Quadruple fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection method for pathogenic virus of porcine intestinal diseases

Abstract

The invention provides a quadruple fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for pathogenic viruses of porcine intestinal diseases, namely a quadruple fluorescent quantitative RT-PCR detection method suitable for detecting diarrhea of pigs in China, and the quadruple fluorescent quantitative RT-PCR detection method can be used for detecting PEDV (Porcine Epidemic Diarrhea), TGEV (Transmissible Gastroenteritis), aPoRVA (Porcine Rotavirus) and PDCoV (Porcine Delta Coronavirus) in one PCR amplification reaction. According to the invention, rapid and high-precision detection of PEDV, TGEV, PoRVA and PDCoV is realized by a real-time fluorescent quantitative PCR technology, and the kit has important significance in prevention of rapid propagation and timely purification treatment of PEDV, TGEV, PoRVA and PDCoV in the future, and has a good application prospect. The method solves the problem that spectrum overlapping of different probes in multiple channels affects signal crossing, avoids false positive, can reduce generation of primer dimers in amplification, and solves the technical problem that segmented annealing is needed due to large annealing temperature difference.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene detection, and particularly relates to a quadruple fluorescence quantitative RT-PCR detection method for porcine enteric disease pathogenic virus. BACKGROUND

[0002] Porcine diarrhea is a common disease, which can be caused by various factors, and the diarrhea symptoms and losses caused by viral infection are particularly serious. The diarrhea symptoms and losses caused by viral infection not only affect the growth and development of pigs, but also can lead to death of pigs in severe cases, thereby bringing huge economic losses to the pig industry. Epidemiological investigation results show that there are four kinds of viral pathogens causing porcine diarrhea in China at present, namely, transmissible gastroenteritis of swine (TGEV), porcine epidemic diarrhea virus (PEDV), porcine delta coronavirus (PDCoV) and porcine rotavirus type A (PoRVA). These viruses have similar clinical symptoms, such as vomiting, diarrhea, dehydration and the like, and can cause death in severe cases. It is difficult to distinguish these viruses through clinical diagnosis methods. Moreover, these viral infections are mostly mixed infections and secondary infections, which makes the differential diagnosis more difficult and delays the disease, so it is urgent to establish a high-throughput, low-cost, high-sensitivity and specific detection method.

[0003] The traditional PCR method has low sensitivity, and needs to be verified by gel electrophoresis after the PCR test, which is time-consuming and requires high personnel operation level and is not accurate, thereby hindering its application in sample analysis. In recent years, the national standards and industry standards for detecting porcine diarrhea virus by real-time fluorescence quantitative PCR have been established. These methods are mainly aimed at a single virus, and the time required for detecting the four viruses at the same time is long and the cost is high. Multiple real-time fluorescence quantitative RT-PCR integrates multiple primer pairs into one reaction system, which improves the detection efficiency while maintaining the sensitivity and specificity of single real-time fluorescence quantitative PCR. In addition, it can be widely applied and reduce the cost.

[0004] Compared with traditional PCR, real-time fluorescence quantitative PCR can monitor the amplification of target genes in real time, with high sensitivity, rapid detection, and more intuitive and accurate results. National and industry standards for real-time fluorescence quantitative PCR for the detection of porcine diarrhea viruses have been established, and these methods are primarily targeted at single viruses. Multiplex real-time fluorescence quantitative RT-PCR integrates multiple primer pairs into a single reaction system, improving detection efficiency while maintaining the sensitivity and specificity of single real-time fluorescence quantitative PCR. Although multiplex PCR amplification detection methods are also used for pathogen detection, the use of conventional multiplex PCR amplification detection methods for the simultaneous detection of four pathogens—porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine rotavirus a (PoRVA), and porcine deltacoronavirus—still presents issues such as signal crosstalk. Summary of the Invention

[0005] The purpose of the present invention is to provide a quadruple fluorescent quantitative RT-PCR detection method for pathogenic viruses of porcine intestinal diseases, that is, a quadruple fluorescent quantitative RT-PCR detection method suitable for detecting diarrhea in pig herds in my country. It can detect porcine epidemic diarrhea (PEDV), porcine transmissible gastroenteritis (TGEV), porcine rotavirus a (PoRVA) and porcine deltacoronavirus (PDCoV) in a single PCR amplification reaction. The method established by the present invention has high sensitivity, strong specificity and good repeatability, and can fully meet the needs of clinical differential diagnosis, thereby making up for the shortcomings of the existing technology.

[0006] The present invention first provides a primer and probe combination for quadruple fluorescent quantitative PCR detection of TGEV, PEDV, PDCoV and PoRVA viruses, which comprises: 1) The primer pairs and probes used to detect porcine transmissible gastroenteritis virus (TGEV) have the following sequence information: TGEV-F: 5'-cactagtatgagcgcaggtaag-3' (SEQ ID NO: 1), TGEV-R: 5'-gggtgcatacagtctcatcaa-3' (SEQ ID NO: 2), TGEV-P: VIC-tatgctggtgawgttgaaggtgtctcc-BHQ1 (SEQ ID NO: 3); 2) The sequence information of primer pairs and probes used to detect porcine epidemic diarrhea virus (PEDV) is as follows: PEDV-F: 5'-ttaaagcacacggtcccatta-3' (SEQ ID NO: 4), PEDV-R: 5'-aggtgcatgcttacccttac-3' (SEQ ID NO: 5), PEDV-P: FAM-aagacctaatgcytccaacatgacacc-BHQ1 (SEQ ID NO: 6); 3) The sequence information of primer pairs and probes used to detect porcine deltacoronavirus (PDCoV) is as follows: PDCoV-F: 5'-tccagcaggtgtcattcatag-3' (SEQ ID NO:7), PDCoV-R: 5'-caacgcatccctaaccataga-3' (SEQ ID NO: 8), PDCoV-P: CY5-acagtctctttatgaaacccawcagcgt-BHQ2 (SEQ ID NO:9); 4) Primer pairs and probes for detecting porcine rotavirus a (PoRVA), the sequence information of which is as follows: PoRVA-F: 5'-ttcagtggttgmtgctcaa-3' (SEQ ID NO: 10), PoRVA-R: 5'-gcaacractgcagcttcaaa-3' (SEQ ID NO: 11), PoRVA-P: ROX-atggagtctactcagcagatggy-BHQ1 (SEQ ID NO: 12).

[0007] Furthermore, the 5' end and 3' end of the probe are respectively connected with different fluorescent reporter groups or fluorescent quencher groups; As a specific description of the embodiment, the probe for detecting porcine transmissible gastroenteritis virus (TGEV) is connected to VIC at its 5' end and to BHQ1 at its 3' end; The probe for detecting porcine epidemic diarrhea virus (PEDV) has FAM linked to its 5' end and BHQ1 linked to its 3' end; The probe for detecting porcine deltacoronavirus (PDCoV) has CY5 attached to its 5' end and BHQ2 attached to its 3' end; The probe for detecting porcine rotavirus a (PoRVA) has ROX linked to its 5' end and BHQ1 linked to its 3' end.

[0008] The use of the above primer and probe combination in the preparation of a kit for quadruple fluorescent quantitative PCR detection of TGEV, PEDV, PDCoV and PoRVA.

[0009] In another aspect, the present invention further provides a kit for quadruple fluorescent quantitative PCR detection of TGEV, PEDV, PDCoV and PoRVA, which comprises the above-mentioned primer and probe combination.

[0010] The present invention provides a method for detecting TGEV, PEDV, PDCoV and PoRVA by quadruple fluorescent quantitative PCR. The method uses RNA of a test sample as a template and employs the above-mentioned primer and probe combination to perform quadruple fluorescent quantitative PCR amplification of TGEV, PEDV, PDCoV and PoRVA. The detection results of TGEV, PEDV, PDCoV and PoRVA are determined based on the CT value.

[0011] As an optimal technical solution, the CT value of TGEV is ≤35 for positive, 35<CT value<40 for suspicious, and CT value ≥40 for negative; the CT value of PEDV is ≤35 for positive, 35<CT value<40 for suspicious, and CT value ≥40 for negative; the CT value of PDCoV is ≤35 for positive, 35<CT value<40 for suspicious, and CT value ≥40 for negative; the CT value of PoRVA is ≤35 for positive, 35<CT value<40 for suspicious, and CT value ≥40 for negative.

[0012] As a preferred technical solution, the reaction conditions for the quadruple fluorescent quantitative PCR amplification of TGEV, PEDV, PDCoV and PoRVA are: reverse transcription at 50°C for 15 min; enzyme inactivation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing and extension at 60°C for 30 s, and 40 cycles.

[0013] As a preferred technical solution, the 25 μL reaction system for the quadruple fluorescent quantitative PCR amplification of TGEV, PEDV, PDCoV and PoRVA is: 5×Hifair MP Buffer 5 μL, Hifair Enzyme Mix 1 μL, 10 μmol / L TGEV-R 0.4 μL, 10 μmol / L TGEV-P 0.2 μL, 10 μmol / L PEDV-F 0.4 μL, 10 μmol / L PEDV-R 0.4 μL, 10 μmol / L PEDV-P 0.2 μL, 10 μmol / L TGEV-F 0.4 μL, 10 μmol / L PEDV-F 0.4 μL, μmol / L PoRVA-R 0.4 μL, 10 μmol / L PoRVA-P 0.2 μL, template 2 μL, ddH2O 13 μL.

[0014] This method uses real-time fluorescence quantitative PCR to achieve rapid, high-precision detection of PEDV, TGEV, PoRVA, and PDCoV. This is of great significance for preventing the rapid spread of PEDV, TGEV, PoRVA, and PDCoV and for their timely decontamination and treatment, and has promising application prospects. This method addresses the signal crosstalk caused by spectral overlap between different probes in multiple channels, preventing false positives. It also reduces the formation of primer dimers during amplification and addresses the technical challenge of segmented annealing due to large annealing temperature differences. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Screening results of primers and probes for quadruple fluorescence quantitative RT-PCR of TGEV, PEDV, PDCoV and PoRVA Figure 2 Figure 2 shows the optimization results of primer and probe concentrations for quadruple fluorescent quantitative RT-PCR of TGEV, PEDV, PDCoV and PoRVA.

[0016] Figure 3 The temperature optimization results of quadruple fluorescence quantitative RT-PCR for TGEV, PEDV, PDCoV and PoRVA are shown.

[0017] Figure 4 The amplification curves and standard curves of the quadruple fluorescence quantitative RT-PCR of TGEV, PEDV, PDCoV and PoRVA are shown in Figure 2. The nucleic acid concentrations are TGEV 10 6-10 copies / μL, PEDV 2×10 5 -10 copies / μL, PDCoV 2×10 3 -10 0 copies / μL, PoRVA 5×10 6 -5×10 0 Amplification curve of copies / μL Figure 5 This is a graph showing the specific results of quadruple fluorescence quantitative RT-PCR for TGEV, PEDV, PDCoV and PoRVA, where 1-7: TGEV, PEDV, PDCoV, PoRVA, PRRSV, CSFV, PCV2; 8: negative control.

[0018] Figure 6 The results of the four-fold fluorescence quantitative RT-PCR sensitivity test of TGEV, PEDV, PDCoV and PoRVA are shown in Table 1. 8 -10 copies / μL, PEDV 2×10 8 -10 copies / μL, PDCoV 2×10 4 -2×10copies / μL, PoRVA 5×10 5 Amplification curve of -5×10 copies / μL. DETAILED DESCRIPTION

[0019] The present invention designs multiple pairs of specific primers and Taqman probes for TGEV, PEDV, PDCoV and PoRVA, and establishes a quadruple fluorescence quantitative RT-PCR method that can simultaneously identify and detect the four viruses, providing a reliable technical means for clinical diagnosis and prevention and control of porcine viral diarrhea diseases in pig herds.

[0020] The TGEV, PDCoV and PoRVA strains (Genebank NO. OQ473581) used in the examples of the present invention were isolated and preserved by the inventor's laboratory, and the PEDV strain was provided by Yangzhou University; the samples tested were 109 diarrhea samples retained in this experiment.

[0021] A Bio-Rad CFX96™ Real-Time System and a thermos scientific fully automated nucleic acid extraction instrument were used. Reagents included the GENFINE FineMag Rapid Magnetic Bead-Based Viral DNA / RNA Extraction Kit, 5× Hifair MPBuffer, Hifair Enzyme Mix, a Monad 96-well fluorescence quantitative PCR plate, and Monad high-transmittance pressure-sensitive qPCR sealing film.

[0022] In the present embodiment, the GENFINE FineMag rapid magnetic bead viral DNA / RNA extraction kit was used to extract nucleic acids of TGEV, PEDV, PDCoV and PoRVA viruses, and the operation was performed according to the instructions.

[0023] For single-plex fluorescent quantitative RT-PCR nucleic acid amplification of TGEV, PEDV, PDCoV, and PoRVA, positive standards for TGEV, PEDV, PDCoV, and PoRVA were amplified using fluorescent quantitative PCR primers and probes, respectively. A negative control was also established. The reaction system (Table 1) was prepared in the dark, centrifuged briefly, and then placed in a fluorescent quantitative PCR instrument for amplification (Table 2).

[0024] Table 1: Fluorescence quantitative RT-PCR amplification reaction system

[0025] Table 2: Fluorescence quantitative RT-PCR reaction procedure

[0026] The present invention is further described below with reference to the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the method, steps or conditions of the present invention are within the scope of the present invention. Experimental methods and reagents without specific conditions in the examples are all based on conventional conditions in the art.

[0027] Example 1: Screening and establishment of quadruple fluorescent quantitative RT-PCR primer pairs for TGEV, PEDV, PDCoV and PoRVA 1. Screening of primers and probes for quadruple fluorescence quantitative RT-PCR of TGEV, PEDV, PDCoV and PoRVA The full-length sequences of 77 PEDV, 63 TGEV, and 19 PDCoV strains from my country were downloaded from the NCBI database and compared with the VP6 gene sequences of 85 PoRVA strains. Based on the alignment results, relatively conserved sequences were identified. Three pairs of primer-probe sequences were designed for PEDV, TGEV, and PDCoV, and two pairs of primer-probe sequences were designed for PoRVA. Primers and probes with the best amplification efficiency for PEDV, TGEV, PDCoV, and PoRVA were selected (Table 3).

[0028] Table 3: Information of primers and probes for quadruple fluorescence quantitative RT-PCR of TGEV, PEDV, PDCoV and PoRVA

[0029] 2. Optimization of quadruple fluorescence quantitative RT-PCR reaction conditions for TGEV, PEDV, PDCoV, and PoRVA Based on the previously optimized single-plex fluorescence quantitative RT-PCR reaction system and annealing temperature for TGEV, PEDV, PDCoV, and PoRVA, different concentrations of primers and probes were added to optimize the reaction system and conditions of the quadruple method (Table 4).

[0030] Table 4: Optimization of primer and probe concentrations for quadruple fluorescent quantitative RT-PCR of TGEV, PEDV, PDCoV, and PoRVA

[0031] 3. Optimization of annealing temperature and cycle number for quadruple fluorescence quantitative RT-PCR of TGEV, PEDV, PDCoV and PoRVA Based on the optimized primer and probe concentrations for the quadruple assay for TGEV, PEDV, PDCoV, and PoRVA, six different annealing temperatures (58°C, 59°C, 60°C, 61°C, 62°C, and 63°C) were set to screen for the optimal reaction temperature. After determining the optimal annealing temperature, the reaction cycle number was set to 40 to explore the optimal number of cycles for quadruple fluorescence quantitative RT-PCR.

[0032] 4. Establishment of standard curves for quadruple fluorescence quantitative RT-PCR of TGEV, PEDV, PDCoV and PoRVA The concentrations of four pathogenic nucleic acids were TGEV 10 6 -10 copies / μL, PEDV 2×10 5 -10 copies / μL, PDCoV 2×10 3 -2×10 copies / μL, PoRVA 5×10 6-5×10 copies / μL was used as the positive template, and this mixture was used as the template (ddH2O was used as the negative control). Amplification was performed according to the optimized quadruple fluorescence quantitative RT-PCR reaction system and conditions, and a standard curve was drawn.

[0033] 5. TGEV, PEDV, PDCoV and PoRVA quadruple fluorescence quantitative RT-PCR specificity test The optimized quadruple fluorescence quantitative RT-PCR system and reaction procedure of PEDV, TGEV and PDcoV were used to amplify the RNA of PEDV, TGEV, PDCoV, PRoV, PRRSV, CSFV and DNA of PCV2 to verify the specificity of the quadruple fluorescence quantitative PCR method.

[0034] 6. Quadruple fluorescence quantitative RT-PCR sensitivity test for TGEV, PEDV, PDCoV and PoRVA At different concentrations (TGEV 10 8 -10 -4 copies / μL, PEDV 2×10 8 -2×10 -4 copies / μL, PDCoV2×10 4 -2×10 -4 copies / μL, PoRVA 5×10 5 -5×10 -4 TGEV, PEDV, PDCoV and PoRVA nucleic acids at a concentration of 500 copies / μL were used as positive templates, and ddH2O was used as a negative control template. Quadruple fluorescent quantitative PCR amplification of TGEV, PEDV, PDCoV and PoRVA was performed according to the optimized system and reaction procedure to determine its sensitivity.

[0035] 7. Repeatability test of quadruple fluorescence quantitative RT-PCR for TGEV, PEDV, PDCoV and PoRVA TGEV, PEDV, PDCoV, and PoRVA nucleic acids were serially diluted 10-fold and mixed in equal proportions. This mixture was used as a template (ddH2O was used as a negative control) and the template was amplified using the optimized quadruple fluorescent quantitative RT-PCR method described above. Each dilution template was repeated three times to verify the intra-group reproducibility of the method. At the same time, the four templates were tested three times at different times, the results were collected and analyzed, and the intra-group and inter-group coefficients of variation of the method were calculated (Table 5).

[0036] Table 5: Intra-group and inter-group repeatability test table

[0037] 8. Application of quadruple fluorescence quantitative PCR for TGEV, PEDV, PDCoV and PoRVA The diarrhea samples stored in this experiment were tested using the quadruple fluorescence quantitative RT-PCR method for TGEV, PEDV, PDCoV and PoRVA (Table 6), and TGEV, PEDV, PDCoV and PoRVA positive nucleic acids were used as positive controls, and ddH2O was used as a negative control.

[0038] Table 6: Clinical sample test results

[0039] In summary, the present invention uses real-time fluorescence PCR technology to achieve high-sensitivity, high-specificity, and low-complexity visual detection of TGEV, PEDV, PDCoV, and PoRVA.

[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A primer and probe combination for quadruple fluorescent quantitative PCR detection of TGEV, PEDV, PDCoV and PoRVA viruses, characterized in that: The primer and probe combination includes: 1) A primer pair and a probe for detecting porcine transmissible gastroenteritis virus (TGEV), wherein the sequences of the primer and the probe are SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; 2) A primer pair and a probe for detecting porcine epidemic diarrhea virus (PEDV), wherein the sequences of the primer and the probe are SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; 3) A primer pair and a probe for detecting porcine deltacoronavirus (PDCoV), wherein the sequences of the primers and the probe are SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; 4) A primer pair and a probe for detecting porcine rotavirus a, wherein the sequences of the primers and the probe are SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

2. The primer and probe combination according to claim 1, wherein The 5' end or 3' end of the probe is connected with different fluorescent reporter groups or fluorescent quencher groups respectively.

3. The primer and probe combination according to claim 1, wherein The probe for detecting porcine transmissible gastroenteritis virus TGEV has a 5' end connected to VIC and a 3' end connected to BHQ1.

4. The primer and probe combination according to claim 1, wherein The probe for detecting porcine epidemic diarrhea virus (PEDV) has a 5' end connected to FAM and a 3' end connected to BHQ1.

5. The primer and probe combination according to claim 1, wherein The probe for detecting porcine deltacoronavirus PDCoV has CY5 connected to its 5' end and BHQ2 connected to its 3' end.

6. The primer and probe combination according to claim 1, wherein The probe for detecting porcine rotavirus a has its 5' end connected to ROX and its 3' end connected to BHQ1.

7. Use of the primer and probe combination according to claim 1 in the preparation of a kit for quadruple fluorescent quantitative PCR detection of TGEV, PEDV, PDCoV and PoRVA.

8. A kit for the detection of TGEV, PEDV, PDCoV and PoRVA by quadruple fluorescence quantitative PCR, characterized in that: The kit comprises the primer and probe combination according to claim 1.

9. A method for detecting TGEV, PEDV, PDCoV and PoRVA by quadruple fluorescence quantitative PCR, characterized in that: The method uses the primer and probe combination described in claim 1 to perform quadruple fluorescent quantitative PCR amplification of TGEV, PEDV, PDCoV and PoRVA, and judges the detection results of TGEV, PEDV, PDCoV and PoRVA according to the CT value.

10. The method according to claim 9, wherein The CT value of TGEV is ≤35 for positive, 35<CT value<40 for suspicious, and CT value ≥40 for negative; the CT value of PEDV is ≤35 for positive, 35<CT value<40 for suspicious, and CT value ≥40 for negative; the CT value of PDCoV is ≤35 for positive, 35<CT value<40 for suspicious, and CT value ≥40 for negative; the CT value of PoRVA is ≤35 for positive, 35<CT value<40 for suspicious, and CT value ≥40 for negative.

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