CN116377133A - Primer, kit and application for dual RPA amplification of infectious bovine rhinotracheitis virus and bovine viral diarrhea virus - Google Patents
Primer, kit and application for dual RPA amplification of infectious bovine rhinotracheitis virus and bovine viral diarrhea virus Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
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Abstract
The invention belongs to the technical field of pathogenic microorganism detection products, and relates to a primer group, a kit and application for dual RPA amplification of infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. The invention provides a primer set for dual RPA amplification of bovine infectious rhinotracheitis virus and bovine viral diarrhea virus, which comprises a forward primer IBRV-F and a reverse primer IBRV-R for amplifying the bovine infectious rhinotracheitis virus, and a forward primer BVDV-F and a reverse primer BVDV-R for amplifying the bovine viral diarrhea virus. The primer set provided by the invention has the advantages of strong specificity and good sensitivity, and can realize double-RPA amplification detection of BVDV and IBRV, and the primer set is efficient and reliable.
Description
Technical Field
The invention belongs to the technical field of pathogenic microorganism detection products, and particularly relates to a primer group for dual RPA amplification of infectious bovine rhinotracheitis virus and bovine viral diarrhea virus, a kit and application.
Background
Infectious bovine rhinotracheitis virus (Infectious bovine rhinotracheitis virus, IBRV), known as bovine herpes virus type I (bovinerepesvirus-1, bhv-1), belongs to the genus varicella (Varicellovirus) of the Herpesviridae (Herpesviridae), alpha-Herpesviridae (Alphaherpesvirinae), and causes infectious bovine rhinotracheitis (Infectious Bovine Rhinotracheitis, IBR). IBR is a very latent acute infectious disease with a wide range of prevalence and cross species transmission, causing a huge economic loss to the cattle industry. The world animal health Organization (OIE) ranks it as a class B infectious disease, and China ranks it as a class B infectious disease. Bovine viral diarrhea virus (Bovine Viral Diarrhea Virus, BVDV) is a sense ribonucleic acid (RNA) virus belonging to the genus pestivirus of the family flaviviridae, and can cause bovine viral diarrhea-Mucosal Disease (Bovine Viral Diarrhea-Mucosal Disease, BVD-MD). BVD is an acute, febrile, contagious disease, and is characterized clinically by fever, diarrhea, mucosal erosion, ulcers, and abortion.
Currently, methods for detecting BVDV and IBRV include serological neutralization tests, cell separation culture, enzyme-linked immunosorbent assay, PCR technology and the like. Serological methods have the disadvantages of time and effort consuming, low accuracy and the like. The detection method of molecular biology has the defects of false positive, environmental pollution, low repeatability and the like. Therefore, there is a need to establish a rapid, accurate and simple detection technique and means for BVD and IBR. Recombinase polymerase amplification (recabinase PolymeraseAmplification, RPA), a nucleic acid detection technique different from PCR, is mainly involved in combining three enzymes, single-stranded nucleic acid Recombinase, single-stranded DNA binding protein, and strand displacement DNA polymerase. The RPA reaction temperature is low in requirement, short in reaction time, simple in operation and free of special equipment. However, no product capable of realizing dual efficient RPA amplification of BVDV and IBRV exists at present.
Disclosure of Invention
The invention aims to provide a primer group, a kit and application for dual RPA amplification of infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. The primer set provided by the invention has the advantages of strong specificity and good sensitivity, and can realize dual RPA amplification detection of BVDV and IBRV, and the primer set is efficient and reliable.
The invention provides a primer group for dual RPA amplification of bovine infectious rhinotracheitis virus and bovine viral diarrhea virus, which comprises a forward primer IBRV-F and a reverse primer IBRV-R for amplifying the bovine infectious rhinotracheitis virus, and a forward primer BVDV-F and a reverse primer BVDV-R for amplifying the bovine viral diarrhea virus;
the nucleotide sequence of the IBRV-F is shown as SEQ ID NO.1, the nucleotide sequence of the IBRV-R is shown as SEQ ID NO.2, the nucleotide sequence of the BVDV-F is shown as SEQ ID NO.3, and the nucleotide sequence of the BVDV-R is shown as SEQ ID NO. 4.
The invention also provides a kit for dual RPA detection of bovine infectious rhinotracheitis virus and bovine viral diarrhea virus, which comprises the primer group for RPA amplification and the reaction reagent.
Preferably, the reaction reagent comprises magnesium acetate aqueous solution, RPA reaction buffer, RPABasic freeze-dried powder and sterile deionized water.
Preferably, the kit further comprises a positive control and a negative control; the positive control is a recombinant plasmid containing a sequence with nucleotide shown as SEQ ID NO.5 and a recombinant plasmid containing a sequence with nucleotide shown as SEQ ID NO. 6; the negative control was sterile deionized water.
The invention also provides application of the primer group in preparation of a kit for detecting or assisting in detecting infectious bovine rhinotracheitis virus and bovine viral diarrhea virus.
The invention also provides a method for detecting infectious bovine rhinotracheitis virus and bovine viral diarrhea virus for non-disease diagnosis and treatment purposes, which comprises the following steps:
the primer group is used for respectively carrying out RPA amplification reaction on a sample to be detected, positive control and negative control to obtain an amplification product; and judging whether the sample to be detected contains the infectious bovine rhinotracheitis virus and the bovine viral diarrhea virus according to the amplification product result.
Preferably, the reaction system of the RPA amplification reaction comprises per 50 μl: 2. Mu.L of the sample to be tested, positive or negative control, 6.4. Mu.L of sterile deionized water, 29.5. Mu.L of LRPA reaction buffer, 2.4. Mu.L of each primer, 2.5. Mu.L of magnesium acetate in water and 4mg of RPABasic lyophilized powder.
Preferably, the condition of the RPA amplification reaction is that the reaction is carried out for 20 to 40 minutes at a temperature of between 37 and 41 ℃.
Preferably, the method of judging based on the result of the amplification product includes an electrophoresis detection method.
Preferably, the judging method includes: after electrophoresis detection, if a 208bp target band appears, judging that the sample contains bovine viral diarrhea virus; if the 141bp target band appears, judging that the sample contains infectious bovine rhinotracheitis; if the target bands of 208bp and 141bp appear, judging that the sample contains bovine viral diarrhea virus and bovine infectious rhinotracheitis virus; if the target bands of 208bp and 141bp do not appear, the sample is judged to be free of bovine viral diarrhea virus and bovine infectious rhinotracheitis virus.
The invention provides a primer group for dual RPA amplification of infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. The primer set has strong specificity and good sensitivity, the kit is simple to operate and quick to detect, and the invention provides a novel method for the on-site detection of bovine viral diarrhea and infectious bovine rhinotracheitis. Specifically, the invention has the following beneficial effects:
(1) The invention provides a primer group for amplifying BVDV and IBRVRPA, which has strong specificity and good sensibility;
(2) The invention screens the optimal primer and the optimal reaction condition for RPA detection and provides a dual RPA detection kit suitable for BVDV and IBRV; the kit has the advantages of low instrument and equipment requirements, simple operation, rapid detection and the like, provides a new method for detecting BVDV and IBRV, and is suitable for on-site detection of ports or samples;
(3) The invention carries out double RPA detection on clinical samples, and the result is consistent with the PCR detection method, which shows that the method is suitable for detecting BVDV and IBRV, and simultaneously, the reliability of the established double RPA detection method is also verified.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a plasmid map diagram of the recombinant plasmids pcDNA3.1-BVDV and pcDNA3.1-IBRV constructed according to the invention; wherein A is pcDNA3.1-BVDV plasmid map; b is a plasmid map spectrogram of pcDNA3.1-IBRV;
FIG. 2 shows the gel electrophoresis patterns for the enzyme digestion and identification of the recombinant plasmids pcDNA3.1-BVDV and pcDNA3.1-IBRV of the invention; wherein A is pcDNA3.1-BVDV restriction enzyme digestion identification gel electrophoresis pattern, in A, M.1kb plus DNAMarker,1.Bam HI is singly cut, and 2.Bam HI-Hand III is doubly cut; b is a digestion identification gel electrophoresis chart of pcDNA3.1-IBRV, in B, M.1kbplus DNAMarker,1.HandIII is digested singly, and 2.hand III-XhoI is digested doubly;
FIG. 3 is a gel electrophoresis diagram of the screening of RPA amplification primers of BVDV 5-UTR gene, IBRVgB gene of the present invention; wherein A is an RPA primer screening electrophoresis chart of BVDV 5-UTR genes, in A, M.50bp DNAMaroer, 1.5-UTR-1 primer amplification result, 2.5-UTR-2 primer amplification result, 3.5-UTR-3 primer amplification result, and 4. Negative control (no primer addition); b is an RPA amplification primer screening electrophoresis chart of the IBRVgB gene, wherein M.50bp DNAMmarker, 1.GB-1 primer amplification result, 2. GB-primer amplification result and 3.GB-3 primer amplification result are shown in B; 4. a negative control;
FIG. 4 shows gel electrophoresis of dual RPA amplification primers of BVDV 5-UTR gene, IBRV gB gene of the invention; wherein M.50bp DNAMaroker; bvdv; ibrv; BVDV+IBRV;4. a negative control;
FIG. 5 shows gel electrophoresis patterns of the temperature optimization of the dual RPA amplification reaction of BVDV and IBRV of the invention; wherein, M.DNA is 50bp marker;1.30 ℃;2.32 ℃;3.35 ℃;4.37 ℃;5.39 ℃;6.42 ℃;7. negative control (4 ℃);
FIG. 6 shows gel electrophoresis patterns of the double RPA amplification reaction time optimization of BVDV and IBRV of the invention; wherein, M.DNA is 50bp marker;1.10min;2.15min;3.20min;4.25min;5.30min;6.40min;7. negative control (0 min);
FIG. 7 shows gel electrophoresis patterns of specific experiments for BVDV and RPA detection of the present invention; wherein M.DNA50bp marker; bvdv; ibrv; BVDV+IBRV; bcov; brv; BNOV; bastv;8. a negative control;
FIG. 8 is a sensitivity test chart of dual RPA detection of BVDV, IBRV of the invention; wherein M.DNA50bp marker;1 to 11: 1X 10 10 copies/μL～1×10 0 cobies/. Mu.L; 12. a negative control;
FIG. 9 is a graph showing the results of the detection of dual RPA clinical samples of BVDV and IBRV of the present invention; wherein 1-10. Niu Shizi clinical samples; 11. a negative control; 12. positive control.
Detailed Description
The invention provides a primer group for dual RPA amplification of bovine infectious rhinotracheitis virus and bovine viral diarrhea virus, which comprises a forward primer IBRV-F and a reverse primer IBRV-R for amplifying the bovine infectious rhinotracheitis virus, and a forward primer BVDV-F and a reverse primer BVDV-R for amplifying the bovine viral diarrhea virus;
the nucleotide sequence of the IBRV-F is shown as SEQ ID NO.1 (5'-AGCTACTCGCCGGAGCGCTTCCAGCAGATC-3'), the nucleotide sequence of the IBRV-R is shown as SEQ ID NO.2 (5'-CTTGGGCACCCAGTCCCAGGCTACCGTCAC-3'), the nucleotide sequence of the BVDV-F is shown as SEQ ID NO.3 (5'-GAGTTCGTTGGATGGCTGAAGCCCTGAGTA-3'), and the nucleotide sequence of the BVDV-R is shown as SEQ ID NO.4 (5'-AGTAGCATTACAGTGGGCCTCTGCAGCACC-3').
The invention also provides a kit for dual RPA detection of bovine infectious rhinotracheitis virus and bovine viral diarrhea virus, which comprises the primer group for RPA amplification and the reaction reagent.
In the invention, the reaction reagent comprises magnesium acetate aqueous solution, RPA reaction buffer solution, RPABasic freeze-dried powder and sterile deionized water.
In the present invention, the kit further comprises a positive control and a negative control; the positive control is a recombinant plasmid containing a nucleotide sequence shown as SEQ ID NO.5 (BVDV, AAGCTTGTATTGGGCAATCAAAAATAATGATTAGGCCTAGGGAACAAACCCTCTCAGCGAAGGCCGAAAGGAGGCTAGCCATGCCCTTAGTAGGACTAGCAAAATGAGGGGGGTAGCAACAGTGGTGAGTTCGTTGGATGGCTGAAGCCCTGAGTACAGGGCAGTCGTCAGTGGTTCGACACCTAGGATGGTAGGTCTCGAGATGCCACGTGGACGAGGGCATGCCCACAGCACATCTTAGCCTGAGCGGGGGTCGCCCAGGTGAAAGCGGTACAGACAGACCGCTACGAATACAGCCTGATAGGGTGCTGCAGAGGCCCACTGCAATGCTACTAAAAATCTCTGCTGTACATGGCACAGGATCC) and a recombinant plasmid containing a nucleotide sequence shown as SEQ ID NO.6 (IBRV, AAGCTTGTACGACTCGTTCGCGCTCTCGACCGGGGACATTATCTACATGTCGCCCTTTTACGGGCTGCGCGAGGGCGCGCACCGCGAGCACACCAGCTACTCGCCGGAGCGCTTCCAGCAGATCGAGGGCTACTACAAGCGCGACATGGCCACGGGCCGGCGCCTCAAGGAGCCGGTCTCGCGGAACTTTTTGCGTACACAGCACGTGACGGTAGCCTGGGACTGGGTGCCCAAGCGCAAAAACGTGTGCTCGCTGGCCAAGTGGCGCGAGGCGGACGAAATGCTGCGAGACGAGAGCCGCGGGAACTTCCGCTTCACGGCCCGCTCGCTCTCGGCGACCTTTGTGAGCGACAGCCACACCTTCGCGTTGCAGAATGTGCCGCTGAGCGACTGCGTGATCGAAGAGGCCGAGGCCGCGGTCGAGCGCGTCTACCGCGAGCGCTACAACGGCACGCACGTGCTGTCGGGCAGCCTGGAGACGTACTTGCTCGAG); the negative control was sterile deionized water.
The invention also provides application of the primer group in preparation of a kit for detecting or assisting in detecting infectious bovine rhinotracheitis virus and bovine viral diarrhea virus.
The invention also provides a method for detecting infectious bovine rhinotracheitis virus and bovine viral diarrhea virus for non-disease diagnosis and treatment purposes, which comprises the following steps:
the primer group is used for respectively carrying out RPA amplification reaction on a sample to be detected, positive control and negative control to obtain an amplification product; and judging whether the sample to be detected contains the infectious bovine rhinotracheitis virus and the bovine viral diarrhea virus according to the amplification product result.
The primer group of the technical scheme is used for respectively carrying out RPA amplification reaction on a sample to be detected, positive control and negative control to obtain an amplification product. In the present invention, the sample to be tested preferably comprises a genomic nucleic acid sample, which preferably comprises DNA or RNA. When the genomic nucleic acid sample is RNA, the present invention preferably reverse transcribes the RNA to obtain cDNA. In the present invention, it is preferable to carry out RPA amplification using DNA or cDNA as a template. In the present invention, the reaction system of the RPA amplification reaction preferably comprises, per 50. Mu.L: 2. Mu.L of the sample to be tested, positive or negative control, 6.4. Mu.L of sterile deionized water, 29.5. Mu.L of LRPA reaction buffer, 2.4. Mu.L of each primer, 2.5. Mu.L of magnesium acetate in water and 4mg of RPABasic lyophilized powder. In the present invention, the conditions for the RPA amplification reaction are preferably 37 to 41℃for 20 to 40 minutes, more preferably 39℃for 20 minutes. In the invention, the positive control and the negative control can ensure the reliability of the detection result.
After the amplification product is obtained, the invention judges whether the sample to be detected contains the infectious bovine rhinotracheitis virus and the bovine viral diarrhea virus according to the result of the amplification product. In the present invention, the method of judging based on the result of the amplified product preferably includes an electrophoresis detection method. In the present invention, the judging method preferably includes: after electrophoresis detection, if a 208bp target band appears, judging that the sample contains bovine viral diarrhea virus; if the 141bp target band appears, judging that the sample contains infectious bovine rhinotracheitis; if the target bands of 208bp and 141bp appear, judging that the sample contains bovine viral diarrhea virus and bovine infectious rhinotracheitis virus; if the target bands of 208bp and 141bp do not appear, the sample is judged to be free of bovine viral diarrhea virus and bovine infectious rhinotracheitis virus.
For further explanation of the present invention, a primer set, a kit and an application for dual RPA amplification of bovine infectious rhinotracheitis virus and bovine viral diarrhea virus, provided by the present invention, will be described in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Design and screening of dual RPA amplification primer set for infectious bovine rhinotracheitis virus and bovine viral diarrhea virus
(1) Template construction
And under the limitation of pathogen sources, artificially synthesizing pathogen genome sequences, and carrying out subsequent experiments by taking the artificially synthesized BVDV 5-UTR gene and IBRVgB gene sequences as templates.
The whole genome sequences of Bovine Viral Diarrhea Virus (BVDV) and bovine infectious rhinotracheitis virus (IBRV) are collected in GeneBank, the sequences are compared and analyzed by using Mega software, genes which are conserved in seeds and specific among seeds are selected as detection target genes, and BVDV 5-UTR genes (GenBank ID: KF 205301.1) and IBRV gB genes (GenBank ID: MK 654723.1) are determined as target genes. Further comparing the target gene sequences, intercepting the relatively conserved 365Bp (BVDV) and 493bp (IBRV) sequences, and determining the target gene to be amplified. Constructing a target gene (SEQ ID NO. 5) into a pcDNA3.1 vector by using BamHI and HindIII restriction sites to obtain a pcDNA3.1-BVDV recombinant vector, wherein the vector map is shown as A in figure 1; the desired gene (SEQ ID NO. 6) was constructed into pcDNA3.1 vector using Hind III and XhoI cleavage sites to give pcDNA3.1-IBRV recombinant vector with the vector pattern shown in B in FIG. 1. After the plasmid is extracted from the constructed recombinant plasmid, double enzyme digestion verification is carried out, the agarose gel electrophoresis result is shown in figure 2, and the result is consistent with the expectation.
In addition, bovine coronavirus (BCoV, genBank ID: KT 318096.1), bovine rotavirus (BRV, genBank ID: M92651.1), niu Nuo such as virus (BNoV, genBank ID: KX 189094.1), bovine astrovirus (BAstV, genBank ID: MH 123914.1) relative conserved gene fragments were constructed between HindIII and XHOI cleavage sites of pcDNA3.1 vector by using a conventional recombinant vector construction method for specific detection of BVDV, IBRV dual RPA.
(2) Primer design
Primer designs of BVDV 5-UTR gene and IBRVgB gene are carried out according to the following principle:
A. the primer length is between 30 and 35 bp;
B. the TM value of the primer is not used as a reference, but the GC content is between 40% and 60%;
C. the primer does not contain a hairpin structure, so that dimers and mismatch are avoided;
D. the first 3 nucleotides at the 5 'end of the primer comprise cytosine or guanine and the last 3 nucleotides at the 3' end comprise cytosine or guanine;
E. specific structures such as palindromic sequences, long strings of polypurine or polypyrimidine (no more than 5), etc., are avoided in the primers.
Primers were synthesized by biological engineering (Shanghai) Inc., and the information about the primer sequences is shown in Table 1.
TABLE 1BVDV, IBRV RPA primer information Table
Example 2
Design and optimization of dual RPA detection kit for infectious bovine rhinotracheitis virus and bovine viral diarrhea virus
(1) Establishment of BVDV (BVDV-television receiver, IBRV (television receiver and television receiver) dual RPA (remote procedure for video) detection method
Reference to
Basic RPA kit (TwitDx Co.) showed that RPA amplification was performed using the extracted recombinant plasmids pcDNA3.1-BVDV and pcDNA3.1-IBRV as templates. The following reaction system of RPA is configured according to the recommended kit: 2 mu L of template, 6.4 mu L of sterile deionized water, 29.5 mu L of LRPA reaction buffer, 2.4 mu L of primer and 2.5 mu L of magnesium acetate aqueous solution are mixed uniformly, and 4mg of RPABasic freeze-dried powder is fully dissolved. RPA reactionThe procedure is: 39 ℃ for 20min. After electrophoresis of the product on a 1.5% agarose gel, the amplification result was observed in a UV gel imaging system and photographed.
(2) BVDV, IBRV dual RPA primer screening
The PRA amplification reaction was performed according to the established PRA method described above by designing 6 pairs of primer sets (Table 1) according to the step (2) of example 1, and agarose electrophoresis results are shown in FIG. 3. The result shows that the primer pair 5-UTR-1F/R, gB-1F/R has the brightest amplified target band and the best amplification effect, so that the 5-UTR-1F/R, gB-1F/R is selected as a post RPA detection primer. As shown in FIG. 4, the dual RPA reaction system successfully amplified a 208bp BVDV band, a 141bp IBRV band.
(3) BVDV (BVDV-IBRV) -dual RPA (reverse transcription factor) detection system optimization
And (3) optimizing the reaction temperature and the reaction time of the RPA detection system according to the screened optimal primer, and respectively setting 6 reaction temperatures (30 ℃, 32 ℃, 35 ℃, 37 ℃, 39 ℃, 42 ℃) and 6 reaction times (10 min, 15min, 20min, 25min, 30min and 40 min) to determine the optimal RPA detection system. The result shows that when the reaction temperature is 37 ℃, the electrophoresis band of the RPA amplification product is the brightest, the BVDV and IBRV gray values are the highest (figure 5), and the amplification effect is optimal; thus, 37℃was used as the BVDV IBRV dual RPA amplification reaction temperature. When the RPA amplification reaction was carried out for 30min, the electrophoresis band of the RPA amplification product was brightest, the gray scale value was highest (FIG. 6), and the target band appeared with the optimal amplification effect. Thus, 30min was chosen as BVDV IBRV dual RPA amplification time.
(4) Specificity of BVDV, IBRV dual RPA detection method
RPA assays were performed on Bovine Viral Diarrhea Virus (BVDV), bovine infectious rhinotracheitis virus (IBRV), bovine Viral Diarrhea Virus (BVDV) + bovine infectious rhinotracheitis virus (IBRV), bovine coronavirus (BCoV), bovine Rotavirus (BRV), niu Nuo, such as virus (BNoV), bovine astrovirus (BAstV), recombinant plasmids, respectively, to determine the specificity of the established RPA assays. The results showed that the target bands only appeared in the pcDNA-BVDV recombinant plasmid, the pcDNA-IBRV recombinant plasmid and the pcDNA-BVDV+pcDNA-IBRV recombinant plasmid (FIG. 7), and none of the other bands, indicating that the established dual PRA detection method has specificity.
(5) Sensitivity of BVDV, IBRV dual RPA detection method
The quantified pcDNA3.1-BVDV and pcDNA3.1-IBRV plasmids were subjected to 11-gradient dilutions, i.e.1X10 10 copies/μL～1×10 0 The RPA detection sensitivity test was performed by electrophoresis detection technique with copies/. Mu.L. BVDV, IBRV dual RPA sensitivity experiments are shown in FIG. 8, the lowest detection threshold of agarose gel electrophoresis is 1×10 1 copies/μL。
Example 3
BVDV (BVDV-IBRV) dual RPA detection application
In order to examine the detection effect of clinical specimens of the BVDV and IBRV dual RPA detection method established by the invention, 30 diarrhea cattle specimens (A, B and C in FIG. 9 are respectively shown as 10 parts) are collected in Ningxia (7 months in 2022), total RNA is extracted by adopting a Trizol method, reverse transcribed cDNA is used as a template, deionized water is used as negative control, artificial recombinant plasmid pcDNA3.1-BVDV is used as positive control, and BVDV and IBRV are detected by utilizing the established dual RPA. And simultaneously detecting samples by using a polymerase chain reaction (PCR method) to verify the RPA detection result. BVDV-PCR detection upstream primer TCTCGACCGGGGACATTATCT (SEQ ID NO. 15); a downstream primer: CATTCTGCAACGCGAAGGTG (SEQ ID NO. 16), the amplification product is 354bp, and the upstream primer for IBRV-PCR detection is AGGTGGTGGCCTTTGACCG (SEQ ID NO. 17); the downstream primer is: TCGTCTCGCAGCATTTCGTC (SEQ ID NO. 18), the amplified product is 483bp. As shown in FIG. 9, the detection rate of RPA of BVDV clinical samples was 33.33%, the detection rate of PCR was 30%, and the detection rate of RPA of IBRV clinical samples was equal to the detection threshold of PCR (36.67%). The gray value calculation result shows that the RPA detection sensitivity is higher than that of PCR.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. A primer set for dual RPA amplification of bovine infectious rhinotracheitis virus and bovine viral diarrhea virus, characterized in that the primer set comprises a forward primer IBRV-F and a reverse primer IBRV-R for amplifying the bovine infectious rhinotracheitis virus, and a forward primer BVDV-F and a reverse primer BVDV-R for amplifying the bovine viral diarrhea virus;
the nucleotide sequence of the IBRV-F is shown as SEQ ID NO.1, the nucleotide sequence of the IBRV-R is shown as SEQ ID NO.2, the nucleotide sequence of the BVDV-F is shown as SEQ ID NO.3, and the nucleotide sequence of the BVDV-R is shown as SEQ ID NO. 4.
2. A kit for dual RPA detection of bovine infectious rhinotracheitis virus and bovine viral diarrhea virus, comprising the primer set for RPA amplification of claim 1 and a reagent.
3. The kit of claim 2, wherein the reaction reagents comprise aqueous magnesium acetate, RPA reaction buffer, RPABasic lyophilized powder, and sterile deionized water.
4. A kit according to claim 2 or 3, wherein the kit further comprises a positive control and a negative control; the positive control is a recombinant plasmid containing a sequence with nucleotide shown as SEQ ID NO.5 and a recombinant plasmid containing a sequence with nucleotide shown as SEQ ID NO. 6; the negative control was sterile deionized water.
5. Use of the primer set of claim 1 for preparing a kit for detecting or assisting in detecting infectious bovine rhinotracheitis virus and bovine viral diarrhea virus.
6. A method for detecting infectious bovine rhinotracheitis virus and bovine viral diarrhea virus for non-disease diagnostic and therapeutic purposes, comprising the steps of:
performing RPA amplification reaction on a sample to be detected, a positive control and a negative control respectively by using the primer group of claim 1 to obtain an amplification product; and judging whether the sample to be detected contains the infectious bovine rhinotracheitis virus and the bovine viral diarrhea virus according to the amplification product result.
7. The method of claim 6, wherein the reaction system of the RPA amplification reaction comprises, per 50 μl: 2. Mu.L of sample to be tested, positive control or negative control, 6.4. Mu.L of sterile deionized water, 29.5. Mu.L of LRPA reaction buffer, 2.4. Mu.L of primers, 2.5. Mu.L of magnesium acetate aqueous solution and 4mg of RPABasic lyophilized powder.
8. The method of claim 6, wherein the conditions for the RPA amplification reaction are 37-41℃for 20-40 min.
9. The method of claim 6, wherein the method of determining based on the result of the amplification product comprises an electrophoretic detection method.
10. The method of claim 9, wherein the method of determining comprises: after electrophoresis detection, if a 208bp target band appears, judging that the sample contains bovine viral diarrhea virus; if the 141bp target band appears, judging that the sample contains infectious bovine rhinotracheitis; if the target bands of 208bp and 141bp appear, judging that the sample contains bovine viral diarrhea virus and bovine infectious rhinotracheitis virus; if the target bands of 208bp and 141bp do not appear, the sample is judged to be free of bovine viral diarrhea virus and bovine infectious rhinotracheitis virus.
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