CN115491438A - Triple RT-PCR method capable of simultaneously detecting bovine viral diarrhea virus, rotavirus and coronavirus and application - Google Patents
Triple RT-PCR method capable of simultaneously detecting bovine viral diarrhea virus, rotavirus and coronavirus and application Download PDFInfo
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Abstract
The invention discloses a triple RT-PCR method capable of simultaneously detecting bovine viral diarrhea virus, rotavirus and coronavirus and application thereof, which comprises the following steps: taking RNA of bovine lymph nodes, intestinal mucosa and viruses as RNA templates, designing specific upstream primers and specific downstream primers as specific primers according to genetic characteristics of three viruses of bovine viral diarrhea virus (SEQ ID NO.1, 187 bp), bovine rotavirus (SEQ ID NO.2, 391 bp) and bovine coronavirus (SEQ ID NO.3, 725 bp), carrying out RT-PCR amplification to obtain amplification products, and judging whether a sample to be detected contains the bovine viral diarrhea virus, the bovine rotavirus and the bovine coronavirus or not according to detection results. The invention is a multiple RT-PCR detection method which is specific, sensitive and rapid and can simultaneously detect 3 RNA viruses of BVDV, BRV and BCoV.
Description
Technical Field
The invention belongs to the technical field of virus detection, and particularly relates to a triple RT-PCR method capable of simultaneously detecting bovine viral diarrhea viruses, rotaviruses and coronavirus and application thereof.
Background
RNA viruses of the genus BVDV (Bovine viral diarrhea virus) of the family Flaviviridae are serologically cross-reactive with Classical Swine Fever Virus (CSFV) and Border sheep virus (BDV). Bovine Rotavirus (BRV) belongs to the genus Rotavirus of the reoviridae family. Bovine Coronavirus (Bovine Coronavirus, BCoV) belongs to the genus Coronavirus (Coronavirus) of the family Coronaviridae (Coronaviridae). The 3 viruses are main viral pathogens causing calf diarrhea and adult bovine winter dysentery. The clinical symptoms of cattle infected with BVDV mainly include diarrhea, mucosal erosion, fever, abortion, fetal deformity and the like. Clinical symptoms after infection of cattle with BRV are mainly manifested by vomiting and diarrhea, and newborn calves are most susceptible. After the cattle are infected with BCoV, the cattle can cause respiratory tract infection besides clinical diarrhea, and newborn calves are most susceptible; whereas, adult cattle infected with BCoV are accompanied by significant respiratory symptoms in addition to acute diarrhea. The 3 pathogens causing the bovine diarrhea are similar in epidemiology, disease characteristics, clinical symptoms and autopsy changes and are not easy to distinguish by naked eyes. The traditional pathogen separation and identification takes longer time, and influences the rapid diagnosis of the pathogen.
At present, there are some publications on the simultaneous detection kit and method for bovine viral diarrhea virus, rotavirus and coronavirus, for example:
1. the patent application CN201910517811.1 discloses a multi-connected RT-PCR detection method for bovine rotavirus, coronavirus and viral diarrhea virus, wherein the detection method takes RNA of a sample to be detected as an RNA template, takes a specific upstream primer and a specific downstream primer designed according to genetic characteristics of the bovine rotavirus, coronavirus and viral diarrhea virus as primers, carries out RT-PCR amplification to obtain an amplification product, then carries out electrophoresis detection on the amplification product through 1.5% agarose gel, records the result, and judges whether the sample to be detected contains the bovine rotavirus, the bovine coronavirus and the viral diarrhea virus or not according to the electrophoresis detection result. The method realizes accurate, rapid and efficient RT-PCR identification and diagnosis of three viral diseases.
2. The patent application CN202110182882.8 discloses a triple fluorescence quantitative PCR kit for simultaneously detecting bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus and application thereof, which belong to the technical field of virus detection and comprise three groups of specific primers and three corresponding probes for the bovine rotavirus, coronavirus and viral diarrhea virus. The primers and the probes for detecting the three viruses have the advantages of strong specificity and high reaction efficiency; the triple real-time fluorescent quantitative PCR kit has the characteristics of high amplification efficiency, strong specificity, stable detection effect and good repeatability, and is suitable for detection and periodic monitoring of dairy cow diarrhea pathogeny, investigation of dairy cow diarrhea epidemiology and the like; the kit using the method can simultaneously detect three kinds of diarrhea pathogens of the dairy cows in a real-time fluorescent quantitative PCR reaction system, saves the detection time, reduces the detection cost, can simultaneously detect a plurality of samples and has high detection efficiency.
3. Patent application CN202210550478.6 discloses a set of triple real-time fluorescent quantitative PCR (Multiplex real-time fluorescent quantitative PCR) primers and probe combinations for simultaneous detection of Bovine Viral Diarrhea Virus (BVDV), bovine Rotavirus (BRV) and Bovine Coronavirus (BCV), wherein the primers and probes are specific for 5' utr gene fragment of BVDV virus, VP6 gene fragment of BRV virus, N gene fragment of BCV virus, and the nucleotide sequences are shown in SEQ ID nos. 1-9. The method can realize qualitative or quantitative detection of three viruses only by carrying out PCR amplification on a sample once, and has the advantages of simple and convenient operation, strong specificity, high sensitivity, good repeatability and the like
4. Patent application CN202110156326.3 discloses a triple RPA detection kit for bovine viral diarrhea virus, bovine coronavirus and bovine rotavirus, which comprises RPA detection primer groups of bovine viral diarrhea virus, bovine coronavirus and bovine rotavirus, which are respectively composed of nucleotide sequences shown in SEQ ID No.1 and SEQ ID No.5, SEQ ID No.8 and SEQ ID No.13, SEQ ID No.15 and SEQ ID No. 18. The kit is based on a recombinase polymerase amplification method, uses cDNA formed by reverse transcription of RNA as a template for amplification reaction, and obtains a detection result by nucleic acid gel electrophoresis. The method can be used for simultaneously detecting the bovine viral diarrhea virus, the bovine coronavirus and the bovine rotavirus, is simple to operate, has higher sensitivity and specificity, and provides a rapid detection reagent for the field screening of the bovine diarrhea virus.
5. Patent application CN202011187035.2 discloses a kit, primers and probes for the simultaneous detection of bovine rotavirus, bovine viral diarrhea virus and bovine coronavirus. The nucleotide sequences of the primers and the probes in the kit are shown as SED ID NO 1-SEQ ID NO 9. The method has the technical advantages of simple operation, strong specificity, high sensitivity and good repeatability, and can simultaneously realize qualitative detection and accurate quantification of BVDV, BRV and BCoV.
In the prior art, the patent applications CN201910517811.1, CN202110182882.8, CN202210550478.6 and CN202110156326.3 all adopt common reverse transcriptases at 50 ℃, and the reaction time is long; while the patent application CN202011187035.2 uses fluorescent probes, although the sensitivity and specificity are stronger than those of the prior art, the cost is much higher.
As can be seen, the prior art discloses a detection method capable of simultaneously detecting 3 viruses of BVDV, BRV and BCoV, but the detection method adopting one-step multiplex RT-PCR is not disclosed yet. With the frequent introduction, distribution and transportation of the Guizhou beef cattle, the pathogeny of the epidemic disease is more complex, and the difficulty in preventing and controlling the epidemic disease is increased. The causes of the bovine diarrhea are very complex, and the infection of pathogens such as toxigenic escherichia coli, salmonella, bovine rotavirus, bovine coronavirus, bovine viral diarrhea-mucosis virus, cryptosporidium, coccidium and the like can cause the diarrhea of beef cattle, and mixed infection often occurs. Therefore, it is important to develop a specific, sensitive, rapid and multiplex RT-PCR detection method capable of simultaneously detecting 3 RNA viruses of BVDV, BRV and BCoV.
Disclosure of Invention
The invention provides a multiple RT-PCR detection method which is specific, sensitive and rapid and can simultaneously detect 3 RNA viruses of BVDV, BRV and BCoV for solving the technical problems, provides effective technical support for early diagnosis, prevention and control of BVDV, BRV and BCoV, and has great significance for diagnosis, prevention and control of 3 viruses of BVDV, BRV and BCoV.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a triple RT-PCR method for simultaneously detecting bovine viral diarrhea virus, rotavirus and coronavirus comprises the following steps: taking RNA of bovine lymph nodes, intestinal mucosa and viruses as RNA templates, referring to BVDV 5' -UTR, BRV VP6 and BCoVN gene sequences in GenBank, designing specific upstream primers and specific downstream primers as specific primers according to genetic characteristics of bovine viral diarrhea viruses (SEQ ID NO.1, 187 bp), bovine rotavirus (SEQ ID NO.2, 391 bp) and bovine coronavirus (SEQ ID NO.3, 725 bp), carrying out RT-PCR amplification to obtain an amplification product, and determining whether a sample to be detected contains the bovine viral diarrhea viruses, the bovine rotavirus and the bovine coronavirus according to a detection result.
Further, the RT-PCR amplification adopts a reverse transcriptase mixture which is transcribed for 10min at 37 ℃, and the reverse transcriptase mixture comprises: 100mM Tris-HCl, 10mM MgCl, pH 8.3 2 、10mM DTT、50mM KCl、0.5mM dTTP、0.4MBq/mL[3H]-dTTP, 1mM poly (A) oligo (dT) 12-18 and reverse transcriptase.
Further, specific upstream primers and specific downstream primers of three viruses of bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus are shown in table 1. The length of the bovine viral diarrhea virus is 187bp, the upstream primer is shown as F1 of SEQ ID NO.1, and the downstream primer is shown as R1 of SEQ ID NO. 1; the length of the bovine rotavirus is 391bp, an upstream primer is shown as F2 of SEQ ID NO.2, and a downstream primer is shown as R2 of SEQ ID NO. 2; the length of the bovine coronavirus is 725bp, an upstream primer is shown as F3 of SEQ ID NO.3, and a downstream primer is shown as R3 of SEQ ID NO. 3.
TABLE 1 primer sequences and fragment sizes
Further, the RT-PCR amplification comprises single RT-PCR amplification and triple RT-PCR amplification.
Further, the single RT-PCR amplification system is: mu.L of each of the upstream and downstream primers, 2X 1Step Buffer 12.5. Mu.L of PrimeScript One Step Enzyme Mix, 1.0. Mu.L of template, and 1.0. Mu.L of 25. Mu.L of a single virus in an RT-PCR reaction system.
Further, the single RT-PCR amplification reaction conditions are as follows: 37 ℃ for 10min;95 deg.C for 5min;94 ℃ for 30s, presetting 5 annealing temperatures for optimization (50.0 ℃, 53.0 ℃, 56.0 ℃, 59.0 ℃ and 62.0 ℃) for 30s,72 ℃ for 45s, and carrying out 30 cycles; 72 deg.C, 10min.
Further, the triple RT-PCR amplification reaction system comprises: 50 μ L of BVDV, BRV and BCoV RT-PCR reaction system, 2.0 μ L of upstream and downstream primers, 2X 1Step Buffer 25.0 μ L, 4.0 μ L of PrimeScript One Step Enzyme Mix, and 1.0 μ L of template, respectively.
Further, the triple RT-PCR amplification reaction conditions are as follows: 37 ℃ for 10min;95 deg.C for 5min;94 ℃ for 30s, presetting 5 annealing temperatures for optimization (48.0 ℃, 50.0 ℃, 52.0 ℃, 54.0 ℃, 56.0 ℃, 58.0 ℃ and 60.0 ℃) for 30s,72 ℃ for 45s, and carrying out 30 cycles in total; 72 deg.C, 10min.
Further, the minimum detectable concentrations of BVDV, BRV and BCoV nucleic acids were 250ng/mL for BVDV, 250ng/mL for BRV and 25ng/mL for BcoV, respectively.
Further, the application of the triple RT-PCR method capable of simultaneously detecting bovine viral diarrhea virus, rotavirus and coronavirus as described above is characterized in that: the triple RT-PCR method is used for detecting bovine diarrhea pathogens, regularly monitoring bovine diarrhea pathogens in a cattle farm and investigating bovine diarrhea epidemiology; the bovine diarrhea pathogen is one or more of bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus.
Study and verification Process
1. Primer design
3 pairs of specific primers were designed by using Snang/mLene, primer5.0 and Oligo primer design software with reference to the 5' -UTR gene sequence of the BVDV (NC _ 001461) reference strain, the VP6 gene of the BRV (JN 790188.1) strain and the N gene sequence of the BCoV (U00735.2) reference strain in NCBI, and the detailed primer sequences are shown in Table 1.
2. Extraction of nucleic acids
Grinding bovine lymph node, adding PBS with equal volume, repeatedly freezing and thawing for 3 times, centrifuging at 4 deg.C at 5000 × g for 10min, and collecting supernatant; repeatedly freezing and thawing intestinal mucosa sample with PBS of the same volume for 3 times, centrifuging at 4 deg.C at 5000 × g for 10min, and collecting supernatant; viral RNA Extraction was performed with reference to the MiniBEST Viral RNA/DNA Extraction KitVer.4.0 nucleic acid Extraction kit instructions. The extracted RNA was stored at-80 ℃.
3.establishment of RT-PCR method
3.1 establishment of Single RT-PCR method
3.1.1 optimization of Single RT-PCR amplification System
25 mu L of single virus RT-PCR reaction system with 1.0 mu L of upstream and downstream primers, 12.5 mu L of 2X 1Step Buffer and 1.0 mu L of template; the RT-PCR reaction condition is 37 ℃ and 10min;95 ℃ for 5min;94 ℃ for 30s, presetting 5 annealing temperatures for optimization (50.0 ℃, 53.0 ℃, 56.0 ℃, 59.0 ℃ and 62.0 ℃) for 30s, and 72 ℃ for 45s, and carrying out 30 cycles; 72 deg.C for 10min.
Construction of 3.1.2pMD-18T-5' -UTR, pMD-18T-VP6 and pMD-18T-N Positive Standard plasmids
BVDV, bcoV and BRV positive RNA are taken as templates, BVDV, BRV and BCoV gene fragments with expected sizes of 187bp, 391bp and 725bp are respectively amplified, a target fragment is connected with a pMD-18T cloning vector, recombinant plasmids are named as pMD-19T-5' -UTR, pMD-19T-VP6 and pMD-19T-N, the constructed recombinant plasmids are sent to a Bowman Bioengineering (Dalian) Limited company for sequencing, the recombinant plasmids with correct sequencing identification are taken as BVDV, BRV and BCoV positive standard plasmids, and the concentration is determined by a nucleic acid protein detector.
3.2 establishment of triple RT-PCR reaction conditions
3.2.1 optimization of triple RT-PCR reaction System and reaction conditions
50 μ L of BVDV, BRV and BCoV RT-PCR reaction system, 2.0 μ L of upstream and downstream primers, 2X 1Step Buffer 25.0 μ L, 4.0 μ L of PrimeScript One Step Enzyme Mix, and 1.0 μ L of template, respectively.
Wherein, positive standard substances of BVDV, BRV and BCoV, BVDV, BRV and BCoV are respectively 2.0 mu L, and RNase Free H 2 O is 3.0 mu L; the RT-PCR reaction condition is 37 ℃ and 10min;95 deg.C for 5min;94 ℃ for 30s, presetting 5 annealing temperatures for optimization (48.0 ℃, 50.0 ℃, 52.0 ℃, 54.0 ℃, 56.0 ℃, 58.0 ℃ and 60.0 ℃) for 30s,72 ℃ for 45s, and carrying out 30 cycles in total; 72 deg.C, 10min.
4. Test of
4.1 specificity assay
The triple RT-PCR method is applied to detect 3 kinds of positive plasmids, salmonella, bovine escherichia coli, bovine pasteurella, mycoplasma bovis and mycobacterium tuberculosis DNA constructed by the method, and negative control is set simultaneously to determine the specificity of the method.
4.2 repeatability test
The established triple RT-PCR method is used for carrying out 3 times of repeated detection on 3 positive plasmid templates and 2 negative samples so as to verify the repeatability of the method.
4.3 susceptibility test
Nucleic acid concentration was measured using positive standard plasmids for BVDV, BRV and BCoV as templates, and then 10-fold serial dilution (1X 10) -1 ～1×10 -8 ) And detecting by using the established triple RT-PCR method to determine the sensitivity of the method.
3 pairs of specific primers are designed by referring to BVDV 5' -UTR, BRV VP6 and BCoVN gene sequences in GenBank. Conditions are optimized by a multiple RT-PCR amplification method, and specificity, sensitivity and repeatability tests are carried out at the same time. Results show that a triple RT-PCR method for simultaneously detecting BVDV, BRV and BCoV is successfully established, gene fragments of BVDV, BRV and BCoV can be specifically amplified, and bovine salmonella, bovine escherichia coli, bovine pasteurella and bovine mycoplasma are not amplified; the minimum detection concentrations for BVDV, BRV and BCoV nucleic acids were 250ng/mL for BVDV, 250ng/mL for BRV and 25ng/mL for BcoV, respectively.
BRVVP6 gene clone plasmid, BVDV 5' -UTR gene clone plasmid, BCoVN gene clone plasmid, salmonella DNA, bovine Escherichia coli DNA, bovine mycoplasma DNA, bovine pasteurella DNA, tubercle bacillus DNA and the like used in the application are all stored in livestock and poultry epidemic disease research laboratory of animal and veterinary research institute of Guizhou province.
198 parts of bovine lymph node and intestinal mucosa samples collected from a green slaughterhouse in Guizhou province are stored at-80 ℃ for later use.
Due to the adoption of the technical scheme, the invention has the following beneficial effects:
(1) In the application, 3 pairs of specific primers are designed by referring to specific genes of BVDV, BRV and BCoV in Gen Bank, the established triple RT-PCR can be used for quickly and accurately detecting the BVDV, BRV and BCoV which are singly or mixedly infected in the same reaction system, and the established triple RT-PCR method has the advantages of strong specificity, high sensitivity and good repeatability for the BVDV, BRV and BCoV and provides a reliable method for diagnosing clinical cases of bovine viral diarrhea viruses, bovine rotavirus and bovine coronavirus.
(2) The triple RT-PCR reaction system has more sample adding steps, and in order to reduce sample adding pollution, the application adopts a one-step RT-PCR reaction system to reduce the sample adding times and reduce the pollution. Simultaneously, the annealing temperature and the reaction system are optimized to reduce the formation of primer dimer and even polymer. According to the method, the bovine salmonella, escherichia coli, bovine pasteurella, mycobacterium tuberculosis and the like which cause the diarrhea pathogen of the beef cattle are selected for specificity detection, the results are negative, and the establishment method has better specificity, so that the method has certain significance for eliminating the bovine salmonella, the escherichia coli, the bovine pasteurella and the mycobacterium tuberculosis in clinical sample detection.
(3) The positive rate of BVDV and BCoV detected by the sample collected in the slaughterhouse is lower than that of the prior art (such as lithocarpus, liu Tuo, zhang, etc., infection condition investigation and BVDV molecular feature analysis [ J ], livestock and veterinarians 2021,53 (09): 83-88.) and BCoV is not detected by the sample.
(4) The optimized reverse transcriptase for completing transcription at 37 ℃ for 10min is 30min faster than the common reverse transcriptase at 50 ℃.
Drawings
In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed in the description of the embodiments or the prior art will be briefly introduced below, it is obvious that the drawings in the following description are only some examples of the present invention, and for a person skilled in the art, without inventive step, other drawings can be obtained according to these drawings:
FIG. 1 shows different annealing temperature RT-PCR amplification bands of BVDV of the present application;
FIG. 2 shows RT-PCR amplification bands at different annealing temperatures for BRV of the present application;
FIG. 3 shows the different annealing temperatures RT-PCR amplification bands of BCoV of the present application;
FIG. 4 is a diagram showing three different annealing temperature RT-PCR amplification bands of BVDV, BRV and BcoV of the present application;
FIG. 5 is a histogram of BVDV, BRV and BcoV triple RT-PCR specificity test of the present application;
FIG. 6 is a schematic representation of the BVDV, BRV and BcoV triple RT-PCR repetitive gene band diagram of the present application;
FIG. 7 shows a BVDV and BcoV triple RT-PCR sensitivity band of the present application;
in the drawings: 1-48.0 ℃;2 to 50.0 ℃;3-52.0 ℃;4-54.0 ℃;5-56.0 ℃;6 to 58.0 ℃;7-60.0 ℃;8-BVDV, BRV and BcoV triple RT-PCR;9-BCoV;10-BRV;11-BVDV; 12-salmonella; 13-Escherichia coli; 14-pasteurella; 15-mycoplasma bovis; 16-tubercle bacillus; 17-BVDV, BRV and BCoV positive standard plasmid mixtureA compound; 18-negative control; 19-10 -1 Carrying out dilution; 20-10 -2 Diluting by times; 21-10 -3 Diluting by times; 22-10 -4 Carrying out dilution; 23-10 -5 Carrying out dilution; 24-10 -6 Carrying out dilution; 25-10 -7 Carrying out dilution; 26-10 -8 And (5) diluting by times.
Detailed Description
The following description will explain the embodiments of the present invention in further detail, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are still within the scope of the present invention as claimed in the claims.
Example 1
A triple RT-PCR method for simultaneously detecting bovine viral diarrhea virus, rotavirus and coronavirus comprises the following steps: the method comprises the steps of taking RNA of bovine lymph nodes, intestinal mucosa and viruses as RNA templates, referring to BVDV 5' -UTR, BRV VP6 and BCoV N gene sequences in GenBank, designing specific upstream primers and specific downstream primers as specific primers according to genetic characteristics of three viruses including bovine viral diarrhea virus (SEQ ID NO.1, 187 bp), bovine rotavirus (SEQ ID NO.2, 391 bp) and bovine coronavirus (SEQ ID NO.3, 725 bp), carrying out RT-PCR amplification to obtain an amplification product, and judging whether a sample to be detected contains the bovine viral diarrhea virus, the bovine rotavirus and the bovine coronavirus according to a detection result.
Further, the RT-PCR amplification adopts a reverse transcriptase mixture with the completion of transcription at 37 ℃ for 10min, and the reverse transcriptase mixture comprises: 100mM Tris-HCl, 10mM MgCl, pH 8.3 2 、10mM DTT、50mM KCl、0.5mM dTTP、0.4MBq/mL[3H]-dTTP, 1mM poly (A) oligo (dT) 12-18 and reverse transcriptase. The lengths and primer sequences of the bovine viral diarrhea virus, the bovine rotavirus and the bovine coronavirus are shown in Table 1, the length of the bovine viral diarrhea virus is 187bp, an upstream primer is shown as F1 of SEQ ID NO.1, and a downstream primer is shown as R1 of SEQ ID NO. 1; the length of the bovine rotavirus is 391bp, an upstream primer is shown as F2 of SEQ ID NO.2, and a downstream primer is shown as R2 of SEQ ID NO. 2; the bovine coronavirus has a length of 725bp, an upstream primer shown as F3 of SEQ ID NO.3, and a downstream primer shown asR3 of SEQ ID NO. 3.
The RT-PCR amplification comprises single RT-PCR amplification and triple RT-PCR amplification. The single RT-PCR amplification system is as follows: mu.L of each of the upstream and downstream primers, 2X 1Step Buffer 12.5. Mu.L of PrimeScript One Step Enzyme Mix, 1.0. Mu.L of template, and 1.0. Mu.L of 25. Mu.L of a single virus in an RT-PCR reaction system. The single RT-PCR amplification reaction conditions are as follows: 37 ℃ for 10min;95 deg.C for 5min;94 ℃ for 30s, presetting 5 annealing temperatures for optimization (50.0 ℃, 53.0 ℃, 56.0 ℃, 59.0 ℃ and 62.0 ℃) for 30s,72 ℃ for 45s, and carrying out 30 cycles; 72 deg.C for 10min. The triple RT-PCR amplification reaction system comprises: 50 μ L of BVDV, BRV and BCoV RT-PCR reaction system, 2.0 μ L of upstream and downstream primers, 2X 1Step Buffer 25.0 μ L, 4.0 μ L of PrimeScript One Step Enzyme Mix, and 1.0 μ L of template, respectively. The reaction conditions of the triple RT-PCR amplification are as follows: 37 ℃ for 10min;95 deg.C for 5min;94 ℃ for 30s, presetting 5 annealing temperatures for optimization (48.0 ℃, 50.0 ℃, 52.0 ℃, 54.0 ℃, 56.0 ℃, 58.0 ℃ and 60.0 ℃) for 30s,72 ℃ for 45s, and carrying out 30 cycles in total; 72 deg.C, 10min. The minimum detection concentrations of the BVDV, BRV and BCoV nucleic acids are 250ng/mL for BVDV, 250ng/mL for BRV and 25ng/mL for BcoV respectively.
The application of the triple RT-PCR method capable of simultaneously detecting the bovine viral diarrhea virus, the rotavirus and the coronavirus is characterized in that: the triple RT-PCR method is used for detecting bovine diarrhea pathogens, regularly monitoring bovine diarrhea pathogens in a cattle farm and investigating bovine diarrhea epidemiology; the bovine diarrhea pathogen is one or more of bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus.
Application example 1
198 bovine lymph nodes and intestinal mucosa samples collected from Guanling of Guizhou province and the Phoenix slaughterhouse can be detected by using the triple RT-PCR method capable of simultaneously detecting BVDV, BRV and BcoV in example 1, and the infection conditions of BVDV, BRV and BcoV are analyzed, wherein details and results are as follows:
1. construction of pMD-18T-5' -UTR, pMD-18T-VP6 and pMD-18T-N Positive Standard plasmids
The constructed positive pMD-18T-5' -UTR, pMD-18T-VP6 and pMD-18T-N standard plasmids are sequenced by Takara bioengineering (Dalian) Co., ltd, and the result shows that the constructed positive standard plasmids have specific sequences containing various viruses.
2. RT-PCR amplification
2.1 optimization of Single RT-PCR amplification System
BVDV, BRV and BcoV were amplified by single RT-PCR in 25. Mu.L system at 50.0 deg.C, 52.0 deg.C, 54.0 deg.C and 56.0 deg.C, and the results are shown in FIGS. 1, 2 and 3.
2.2 establishment of triple RT-PCR reaction conditions
2.2.1 optimization of triple RT-PCR reaction conditions
The triple RT-PCR amplification effect is best when the annealing temperature is 56.0 ℃, specific bands can appear, and no specific amplification exists, as shown in figure 4.
2.2.2 triple PCR specificity assay
The established triple RT-PCR reaction conditions and system are applied to carry out specificity tests, and the results show that: specific bands which are consistent with the size of the target fragment are amplified by the BVDV, BRV and BCoV gene plasmid templates, and bands are not amplified by the salmonella, bovine escherichia coli, bovine pasteurella, mycoplasma bovis and mycobacterium tuberculosis DNA templates, which is shown in figure 5.
2.2.3 triple PCR repeatability test
The RT-PCR was repeated 3 times to amplify uniform target gene bands, as shown in FIG. 6.
2.2.4 triple PCR sensitivity
The 10-fold gradient dilution of the nucleic acids of 3 BVDV, BRV and BCoV resulted in a minimum detectable concentration of 250ng/mL for BVDV, 250ng/mL for BRV and 25ng/mL for BCoV, as shown in FIG. 7.
2.2.5 test results for clinical samples
The results of the detection of 198 clinical specimens by using the established triple RT-PCR method show that: in 198 samples, the triple infection positive rate of BVDV, BRV and BCoV is 0% (0/198), the positive rate of BVDV is 13.13% (26/198), the positive rate of BRV is 7.57% (15/198) and the positive rate of BCoV is 0; the mixed infection rate of BVDV and BRV is 2.02% (4/198). Meanwhile, single RT-PCR is adopted for rechecking, and as a result, the triple RT-PCR method and the single RT-PCR rechecking rate are 100 percent. The results are detailed in table 2.
TABLE 2 table of test results of clinical samples
| Sampling site | Guanling county | Fenggang county | Total up to |
| Detecting the amount | 98 | 100 | 198 |
| Positive rate of BVDV detection | 15.30％ | 11％ | 13.13％ |
| Positive rate of BRV test | 5.10％ | 10％ | 7.57％ |
| Positive rate of BCoV detection | 0 | 0 | 0 |
| Blending of BVDV and BRVPositive rate of infection | 1.51％ | 1.0％ | 2.02％ |
| Positive rate of mixed BVDV, BRV and BCoV infection | 0 | 0 | 0 |
In summary, the positive rate of BVDV and BCoV detected by the sample collected in the slaughterhouse is lower than that detected by the prior art (such as Lithocarpus, liu Tuo, zhang, etc., infection condition survey and BVDV molecular feature analysis [ J ], livestock and veterinarian 2021,53 (09): 83-88.) in partial cattle farms in Guizhou province), and BCoV is not detected.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (10)
1. The triple RT-PCR method capable of simultaneously detecting the bovine viral diarrhea viruses, rotaviruses and coronaviruses is characterized by comprising the following steps: the RNA of bovine lymph node, intestinal mucosa and virus is taken as an RNA template, BVDV 5' -UTR, BRV VP6 and BCoVN gene sequences in GenBank are referred to, genetic characteristics of bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus are taken as specific primers, a specific upstream primer and a specific downstream primer are designed to be taken as specific primers, RT-PCR amplification is carried out to obtain an amplification product, and a detection result is obtained to judge whether a sample to be detected contains the bovine viral diarrhea virus, the bovine rotavirus and the bovine coronavirus.
2. The triple RT-PCR method for simultaneous detection of bovine viral diarrhea virus, rotavirus and coronavirus according to claim 1, wherein: the RT-PCR amplification adopts a reverse transcriptase mixture which is transcribed for 10min at 37 ℃, and the reverse transcriptase mixture comprises: 100mM Tris-HCl, 10mM MgCl, pH 8.3 2 、10mM DTT、50mM KCl、0.5mM dTTP、0.4MBq/mL[3H]-dTTP, 1mM poly (A) oligo (dT) 12-18 and reverse transcriptase.
3. The triple RT-PCR method for simultaneous detection of bovine viral diarrhea virus, rotavirus and coronavirus according to claim 1, wherein: the length of the bovine viral diarrhea virus is 187bp, the upstream primer is shown as F1 of SEQ ID NO.1, and the downstream primer is shown as R1 of SEQ ID NO. 1;
the length of the bovine rotavirus is 391bp, an upstream primer is shown as F2 of SEQ ID NO.2, and a downstream primer is shown as R2 of SEQ ID NO. 2;
the length of the bovine coronavirus is 725bp, an upstream primer is shown as F3 of SEQ ID NO.3, and a downstream primer is shown as R3 of SEQ ID NO. 3.
4. The triple RT-PCR method for simultaneous detection of bovine viral diarrhea virus, rotavirus, coronavirus according to claim 1 or 2, characterized in that: the RT-PCR amplification comprises single RT-PCR amplification and triple RT-PCR amplification.
5. The triple RT-PCR method for simultaneous detection of bovine viral diarrhea virus, rotavirus and coronavirus according to claim 4, wherein: the single RT-PCR amplification system is as follows: mu.L of each of the upstream and downstream primers was 1.0. Mu.L, 2X 1Step Buffer 12.5. Mu.L of the upstream and downstream primers and 1.0. Mu.L of the template in an RT-PCR reaction system with a single virus.
6. The triple RT-PCR method for simultaneous detection of bovine viral diarrhea virus, rotavirus and coronavirus according to claim 4, wherein: the single RT-PCR amplification reaction conditions are as follows: 37 ℃ for 10min;95 deg.C for 5min; carrying out optimization on 30s at 5 annealing temperatures of 94 ℃, 30s, 50.0 ℃, 53.0 ℃, 56.0 ℃, 59.0 ℃ and 62.0 ℃ in advance, and carrying out 30 cycles at 72 ℃ and 45s in total; 72 deg.C for 10min.
7. The triple RT-PCR method for simultaneous detection of bovine viral diarrhea virus, rotavirus and coronavirus according to claim 4, wherein: the triple RT-PCR amplification reaction system comprises: 50 μ L of BVDV, BRV and BCoV RT-PCR reaction system, 2.0 μ L of upstream and downstream primers, 2X 1Step Buffer 25.0 μ L, 4.0 μ L of PrimeScript One Step Enzyme Mix, and 1.0 μ L of template, respectively.
8. The triple RT-PCR method for simultaneous detection of bovine viral diarrhea virus, rotavirus and coronavirus according to claim 4, wherein: the reaction conditions of the triple RT-PCR amplification are as follows: 37 ℃ for 10min;95 deg.C for 5min; carrying out optimization at 5 annealing temperatures of 94 ℃, 30s, 48.0 ℃, 50.0 ℃, 52.0 ℃, 54.0 ℃, 56.0 ℃, 58.0 ℃ and 60.0 ℃ for 30s,72 ℃ and 45s for 30 cycles; 72 deg.C, 10min.
9. The triple RT-PCR method for simultaneous detection of bovine viral diarrhea virus, rotavirus and coronavirus according to claim 4, wherein: the minimum detection concentrations of the BVDV, BRV and BCoV nucleic acids are 250ng/mL for BVDV, 250ng/mL for BRV and 25ng/mL for BcoV respectively.
10. The use of the triple RT-PCR method for simultaneous detection of bovine viral diarrhea virus, rotavirus, coronavirus according to claims 1-9, wherein: the triple RT-PCR method is used for detecting bovine diarrhea pathogens, regularly monitoring bovine diarrhea pathogens in a cattle farm and investigating bovine diarrhea epidemiology; the bovine diarrhea pathogen is one or more of bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus.
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| CN110643739A (en) * | 2019-10-15 | 2020-01-03 | 云南省畜牧兽医科学院 | One-step triple RT-PCR detection primer and kit for distinguishing CHUV, BCV and DAV |
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| CN116574844A (en) * | 2023-03-01 | 2023-08-11 | 宁夏大学 | RPA primer pair, kit and detection method for detecting bovine viral diarrhea virus |
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