mmol / L Tri at pH 8.8, 100 mmol / L KCl and 1% DMSO, 300 μmol / L dNTPs and 3 mmol / L MgCl2, wherein the dNTPs include any one or a combination of at least two of dATP, dGTP, dCTP and dTTP.

[0011] Preferably, the enzyme mixture comprises 5 U of hot-start Taq enzyme and 2.5 U of M-MLV enzyme.

[0012] Preferably, the kit further includes a positive control and a negative control. The positive control includes a porcine epidemic diarrhea virus plasmid, a porcine delta coronavirus plasmid, a pathogenic Escherichia coli plasmid, a Clostridium perfringens plasmid, and a lyophilization protectant. The negative control includes TE water.

[0013] Preferably, the freeze-drying protectant is a mixture of glycerol, trehalose and glycine.

[0014] Preferably, the reaction reagent and the positive control are lyophilized powder reagents.

[0015] A second aspect of the present invention provides a method for detecting multiple pathogens causing diarrhea in piglets, using the kit for detecting multiple pathogens causing diarrhea in piglets as described in any of the preceding claims, the detection method comprising the following steps: S1. Extract nucleic acid from the sample to be tested; S2. Mix the specific primers and probes for porcine epidemic diarrhea virus, porcine delta coronavirus, pathogenic Escherichia coli in piglets, Clostridium perfringens, buffer solution, enzyme mixture and freeze-drying protectant, pre-freeze, vacuum dry to obtain the reaction reagent, and store in the dark. S3. Mix porcine epidemic diarrhea virus plasmid, porcine delta coronavirus plasmid, pathogenic Escherichia coli plasmid of piglets, Clostridium perfringens plasmid and freeze-drying protectant, pre-freeze, vacuum dry to obtain positive control, and store in the dark; use TE water as negative control. S4. Mix the reaction reagents with the nucleic acid of the sample to be tested, the positive control, and the negative control separately, and perform multiplex quantitative PCR detection. S5. Determine the results based on the collected fluorescence curves and Ct values.

[0016] Preferably, in steps S2 and S3, the cells are pre-frozen at -80℃ and stored at 2-8℃ in the dark; in step S4, the PCR amplification conditions are: reverse transcription at 50℃ for 10 min, pre-denaturation at 93℃ for 2 min, denaturation at 93℃ for 10 s, fluorescence collection at 60℃ for 35 s, and the cycle is repeated 40 times.

[0017] This invention provides a reagent kit and detection method for detecting multiple pathogens causing diarrhea in piglets. Compared with the prior art, the advantages of this invention are: (1) The kit of the present invention contains four common pathogens of piglet diarrhea: porcine epidemic diarrhea virus, porcine delta coronavirus, pathogenic Escherichia coli of piglets, and Clostridium perfringens. Through quadruple fluorescent PCR reaction, only one amplification is needed to simultaneously detect the four target genes of porcine epidemic diarrhea virus, porcine delta coronavirus, pathogenic Escherichia coli of piglets, and Clostridium perfringens. One reaction can accurately distinguish the four pathogens, reduce detection costs, greatly shorten detection time, improve the accuracy of subsequent medication, and reduce economic losses for farmers.

[0018] (2) The kit of the present invention has extremely high specificity, sensitivity and repeatability, and there is no interference between the primers and probes: it is negative for common classical swine fever virus, swine pseudorabies virus, swine circovirus, swine reproductive and respiratory syndrome virus, swine salmonella, swine streptococcus, swine transmissible gastroenteritis virus, swine rotavirus and other swine pathogens, and is positive only for four target genes: swine epidemic diarrhea virus, swine delta coronavirus, pathogenic Escherichia coli of piglets and Clostridium perfringens, showing good specificity; the detection sensitivity of the target genes can reach 100 copies / mL, showing good sensitivity; the intra-assay coefficient of variation (CV value) of repeatable samples is <5%, showing good repeatability; the swine epidemic diarrhea virus reagent, swine delta coronavirus reagent, pathogenic Escherichia coli of piglets reagent and Clostridium perfringens reagent can only detect the corresponding pathogen samples positive, and are negative for other samples, and there is no mutual interference between different primers and probes.

[0019] (3) The kit of the present invention is a lyophilized powder kit, which has good room temperature storage stability. Compared with ordinary liquid test kits that need to be stored at -20℃, it is more stable and can be stored at room temperature for a long time without cold chain transportation. Its storage stability and transportation convenience are more prominent, and it has great market promotion and application value. Detailed Implementation

[0020] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit this application. Unless otherwise specified, the raw materials and apparatus used in this invention are all conventional commercially available products. Unless otherwise specified, the methods used in this invention are all conventional methods.

[0021] To demonstrate the reliability of the effectiveness of this invention, the invention is described below with reference to Examples 1-8. Unless otherwise specified, all reagents and biological materials used in Examples 1-8 are commercially available products.

[0022] (a) Reagent kit for detecting multiple pathogens causing diarrhea in piglets Example 1: Kit for detecting multiple pathogens causing diarrhea in piglets This embodiment provides a kit for detecting multiple pathogens causing diarrhea in piglets, including reaction reagents, positive controls, and negative controls. The preparation of the kit includes the following steps: (1) Preparation of reaction reagents The reaction reagents include specific primers and probes for porcine epidemic diarrhea virus, specific primers and probes for porcine delta coronavirus, specific primers and probes for pathogenic Escherichia coli in piglets, specific primers and probes for Clostridium perfringens, buffer solutions, enzyme mixtures, and lyophilization protectants.

[0023] The buffer solution comprises 30 mmol / L Tri at pH 8.8, 100 mmol / L KCl and 1% DMSO, 300 μmol / L dNTPs, and 3 mmol / L MgCl2. The dNTPs include any one or a combination of at least two of dATP, dGTP, dCTP, and dTTP. The enzyme mixture comprises 5 U of hot-start Taq enzyme and 2.5 U of M-MLV enzyme. The lyophilization protectant is a mixture of glycerol, trehalose, and glycine.

[0024] The specific steps are as follows: Take a 10 mL volumetric flask and add 96 μL of 0.5 mol / L Trizma® HCl, 504 μL of 0.5 mol / L Trizma® Base, 30 μL of 1 mol / L MgCl2, 1000 μL of 1 mol / L KCl, 100 μL of DMSO, and dNTPs respectively. 135 μL of 100 μL each of 50 μmol / L porcine epidemic diarrhea virus (PEDV) upstream and downstream primers, 20 μL of 50 μmol / L PEDV probe, 100 μL each of 50 μmol / L porcine delta coronavirus upstream and downstream primers, 20 μL of 50 μmol / L porcine delta coronavirus probe, 42 μL each of 50 μmol / L swine pathogenic Escherichia coli upstream and downstream primers, 20 μL of 50 μmol / L swine pathogenic Escherichia coli probe, 42 μL each of 50 μmol / L Clostridium perfringens upstream and downstream primers, 20 μL of 50 μmol / L Clostridium perfringens probe, 1 μL of hot-start Taq enzyme, 0.5 μL of M-MLV enzyme, 500 μL of glycerol, 2000 μL of 10% trehalose, and 800 μL of 10% glycine were added to bring the volume to 10 mL with double-distilled water. The mixture was then inverted to mix thoroughly. Transfer the liquid to a 10mL beaker, dispense 25μL / well into eight-tube bundles, pre-freeze at -80℃ for 6 hours, then vacuum dry for 6 hours. After the time is up, remove the eight-tube bundles, cap them, and store at 2-8℃ away from light for later use.

[0025] The nucleotide sequences of the upstream and downstream specific primers and probes for porcine epidemic diarrhea virus, porcine delta coronavirus, pathogenic Escherichia coli in piglets, and Clostridium perfringens are as follows: Upstream primer for porcine epidemic diarrhea virus: 5'-TAGTGGTACATTGCTTGTAG-3'; Porcine epidemic diarrhea virus downstream primer: 5'-GAGTAGTCGCCGTGTTTTGA-3'; Porcine epidemic diarrhea virus probe: 5'-FAM-ATGCTTCATCTGGCACTGGT-BHQ1-3'; Upstream primer for porcine delta coronavirus: 5'-TTTTGCCGTACTGACCTTCA-3'; Porcine delta coronavirus downstream primer: 5'-TTGGCCAGCTCTTGCCCATG-3'; Porcine delta coronavirus probe: 5'-HEX-ATGGATCCAATGGGTACATGGA-BHQ1-3'; Upstream primer for pathogenic Escherichia coli in piglets: 5'-TGCGGCACAACAGGCGGCGA-3'; Downstream primer for pathogenic Escherichia coli in piglets: 5'-CATAGAACGGTAATAAGAAG-3'; Pathogenic Escherichia coli probe for piglets: 5'-ROX-GCTCTTGGTATCGCTGGTAA-BHQ2-3'; upstream primer for Clostridium perfringens: 5'-AATATACAGCATTCCCAGAT-3'; Clostridium perfringens downstream primer: 5'-GTAAATACCACCAAAACCAA-3'; Clostridium perfringens probe: 5'-Cy5-ATGGAAAAGTTGTAGTAGAC-BHQ2-3'.

[0026] (2) Preparation of negative control Negative control standards include TE water.

[0027] The specific steps are as follows: Take a 10mL volumetric flask, add TE water to bring the volume to 10mL, dispense into centrifuge tubes at 500µL / vial, and store at 2-8℃ for later use.

[0028] (3) Preparation of positive control Positive controls include porcine epidemic diarrhea virus plasmid, porcine delta coronavirus plasmid, pathogenic Escherichia coli plasmid, Clostridium perfringens plasmid, and lyophilization protectant.

[0029] The freeze-drying protectant is a mixture of glycerol, trehalose, and glycine.

[0030] The specific steps are as follows: Take a 10mL volumetric flask and add 1.0×10⁻⁶ ppm of the solution. 5 100 µL each of porcine epidemic diarrhea virus plasmid, porcine delta coronavirus plasmid, pathogenic Escherichia coli plasmid, and Clostridium perfringens plasmid; 500 µL of glycerol; 2000 µL of 10% trehalose; and 800 µL of 10% glycine. Dilute to 10 mL with TE buffer, invert to mix thoroughly, and obtain a concentration of 1.0 × 10⁻⁶. 3 Prepare a positive control sample in copies / mL. Transfer the liquid to a 10mL beaker, aliquot into centrifuge tubes at 50µL / tube, pre-freeze at -80℃ for 6 hours, then vacuum dry for 6 hours. After 6 hours, remove the centrifuge tubes, cap them, and store at 2-8℃ protected from light for later use.

[0031] (II) Detection methods for multiple pathogens causing diarrhea in piglets Example 2: Detection methods for multiple pathogens causing diarrhea in piglets This embodiment provides a method for detecting multiple pathogens causing diarrhea in piglets, which is a multiplex fluorescent PCR detection method, including the following steps: S1. Nucleic acid extraction: Nucleic acid extraction was performed on the test samples using a nucleic acid extraction kit (a commercially available product purchased directly). After extraction, the purity of the nucleic acid samples was detected using a micro-ultraviolet spectrophotometer, and its OD260 / OD280 should be between 1.6 and 2.0.

[0032] S2. Reagent preparation: The reaction reagents, positive control, and negative control were prepared using the method described in Example 1.

[0033] S3. Sample loading and instrumentation: (1) Dissolution of positive control: Add 250 μL of negative control to positive control, mix well and set aside.

[0034] (2) Sample addition: Remove the eight-tube set containing the reaction reagents and add 25 μL of the test sample nucleic acid, 25 μL of the positive control, and 25 μL of the negative control, respectively. After sealing the tubes, centrifuge briefly and then place them in a fluorescence PCR amplification instrument for multiplex quantitative PCR detection.

[0035] (3) On-machine testing: ①PCR amplification conditions: 50℃ reverse transcription for 10 min, 93℃ pre-denaturation for 2 min; 93℃ denaturation for 10 s, 60℃ fluorescence collection for 35 s, 40 cycles.

[0036] ② The detection modes are shown in Table 1.

[0037] Table 1. Detection modes of multiplex quantitative PCR.

[0038] S4. Validity judgment: The results are determined by collecting fluorescence curves and Ct values. Negative controls should have no Ct value or a value of 0, while positive controls should have a Ct value ≤ 35; otherwise, the test results are invalid.

[0039] S5. Result Interpretation: The results of the test samples were interpreted according to Table 2.

[0040] Table 2. Result Interpretation Table for Multiplex Quantitative Real-Time PCR Detection

[0041] Example 3 Specificity Detection (1) Specific samples: Eight specific samples were prepared: Sample 1 was classical classical swine fever virus; Sample 2 was porcine pseudorabies virus; Sample 3 was porcine circovirus type 2; Sample 4 was porcine reproductive and respiratory syndrome virus; Sample 5 was porcine salmonella; Sample 6 was porcine streptococcus; Sample 7 was porcine transmissible gastroenteritis virus; and Sample 8 was porcine rotavirus. Physiological saline was used as a negative control, and porcine epidemic diarrhea virus was used as a positive control.

[0042] (2) Experimental procedure: Using the kit from Example 1 and the multiplex fluorescent PCR detection method from Example 2, the above eight specific samples were tested. The results are shown in Table 3.

[0043] Table 3. Detection results of four fluorescence channels in specific samples 1-8

[0044] (3) Experimental results: As shown in Table 3, the test results of all 8 specific samples were negative, indicating that the kit of the present invention has good specificity and no cross-reaction with other porcine pathogens.

[0045] Example 4 Repeatability Test (1) Repeated samples: Take a 10mL volumetric flask and add 1.0×10⁻⁶ ppm of the solution. 7 10 µL each of porcine epidemic diarrhea virus plasmid, porcine delta coronavirus plasmid, pathogenic Escherichia coli plasmid of piglets, and Clostridium perfringens plasmid were diluted to 10 mL with TE buffer to obtain a concentration of 1.0 × 10⁻⁶. 4 Reproducible samples of copies / mL.

[0046] (2) Experimental procedure: Using the kit from Example 1, the multiplex fluorescent PCR detection method from Example 2 was used to repeatedly test the prepared reproducible samples 10 times. The detection results are shown in Table 4.

[0047] Table 4. Results of 10 tests on the four fluorescence channels of repeatable samples.

[0048] In Table 4, 1-10 represent the CT values ​​of repeatable samples from the 1st to the 10th tests, respectively. (3) Experimental results: As shown in Table 4, the intra-batch coefficient of variation (CV) of the reproducible samples is <5%, indicating that the kit of the present invention has good reproducibility.

[0049] Example 5 Sensitivity Detection (1) Sensitivity sample: Take a 10mL volumetric flask and add 1.0×10⁻⁶ ppm of the solution. 7 1 mL each of porcine epidemic diarrhea virus plasmid, porcine delta coronavirus plasmid, pathogenic Escherichia coli plasmid of piglets, and Clostridium perfringens plasmid were diluted to 10 mL with TE buffer to obtain a concentration of 1.0 × 10⁻⁶. 6 Sensitivity sample 1 (copies / mL).

[0050] Take a 10 mL volumetric flask, add 1 mL of sensitivity sample 1, and dilute to 10 mL with TE water to obtain a concentration of 1.0 × 10⁻⁶. 5 Sensitivity sample 2, copies / mL.

[0051] Take a 10 mL volumetric flask, add 1 mL of sensitivity sample 2, and dilute to 10 mL with TE water to obtain a concentration of 1.0 × 10⁻⁶. 4 Sensitivity sample 3 (copies / mL).

[0052] Take a 10 mL volumetric flask, add 1 mL of sensitivity sample 3, and dilute to 10 mL with TE water to obtain a concentration of 1.0 × 10⁻⁶. 3 Sensitivity sample 4, copies / mL.

[0053] Take a 10 mL volumetric flask, add 1 mL of sensitivity sample 4, and dilute to 10 mL with TE water to obtain a concentration of 1.0 × 10⁻⁶. 2 Sensitivity sample 5, copies / mL.

[0054] Take a 10 mL volumetric flask, add 1 mL of sensitivity sample 5, and dilute to 10 mL with TE water to obtain a concentration of 1.0 × 10⁻⁶. 1 Sensitivity sample 6, copies / mL.

[0055] (2) Experimental procedure: Using the kit from Example 1, the six prepared sensitivity samples were tested using the multiplex fluorescent PCR detection method from Example 2. The results were analyzed to verify the sensitivity of the kit. The results are shown in Table 5.

[0056] Table 5. Detection results of the four fluorescence channels for sensitivity samples 1-6.

[0057] (3) Experimental results: As shown in Table 5, the concentration is 1.0 × 10⁻⁶. 2The sensitivity of sample 5, which was 100 copies / mL, was still positive, indicating that the detection sensitivity of the kit and the multiplex quantitative PCR detection method of the present invention is 100 copies / mL.

[0058] Example 6: Detection of interference between primers and probes (1) Interference samples: Prepare 8 interfering samples, among which interfering sample 1 has a concentration of 1.0 × 10⁻⁶. 5 Porcine epidemic diarrhea virus (PEDV) sample of copies / mL, interfering sample 2 with a concentration of 1.0 × 10⁻⁶. 3 Porcine epidemic diarrhea virus (PEDV) samples of copies / mL, with interfering sample 3 having a concentration of 1.0 × 10⁻⁶. 5 Porcine delta coronavirus sample (copies / mL), interfering sample 4 was at a concentration of 1.0 × 10⁻⁶. 3 Porcine delta coronavirus samples of copies / mL, interfering sample 5 with a concentration of 1.0 × 10⁻⁶. 5 The sample contained pathogenic Escherichia coli in piglets at a concentration of 1.0 × 10⁶ copies / mL. Interfering sample 6 had a concentration of 1.0 × 10⁶. 3 The sample contained pathogenic Escherichia coli in piglets at a concentration of copies / mL. Interference sample 7 had a concentration of 1.0 × 10⁻⁶. 5 The sample of Clostridium perfringens was measured in copies / mL, and interfering sample 8 had a concentration of 1.0 × 10⁸. 3 Copies / mL of Clostridium perfringens samples.

[0059] (2) Preparation of reaction reagents: Prepare the following four reaction reagents, each with one and only one set of primers and probes.

[0060] ① Porcine epidemic diarrhea virus reagent: Take a 10 mL volumetric flask and add the following solutions: 96 μL of 0.5 mol / L Trizma® HCl, 504 μL of 0.5 mol / L Trizma® Base, 30 μL of 1 mol / L MgCl2, 1000 μL of 1 mol / L KCl, 100 μL of DMSO, 135 μL of dNTPs, 100 μL each of 50 μmol / L porcine epidemic diarrhea virus (PEDV) upstream and downstream primers, 20 μL of 50 μmol / L PEDV probe, 1 μL of hot-start Taq enzyme, 0.5 μL of M-MLV enzyme, 500 μL of glycerol, 2000 μL of 10% trehalose, and 800 μL of 10% glycine. Make up the volume to 10 mL with double-distilled water. Invert the flask to mix thoroughly. Transfer the liquid to a 10mL beaker, dispense 25μL / well into eight-tube bundles, pre-freeze at -80℃ for 6 hours, then vacuum dry for 6 hours. After the time is up, remove the eight-tube bundles, cap them, and store at 2-8℃ away from light for later use.

[0061] ② Porcine delta coronavirus reagent: Take a 10 mL volumetric flask and add 96 μL of 0.5 mol / L Trizma® HCl, 504 μL of 0.5 mol / L Trizma® Base, 30 μL of 1 mol / L MgCl2, 1000 μL of 1 mol / L KCl, 100 μL of DMSO, 135 μL of dNTPs, 100 μL each of 50 μmol / L porcine delta coronavirus forward and reverse primers, 20 μL of 50 μmol / L porcine delta coronavirus probe, 1 μL of hot-start Taq enzyme, 0.5 μL of M-MLV enzyme, 500 μL of glycerol, 2000 μL of 10% trehalose, and 800 μL of 10% glycine. Make up the volume to 10 mL with double-distilled water. Invert to mix thoroughly. Transfer the liquid to a 10 mL beaker, aliquot into 8-tube strips at 25 μL / well, pre-freeze at -80°C for 6 hours, and then vacuum dry for 6 hours. After the time is up, remove the eight-tube assembly, put the cap back on, and store it at 2-8℃ away from light for later use.

[0062] ③ Reagent for pathogenic Escherichia coli in piglets: Take a 10 mL volumetric flask and add the following solutions: 96 μL of 0.5 mol / L Trizma® HCl, 504 μL of 0.5 mol / L Trizma® Base, 30 μL of 1 mol / L MgCl2, 1000 μL of 1 mol / L KCl, 100 μL of DMSO, 135 μL of dNTPs, 42 μL each of upstream and downstream primers for 50 μmol / L pathogenic Escherichia coli in piglets, 20 μL of a probe for 50 μmol / L pathogenic Escherichia coli in piglets, 1 μL of hot-start Taq enzyme, 0.5 μL of M-MLV enzyme, 500 μL of glycerol, 2000 μL of 10% trehalose, and 800 μL of 10% glycine. Make up the volume to 10 mL with double-distilled water. Invert the flask to mix thoroughly. Transfer the liquid to a 10mL beaker, dispense 25μL / well into eight-tube bundles, pre-freeze at -80℃ for 6 hours, then vacuum dry for 6 hours. After the time is up, remove the eight-tube bundles, cap them, and store at 2-8℃ away from light for later use.

[0063] ④ Clostridium perfringens reagent: Take a 10 mL volumetric flask and add 96 μL of 0.5 mol / L Trizma® HCl, 504 μL of 0.5 mol / L Trizma® Base, 30 μL of 1 mol / L MgCl2, 1000 μL of 1 mol / L KCl, 100 μL of DMSO, 135 μL of dNTPs, 42 μL each of 50 μmol / L Clostridium perfringens upstream and downstream primers, 20 μL of 50 μmol / L Clostridium perfringens probe, 1 μL of hot-start Taq enzyme, 0.5 μL of M-MLV enzyme, 500 μL of glycerol, 2000 μL of 10% trehalose, and 800 μL of 10% glycine. Make up the volume to 10 mL with double-distilled water. Invert to mix thoroughly. Transfer the liquid to a 10mL beaker, dispense 25μL / well into eight-tube bundles, pre-freeze at -80℃ for 6 hours, then vacuum dry for 6 hours. After the time is up, remove the eight-tube bundles, cap them, and store at 2-8℃ away from light for later use.

[0064] (3) Experimental procedure: ① Nucleic acid extraction: Nucleic acid was extracted from the above 8 interfering samples using a nucleic acid extraction kit (a commercially available product purchased directly). After extraction, the purity of the nucleic acid samples was detected using a micro-ultraviolet spectrophotometer, and their OD260 / OD280 were all between 1.6 and 2.0.

[0065] ② Sample loading and instrumentation: Eight 25 μL aliquots of nucleic acid extracted from the test samples were added to eight-tube strips containing the four reaction reagents prepared above. After sealing the tubes, the strips were slightly centrifuged and then placed in a fluorescence PCR amplification instrument for multiplex quantitative PCR detection. The PCR amplification conditions and detection mode were the same as in Example 2. The detection results are shown in Table 6.

[0066] Table 6. Detection results of the four fluorescence channels of interference samples 1-8

[0067] (4) Experimental results: As shown in Table 6, the porcine epidemic diarrhea virus reagent, porcine delta coronavirus reagent, pathogenic Escherichia coli reagent for piglets, and Clostridium perfringens reagent can only detect positive results for their respective pathogen samples, while all other samples are negative. This indicates that there is no mutual interference between different primers and probes in the kit of the present invention.

[0068] Example 7: Stability Test of Lyophilized Powder Reagent Kit (1) Stability sample: Take a 10mL volumetric flask and add 1.0×10⁻⁶ ppm of the solution. 3 1 mL each of porcine epidemic diarrhea virus plasmid, porcine delta coronavirus plasmid, pathogenic Escherichia coli plasmid of piglets, and Clostridium perfringens plasmid were diluted to 10 mL with TE buffer to obtain a concentration of 1.0 × 10⁻⁶. 2 Stability samples of copies / mL.

[0069] (2) Experimental procedure: The lyophilized powder kit prepared in Example 1 was placed at room temperature, and the stability of the prepared samples was tested using the multiplex fluorescent PCR detection method described in Example 2 at 1, 3, 6, 9, and 12 months after placement to obtain the CT values. The test results are shown in Table 7.

[0070] Table 7. Results of storage stability test for the lyophilized powder kit

[0071] (3) Experimental results: As shown in Table 7, the kit of the present invention can still stably and accurately detect samples with the limit of detection as positive after being stored at room temperature for 12 months, and the CT values ​​are not significantly different, indicating that the lyophilized powder kit of the present invention has good stability at room temperature.

[0072] Example 8: Clinical Sample Testing Application (1) Clinical samples of piglets with diarrhea: Weifang Huazhuo Biotechnology Co., Ltd. collected 153 samples of piglet diarrhea from 37 different farms.

[0073] (2) Experimental procedure: ① Nucleic acid extraction: Nucleic acid was extracted from the 153 piglet diarrhea samples using a nucleic acid extraction kit (a commercially available product purchased directly). The purity of the nucleic acid samples was then tested using a micro-ultraviolet spectrophotometer. The OD260 / OD280 of the 153 piglet diarrhea samples were all between 1.6 and 2.0.

[0074] ②Detection method: Using the kit from Example 1 and the multiplex fluorescent PCR detection method from Example 2, the above 153 piglet diarrhea samples were tested. Simultaneously, verification tests were performed using current national standards, industry standards, and local standards (GB / T36871-2018 Multiplex RT-PCR Detection Method for Porcine Transmissible Gastroenteritis Virus, Porcine Epidemic Diarrhea Virus, and Porcine Rotavirus; DB31 / T 1512-2024 Identification Technical Requirements for Porcine Epidemic Diarrhea Virus, Porcine Transmissible Gastroenteritis Virus, and Porcine Delta Coronavirus; NY / T 2839-2015 Isolation and Identification Techniques for Escherichia coli Causing Yellow Scours in Piglets; NY / T 4656-2025 Technical Procedures for Isolation and Identification of Clostridium perfringens from Animals). The test results are shown in Table 8.

[0075] Table 8. Detection results of clinical piglet diarrhea samples

[0076] (3) Experimental results: As shown in Table 8, the detection results of porcine epidemic diarrhea virus, porcine delta coronavirus, pathogenic Escherichia coli, and Clostridium perfringens in clinical piglet diarrhea samples by the kit of the present invention are consistent with the detection results of the current national standard methods (GB / T 36871-2018 Multiplex RT-PCR Detection Method for Porcine Transmissible Gastroenteritis Virus, Porcine Epidemic Diarrhea Virus and Porcine Rotavirus; DB31 / T1512-2024 Identification Technical Requirements for Porcine Epidemic Diarrhea Virus, Porcine Transmissible Gastroenteritis Virus and Porcine Delta Coronavirus; NY / T 2839-2015 Isolation and Identification Technology for Escherichia coli that Causes Yellow Scours in Piglets; NY / T 4656-2025 Technical Procedures for Isolation and Identification of Clostridium perfringens from Animals), with a compliance rate of 100%, indicating that the kit of the present invention exhibits excellent clinical detection accuracy.

[0077] In summary, this invention provides a reagent kit and detection method for detecting multiple pathogens causing diarrhea in piglets, which, compared with existing technologies: (1) The kit of the present invention contains four common pathogens of piglet diarrhea: porcine epidemic diarrhea virus, porcine delta coronavirus, pathogenic Escherichia coli of piglets, and Clostridium perfringens. Through quadruple fluorescent PCR reaction, only one amplification is needed to simultaneously detect the four target genes of porcine epidemic diarrhea virus, porcine delta coronavirus, pathogenic Escherichia coli of piglets, and Clostridium perfringens. One reaction can accurately distinguish the four pathogens, reduce detection costs, greatly shorten detection time, improve the accuracy of subsequent medication, and reduce economic losses for farmers.

[0078] (2) The kit of the present invention has extremely high specificity, sensitivity and repeatability, and there is no interference between the primers and probes: it is negative for common classical swine fever virus, swine pseudorabies virus, swine circovirus, swine reproductive and respiratory syndrome virus, swine salmonella, swine streptococcus, swine transmissible gastroenteritis virus, swine rotavirus and other swine pathogens, and is positive only for four target genes: swine epidemic diarrhea virus, swine delta coronavirus, pathogenic Escherichia coli of piglets and Clostridium perfringens, showing good specificity; the detection sensitivity of the target genes can reach 100 copies / mL, showing good sensitivity; the intra-assay coefficient of variation (CV value) of repeatable samples is <5%, showing good repeatability; the swine epidemic diarrhea virus reagent, swine delta coronavirus reagent, pathogenic Escherichia coli of piglets reagent and Clostridium perfringens reagent can only detect the corresponding pathogen samples positive, and are negative for other samples, and there is no mutual interference between different primers and probes.

[0079] (3) The kit of the present invention is a lyophilized powder kit, which has good room temperature storage stability. Compared with ordinary liquid test kits that need to be stored at -20℃, it is more stable and can be stored at room temperature for a long time without cold chain transportation. Its storage stability and transportation convenience are more prominent, and it has great market promotion and application value.

[0080] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application, and should all be included within the protection scope of this application.

Claims

1. A kit for detecting multiple pathogens causing diarrhea in piglets, characterized in that, The reagents include reaction reagents, which include specific primers and probes for porcine epidemic diarrhea virus, specific primers and probes for porcine delta coronavirus, specific primers and probes for pathogenic Escherichia coli in piglets, specific primers and probes for Clostridium perfringens, buffer solutions, enzyme mixtures, and lyophilization protectants.

2. The kit for detecting multiple pathogens causing diarrhea in piglets according to claim 1, characterized in that, The porcine epidemic diarrhea virus-specific primers, the porcine delta coronavirus-specific primers, the porcine pathogenic Escherichia coli-specific primers, and the Clostridium perfringens-specific primers all include upstream and downstream primers; the 5' end of the porcine epidemic diarrhea virus probe, the porcine delta coronavirus probe, the porcine pathogenic Escherichia coli probe, and the Clostridium perfringens probe includes any one of the FAM, HEX, ROX, or Cy5 fluorescent reporter groups, and the 3' end includes any one of the BHQ1 or BHQ2 fluorescent quencher groups; The nucleotide sequences of the upstream and downstream specific primers and probes are as follows: Upstream primer for porcine epidemic diarrhea virus: 5'-TAGTGGTACATTGCTTGTAG-3'; Porcine epidemic diarrhea virus downstream primer: 5'-GAGTAGTCGCCGTGTTTTGA-3'; Porcine epidemic diarrhea virus probe: 5'-FAM-ATGCTTCATCTGGCACTGGT-BHQ1-3'; Upstream primer for porcine delta coronavirus: 5'-TTTTGCCGTACTGACCTTCA-3'; Porcine delta coronavirus downstream primer: 5'-TTGGCCAGCTCTTGCCCATG-3'; Porcine delta coronavirus probe: 5'-HEX-ATGGATCCAATGGGTACATGGA-BHQ1-3'; Upstream primer for pathogenic Escherichia coli in piglets: 5'-TGCGGCACAACAGGCGGCGA-3'; Downstream primer for pathogenic Escherichia coli in piglets: 5'-CATAGAACGGTAATAAGAAG-3'; Pathogenic Escherichia coli probe for piglets: 5'-ROX-GCTCTTGGTATCGCTGGTAA-BHQ2-3'; upstream primer for Clostridium perfringens: 5'-AATATACAGCATTCCCAGAT-3'; Clostridium perfringens downstream primer: 5'-GTAAATACCACCAAAACCAA-3'; Clostridium perfringens probe: 5'-Cy5-ATGGAAAAGTTGTAGTAGAC-BHQ2-3'.

3. The kit for detecting multiple pathogens causing diarrhea in piglets according to claim 2, characterized in that, The concentrations of the upstream and downstream primers for the porcine epidemic diarrhea virus and the porcine delta coronavirus are both 500 nmol / L; the concentrations of the upstream and downstream primers for the pathogenic Escherichia coli and Clostridium perfringens are both 210 nmol / L; and the concentrations of the porcine epidemic diarrhea virus probe, the porcine delta coronavirus probe, the pathogenic Escherichia coli probe, and the Clostridium perfringens probe are all 100 nmol / L.

4. The kit for detecting multiple pathogens causing diarrhea in piglets according to claim 1, characterized in that, The buffer solution comprises 30 mmol / L Tri at pH 8.8, 100 mmol / L KCl and 1% DMSO, 300 μmol / L dNTPs and 3 mmol / L MgCl2, wherein the dNTPs include any one or a combination of at least two of dATP, dGTP, dCTP and dTTP.

5. The kit for detecting multiple pathogens causing diarrhea in piglets according to claim 1, characterized in that, The enzyme mixture comprises 5 U of hot-start Taq enzyme and 2.5 U of M-MLV enzyme.

6. The kit for detecting multiple pathogens causing diarrhea in piglets according to claim 1, characterized in that, The kit also includes positive and negative controls. The positive controls include porcine epidemic diarrhea virus plasmid, porcine delta coronavirus plasmid, pathogenic Escherichia coli plasmid, Clostridium perfringens plasmid, and lyophilization protectant. The negative control includes TE water.

7. The kit for detecting multiple pathogens causing diarrhea in piglets according to claim 1 or 6, characterized in that, The freeze-drying protectant is a mixture of glycerol, trehalose, and glycine.

8. The kit for detecting multiple pathogens causing diarrhea in piglets according to claim 1 or 6, characterized in that, The reaction reagents and the positive control are lyophilized powder reagents.

9. A method for detecting multiple pathogens causing diarrhea in piglets, characterized in that, The detection method using the kit for detecting multiple pathogens of piglet diarrhea as described in any one of claims 1-8 includes the following steps: S1. Extract nucleic acid from the sample to be tested; S2. Mix the specific primers and probes for porcine epidemic diarrhea virus, porcine delta coronavirus, pathogenic Escherichia coli in piglets, Clostridium perfringens, buffer solution, enzyme mixture and freeze-drying protectant, pre-freeze, vacuum dry to obtain the reaction reagent, and store in the dark. S3. Mix porcine epidemic diarrhea virus plasmid, porcine delta coronavirus plasmid, pathogenic Escherichia coli plasmid of piglets, Clostridium perfringens plasmid and freeze-drying protectant, pre-freeze, vacuum dry to obtain positive control, and store in the dark; use TE water as negative control. S4. Mix the reaction reagents with the nucleic acid of the sample to be tested, the positive control, and the negative control separately, and perform multiplex quantitative PCR detection. S5. Determine the results based on the collected fluorescence curves and Ct values.

10. The method for detecting multiple pathogens causing diarrhea in piglets according to claim 9, characterized in that, In steps S2 and S3, the sample was pre-frozen at -80℃ and stored at 2-8℃ in the dark. In step S4, the PCR amplification conditions were: reverse transcription at 50℃ for 10 min, pre-denaturation at 93℃ for 2 min, denaturation at 93℃ for 10 s, fluorescence collection at 60℃ for 35 s, and the cycle was repeated 40 times.

Images