Primer group and kit for multiple PCR (Polymerase Chain Reaction) detection of bovine diarrhea virus

Abstract

The invention belongs to the technical field of gene detection, and relates to a primer group and a kit for multiple PCR (Polymerase Chain Reaction) detection of a bovine diarrhea virus. The SapV, BNoV and BEV triple PCR detection technology established in the invention has good specificity and repeatability, can be used for rapid detection of cattle farm diarrhea virus mixed infection, and lays a technical foundation for bovine viral diarrhea pathogen epidemiological investigation.

Description

Technical Field

[0001] The invention belongs to the technical field of gene detection and relates to a primer set and a kit for multiplex PCR detection of bovine diarrhea virus. Background Art

[0002] Bovine diarrhea (BED) is a major disease that hinders the development of the cattle industry. Its pathogens are complex and diverse, with viral infections being a key factor leading to large-scale outbreaks and high mortality rates, particularly in calves. Currently, single or mixed infections with bovine enterovirus (BEV), bovine rotavirus (BRoV), bovine kobuvirus (BKoV), bovine norovirus (BNoV), bovine viral diarrhea virus (BVDV), and sapovirus (SapV) can all cause severe diarrhea in cattle. BEV belongs to the Picornaviridae family, while BNoV and SapV belong to the Caliciviridae family. All three viruses are single-stranded positive-sense RNA viruses without an envelope. Infected calves are prone to malignant diarrhea, characterized by severe vomiting, watery diarrhea, dehydration and other symptoms. The mortality rate is high, and surviving individuals often experience growth retardation. Infected adult cattle show decreased production performance and reduced milk production.

[0003] Epidemiological surveys have shown that viruses such as SapV, BNoV, and BEV often exist in the form of mixed infections, which not only aggravates damage to the calves' bodies but also increases the difficulty of prevention and control. The frequent occurrence of mixed infection cases not only poses challenges to the prevention and control of the epidemic and clinical treatment in the cattle industry, but also puts higher demands on pathogen detection and diagnosis. Although extensive and in-depth research has been carried out on the PCR detection, diagnosis, and prevention and control technologies of BNoV, the early rapid detection technology for mixed infections of BNoV and other viruses still needs to be improved. Therefore, the establishment of a triple PCR technology for the three diarrhea viruses SapV, BNoV, and BEV can achieve early and accurate detection of complex pathogens of cattle diarrhea, providing key technical support for the early formulation of treatment plans, subsequent prevention and control, and epidemiological investigations. Summary of the Invention

[0004] The present invention addresses the deficiencies of the existing technology and aims to develop a PCR method that can simultaneously detect sapovirus (SapV), bovine norovirus (BNoV), and bovine enterovirus (BEV). To this end, the present invention provides a primer set and kit for multiplex PCR detection of bovine diarrhea virus.

[0005] In one aspect, the present invention relates to a primer set for multiplex PCR detection of bovine diarrhea virus, comprising:

[0006] Primers for detecting the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 for saporovirus;

[0007] Primers for detecting the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 for bovine norovirus;

[0008] Primers for detecting the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 of bovine enterovirus.

[0009] Nucleotide sequence variants that have more than 90% (eg, 92%, 94%, 96% or 98%) homology to the above-mentioned nucleotide sequence and have the same function are within the scope of protection of the present invention.

[0010] As is well known to those skilled in the art, multiplex PCR allows for the amplification of multiple DNA targets in a single reaction system. Key to this is the need for primer design to balance the compatibility of multiple primer sets. Primers can interfere with each other, forming numerous dimers that reduce kit performance; nonspecific amplification can produce numerous non-target amplicons, compromising subsequent sequencing; accurate quantification is impossible; amplification uniformity cannot be guaranteed; and scalability and flexibility are limited. These issues make primer design for multiplex PCR impossible using conventional design methods.

[0011] On the other hand, the present invention relates to the use of the primer set in preparing a multiplex PCR detection kit for bovine diarrhea virus.

[0012] Furthermore, in the application provided by the present invention, the bovine diarrhea virus is selected from at least one of sapovirus, bovine norovirus, and bovine enterovirus.

[0013] On the other hand, the present invention relates to a bovine diarrhea virus multiplex PCR detection kit, which comprises the primer set.

[0014] Furthermore, the bovine diarrhea virus multiplex PCR detection kit provided by the present invention also includes a detection reagent and an RNA extraction reagent.

[0015] Furthermore, in the bovine diarrhea virus multiplex PCR detection kit provided by the present invention, the detection reagents include Taq Master Mix and ddH2O.

[0016] In another aspect, the present invention relates to a method for detecting bovine diarrhea virus for non-disease diagnosis purposes, comprising:

[0017] The bovine diarrhea virus multiplex PCR detection kit is used to perform PCR amplification to detect the bovine diarrhea virus in the sample.

[0018] Furthermore, in the bovine diarrhea virus detection method for non-disease diagnosis purposes provided by the present invention, the sample is a cDNA sample derived from bovine feces.

[0019] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0020] SapV, BNoV, and BEV are all RNA viruses with high gene mutation rates. Therefore, the selection of highly conserved detection genes is the key to this application. Based on previous studies and sequence alignment, three well-conserved target genes were selected: SapV RdRp, BNoV RdRp, and BEV 5′-UTR genes. The results showed that at the optimal annealing temperature of 50°C and the primer concentrations of SapV, BNoV, and BEV were 2.5 μmol·L -1 , 5 μmol·L -1 , 2.5 μmol·L -1 Under the following conditions, the primers of the present application can specifically and simultaneously amplify the three viral target genes of SapV, BNoV, and BEV, and there is no mutual interference in the detection of clinical samples, indicating that the selection of the target genes detected by the present application and the primers designed have met the expectations of the established method. In order to determine the feasibility of the clinical application of triple PCR technology, the present application conducted single and triple PCR detection on 200 clinical fecal samples in Yulin area. The results showed that the coincidence rates of triple PCR and single PCR (SapV, BNoV, BEV) were 96.72% (59 / 61), 96.00% (24 / 25), and 94.38% (84 / 89), respectively, and achieved the purpose of simultaneously detecting mixed infection of three viruses, indicating that the present technology can be used for laboratory diagnosis of clinical samples. The phenomenon of mixed infection of multiple pathogens is common in the clinical practice of bovine diarrhea, which fully reflects the urgent need and important value of this technology in clinical application. This technology can effectively assist clinical veterinarians to diagnose the pathogens of bovine diarrhea viruses more accurately, provide key technical support for the effective prevention and control of bovine diarrhea, and has significant practical significance. BEV, BNoV, and SapV are three pathogens that cause cattle diarrhea, and mixed infections can occur. Early and rapid detection and diagnosis are key to their prevention and control. This application successfully established a SapV / BNoV / BEV triple PCR detection technology, which has good specificity, high sensitivity, and strong repeatability. It can detect lower titers of viruses and can also perform low-cost batch detection of all three viruses simultaneously. This technology lays a technical foundation for cattle farms to carry out early diagnosis and epidemiological surveys of SapV / BNoV / BEV, and also provides strong technical support for the healthy and rapid development of the cattle industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Figure 1 shows the results of single and triplex PCR amplification. M represents DL2000 DNA Marker; 1 represents triplex PCR amplification; 2 represents SapV; 3 represents BNoV; 4 represents BEV; and 5 represents the negative control.

[0023] Figure 2 The figure shows the experimental results of optimizing the annealing temperature for triplex PCR. M represents the DL2000 DNA Marker; 1 represents the experimental results at an annealing temperature of 50.0°C; 2 represents the experimental results at an annealing temperature of 52.2°C; 3 represents the experimental results at an annealing temperature of 56.2°C; 4 represents the experimental results at an annealing temperature of 58.8°C; 5 represents the experimental results at an annealing temperature of 61.1°C; 6 represents the experimental results at an annealing temperature of 62.8°C; and 7 represents the negative control.

[0024] Figure 3 The figure shows the results of the triple PCR primer concentration optimization experiment. M represents DL2000 DN AMarker; 1 represents the concentration of SapV, BRoV, and BEV (F / R) primers, each of which has a concentration of 10 μmol·L -1 , 10 μmol·L -1 , 10 μmol·L -1 2 is the experimental results when the concentrations of SapV, BRoV, and BEV (F / R) primers were 10 μmol·L -1 , 10 μmol·L -1 , 5 μmol·L -1 3 is the experimental results when the concentrations of Sap V, BRoV, and BEV (F / R) primers were 5 μmol·L -1 , 10 μmol·L -1 , 5 μmol·L -1 4 is the experimental results when the concentrations of SapV, BRoV, and BEV (F / R) primers were 5 μmol·L -1 , 5 μmol·L -1 , 5 μmol·L -1 5 is the experimental result when the concentration of SapV, BRoV, and BEV (F / R) primers were 5 μmol·L-1 , 5 μmol·L -1 , 2.5 μmol·L -1 6 is the experimental results when the concentrations of SapV, BRoV, and BEV (F / R) primers were 2.5 μmol·L -1 , 5 μmol·L -1 , 2.5 μmol·L -1 7 is the negative control.

[0025] Figure 4 The results of the specificity detection experiment are shown in Figure 1. M represents DL2000 DNA Marker; 1 represents SapV / .BNoV / BEV; 2 represents SapV; 3 represents BNoV; 4 represents.BEV; 5 represents BVDV; 6 represents BRoV; 7 represents BKoV; 8 represents.E coli; 9 represents Cp; and 10 represents the negative control.

[0026] Figure 5 The sensitivity test results are shown in Figure 1. M represents DL2000 DNA Marker; 1 represents 10 -1 Test results after dilution; 2 is 10 -2 Test results after dilution; 3 is 10 -3 Test results after dilution; 4 is 10 -4 Test results after dilution; 5 is 10 -5 Test results after dilution; 6 is 10 -6 Test results after dilution; 7 is 10 -7 Test results after dilution; 8 is 10 -8 Test results after dilution; 9 is the negative control.

[0027] Figure 6 This is a graph showing the results of an intra-batch reproducibility test. M represents the DL2000 DNA Marker; 1 to 3 represent three replicates of the first test in the intra-batch reproducibility test; 4 to 6 represent three replicates of the second test in the intra-batch reproducibility test; and 7 to 8 represent three replicates of the negative control in the intra-batch reproducibility test.

[0028] Figure 7 This is a graph showing the results of an inter-batch reproducibility test. M represents the DL2000 DNA Marker; 1 to 3 represent three replicates of Template 1 in the inter-batch reproducibility test; 4 to 6 represent three replicates of Template 2 in the inter-batch reproducibility test; and 7 to 9 represent three replicates of the negative control in the inter-batch reproducibility test.

[0029] Figure 8 The following are the test results of some clinical samples. M represents DL2000 DNA Marker; 1 to 20 represent the test results of different clinical samples; 21 represents the negative control. DETAILED DESCRIPTION

[0030] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0031] In the following examples, pUC57-BVDV-5′-UTR, pUC57-BRoV-Vp6, pUC57-BKoV-3D, pUC57-SapV-RdRp, pUC57-BNoV-RdRp, and pUC57-BEV-5′-UTR plasmids, Escherichia coli (E. coli), and Clostridium perfringens (Cp) strains were stored by the Key Laboratory of Animal Disease Diagnosis and Detection of Yulin University; DL2000 DNA Marker was purchased from Takara Biotechnology (Dalian) Co., Ltd.; 2× Taq PCR Master Mix and SanPrep column-based plasmid DNA miniprep kit were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; FastKing The cDNA first-strand synthesis kit was purchased from Tiangen Biochemical Technology Co., Ltd.; the low-temperature centrifuge (model 5424ANO70408) and PCR amplifier (model 6325YI602122) were purchased from Shanghai Xinliang Industrial Co., Ltd.; and the electrophoresis apparatus (model DYCP-3DN) was purchased from Bio-Rad Biomedical Products (Shanghai) Co., Ltd.

[0032] Example 1

[0033] This example provides information about primers.

[0034] (1) Primer design and synthesis

[0035] Based on the conserved gene sequences of SapV, BRoV, and BEV published in the NCBI database, specific primers for detecting the three diarrheal viruses were designed using Oligo 7. The primers were synthesized by Invitrogen (Shanghai) Trading Co., Ltd. Primer information is shown in Table 1.

[0036] Table 1: Primer information

[0037]

[0038] (2) Extraction of positive plasmids and nucleic acids

[0039] The pUC57-BVDV-5′-UTR, pUC57-BRoV-Vp6, pUC57-BKoV-3D, pUC57-SapV-RdRp, pUC57-BNoV-RdRp, and pUC57-BEV-5′-UTR plasmids were transformed into DH5α competent cells. After enrichment, the recombinant plasmids were extracted using a SanPrep column-based plasmid DNA miniprep kit and stored at -20°C. After enrichment of E. coli and Cp, DNA was extracted using a nucleic acid DNA / RNA extraction kit and stored at -20°C.

[0040] Example 2

[0041] This example provides an optimization process for the conditions of the triple PCR detection method.

[0042] (1) Single PCR and triple PCR amplification

[0043] SapV (40.17 ng·μL -1 )、BNoV(38.47ng·μL -1 )、BEV(45.38ng·μL -1 ) positive plasmid as template, 1 μL of the mixture was used as template to establish 2× Taq Master Mix 5 μL, plasmid cDNA 1 μL, upstream and downstream primers (10 μmol·L -1 ) 0.5 μL each, ddH2O 1 μL, and a medium-volume triple PCR reaction system was established using the positive plasmids of SapV, BRoV, and BEV as templates, respectively. 2× Taq Master Mix 5 μL, plasmid cDNA 0.5 μL, upstream and downstream primers (10 μmol·L -1 ) and 3.5 μL of ddH2O. A single PCR reaction system was prepared by pre-denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. 10 g·L -1 The results were observed after agarose gel electrophoresis.

[0044] like Figure 1 As shown, both single PCR and triple PCR methods can amplify the target fragments of SapV (827 bp), BNoV (532 bp), and BEV (300 bp), which are consistent with the expected sizes.

[0045] (2) Optimization of triple PCR annealing temperature

[0046] The same triple PCR reaction system was established, and 50.0°C, 52.2°C, 56.2°C, 58.8°C, 61.1°C, and 62.8°C were set as annealing temperatures to confirm the optimal annealing temperature for triple PCR.

[0047] like Figure 2 As shown, the amplification effect is best at 50°C.

[0048] (3) Optimization of triple PCR primer concentration

[0049] A reaction system with an optimal annealing temperature was established using a medium volume of mixed SapV, BNoV, and BEV positive plasmids as templates. Six combinations of final SapV, BNoV, and BEV primer (F / R) concentrations were set (Table 2). The optimal primer concentrations for triplex PCR were determined. PCR amplification was performed using the optimal annealing temperature.

[0050] Table 2: Primer combinations at different concentrations

[0051]

[0052] like Figure 3 As shown in the figure, under the optimal annealing temperature of 50°C, the primer concentrations of SapV, BNoV, and BEV were 2.5 μmol·L -1 , 5 μmol·L -1 , 2.5 μmol·L -1 When combined with PCR products, triplex PCR has the best reaction efficiency.

[0053] Example 3

[0054] This example provides a specificity test, sensitivity evaluation, and repeatability test for the triple PCR detection method.

[0055] (1) Specificity test

[0056] To evaluate the specificity of the established triplex PCR method, PCR amplification was performed using a medium volume of a mixture of SapV, BNoV, and BEV positive plasmids, SapV, BNoV, BEV, BVDV, BRoV, and BKoV positive plasmids, E. coli DNA, and Cp DNA as templates, and ddH2O as a negative control. The optimal annealing temperature and primer concentrations were used for PCR amplification.

[0057] like Figure 4 As shown, the single and mixed positive plasmids of SapV, BNoV, and BEV could amplify the target bands, while the positive plasmids of BVDV, BRoV, BKoV, E. coli and Cp DNA could not amplify the target bands.

[0058] (2) Sensitivity test

[0059] The SapV, BNoV, and BEV positive plasmids mixed in a medium volume were diluted 10-fold in series and PCR amplified using the optimal annealing temperature and primer concentration combination.

[0060] like Figure 5 As shown in Figure 2, the minimum concentrations of BVDV, BNoV, and BKoV that triple PCR could detect were 4.14×10 - 6 ng·μL -1 , 3.83×10 -4 ng·μL -1 , 4.55×10 -6 ng·μL -1 .

[0061] (3) Repeatability test

[0062] An equal volume of SapV, BNoV, and BEV positive mixed plasmids was used as template, and ddH2O was used as a negative control. The established triple PCR method was used to detect the same template twice within a short period of time, with each sample repeated three times to evaluate the intra-batch reproducibility of the method. An equal volume of BVDV, BRoV, and BKoV positive plasmids extracted twice at different times was mixed as template 1 and template 2, respectively, with ddH2O as a negative control. The established triple PCR method was used to detect each sample repeated three times to evaluate the inter-batch reproducibility of the method.

[0063] Depend on Figure 6 and Figure 7 It can be seen that the intra-batch repeatability test ( Figure 6 ) and batch-to-batch ( Figure 7 ) Repeatability test showed that both mixed templates could specifically amplify the expected target bands without any mixed bands.

[0064] (4) Detection of clinical samples: cDNA was obtained from clinical samples and tested using the single PCR method and the established triple PCR method. The coincidence rate (coincidence rate = triple PCR method positive rate / single PCR method positive rate × 100%) was calculated to evaluate the clinical application effect of the established triple PCR method.

[0065] Clinical samples (30, 80, and 90 samples, respectively) of cattle feces were randomly collected from Yuyang, Shenmu, and Qingjian counties in Yulin City, Shaanxi Province, for a total of 200 samples. These samples were refrigerated in nucleic acid preservation solution and sent to the Key Laboratory of Animal Disease Diagnosis and Testing at Yulin University for testing. RNA was extracted from fecal swabs according to the RNAiso Plus instruction manual and reverse-transcribed into cDNA using the FastKing cDNA First-Strand Synthesis Kit. The extracted RNA was then stored at -20°C until further use.

[0066] like Figure 8 As shown, 200 cattle fecal samples were tested using the established triple PCR method. The results showed that the detection rates of SapV, BNoV, and BEV were 29.50% (59 / 200), 12.00% (24 / 200), and 42.00% (84 / 200), respectively. The positive detection rates of single PCR for SapV, BNoV, and BEV were 10.5% (21 / 200), 5.0% (10 / 200), and 20.5% (41 / 200), respectively. The results showed that the detection rates of SapV / BNoV, SapV / BEV, BNoV / BEV, and SapV / BNoV / BEV were 0.5% (1 / 200), 17.0% (34 / 200), 4.00% (8 / 200), and 2.0% (4 / 200), respectively. Mixed infections accounted for 39.49% (47 / 119) of the positive cases. The concordance rates of triple PCR and single PCR (SapV, BNoV, BEV) were 96.72% (59 / 61), 96.00% (24 / 25), and 94.38% (84 / 89), respectively.

[0067] The embodiments described above are some of the embodiments of the present invention, rather than all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but rather represents only selected embodiments of the present invention. All other embodiments obtained without creative effort and through deduction and substitution by a person of ordinary skill in the art based on the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A primer set for multiplex PCR detection of bovine diarrhea virus, characterized in that: include: Primers for detecting the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 for saporovirus; Primers for detecting the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 for bovine norovirus; Primers for detecting the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 of bovine enterovirus.

2. Use of the primer set of claim 1 in preparing a multiplex PCR detection kit for bovine diarrhea virus.

3. The use according to claim 2, characterized in that The bovine diarrhea virus is selected from at least one of sapovirus, bovine norovirus, and bovine enterovirus.

4. A multiplex PCR detection kit for bovine diarrhea virus, characterized in that: Comprising the primer set according to claim 1.

5. The bovine diarrhea virus multiplex PCR detection kit according to claim 4, characterized in that: Also included are detection reagents and RNA extraction reagents.

6. The bovine diarrhea virus multiplex PCR detection kit according to claim 5, characterized in that: The detection reagent includes Taq Master Mix and ddH2O.

7. A method for detecting bovine diarrhea virus for non-disease diagnosis purposes, characterized in that: include: The bovine diarrhea virus multiplex PCR detection kit according to any one of claims 4 to 6 is used to perform PCR amplification to detect the bovine diarrhea virus in the sample.

8. The method for detecting bovine diarrhea virus for non-disease diagnosis purposes according to claim 7, characterized in that: The sample is a cDNA sample derived from cow feces.

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