CN110157836B - Primer, probe and method for detecting IBRV and BVDV - Google Patents
Primer, probe and method for detecting IBRV and BVDV Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
The invention provides a primer, a probe and a method for detecting IBRV and BVDV. The primer and/or probe composition provided by the invention comprises the following SEQ ID No: 1-SEQ ID No.: 6, and at least one nucleotide sequence as set forth in seq id no. The primer and/or probe composition provided by the invention is utilized to realize the simultaneous detection of bovine infectious rhinotracheitis virus (IBRV) and Bovine Viral Diarrhea Virus (BVDV) through a PCR reaction comprising a Taqman double fluorescent quantitative PCR reaction. The primer and/or probe composition and the detection method provided by the invention have the advantages of high sensitivity, high specificity, good repeatability, small interference and good clinical detection effect, can detect and distinguish IBRV and BVDV rapidly, accurately and sensitively, and provide a better method for clinically distinguishing and diagnosing IBRV and BVDV infected cattle.
Description
Technical Field
The invention belongs to the technical field of virus detection, and in particular relates to a primer, a probe and a method for detecting Infectious Bovine Rhinotracheitis Virus (IBRV) and Bovine Viral Diarrhea Virus (BVDV)
Background
Infectious bovine rhinotracheitis virus (Infectious bovine rhino-trachitis virus, IBRV) and bovine viral diarrhea virus (bovine viral diarrheamucosal disease virus, BVDV) are two common viruses that endanger the cattle industry, and infection by infectious bovine rhinotracheitis virus can cause symptoms such as inflammation of the upper bovine respiratory tract and tracheal mucosa, dyspnea, runny nose, and the like, and cause acute, febrile, contagious infections and the like, including inflammation such as conjunctivitis, immature Niu Naomo inflammation, pustular vulvitis, vaginitis, balanitis, mastitis, metritis and the like, and serious abortion in cattle. The virus was first found and reported in China, and is currently popular worldwide in 1980, and the economic loss caused by the virus is quite large. Bovine viral diarrhea viruses commonly cause bovine diarrhea, and the viruses can be transmitted through various pathways such as bovine blood and bovine excreta. It has been reported that some BVDV strains (1 a and 1b, biological non-cytopathogenic genes) are often associated with the occurrence of respiratory diseases and that the virus has been isolated in the bovine lung, that type 2 virus causes severe interstitial pneumonia in calves, thrombocytopenia, bone marrow necrosis and diarrhea, and that once the calf or cow that is continuously infected with BVDV develops rapidly bacterial pneumonia. The virus causes high incidence rate of cattle, but has low mortality rate, and the sick cattle is a carrier of the virus, can carry the virus for life and expel the virus, and brings great harm to the cattle raising industry.
In recent years, with large-scale cattle cultivation, more and more cases of mixed infection of cattle with two pathogens clinically appear, and certain difficulty is brought to diagnosis, and the common characteristics of the two diseases are that the mucosa inflammation and abortion of the cattle are caused, and the accurate judgment is difficult to be made according to clinical symptoms, and detection and differentiation are needed through a laboratory method. The current methods for diagnosing the two pathogens mainly comprise virus separation and identification, a PCR method, a single fluorescent PCR method, an ELISA method and the like, but the established methods can not meet the requirements of rapid and accurate differential diagnosis in clinic more or less.
Disclosure of Invention
The invention provides a primer and a probe for detecting Infectious Bovine Rhinotracheitis Virus (IBRV) and Bovine Viral Diarrhea Virus (BVDV), and establishes a Taqman double-fluorescence quantitative PCR method for simultaneously detecting and identifying the IBRV and the BVDV, which can meet the requirements of rapid and accurate clinical detection and diagnosis of the two viruses.
It is an object of the present invention to provide a primer and/or probe composition comprising at least one of the following 1) -6):
1) SEQ ID No. in the sequence Listing: 1, a nucleotide sequence shown in 1; or the SEQ ID No: 1 and the nucleotide sequence shown in the sequence table is subjected to substitution and/or deletion and/or addition of one or more nucleotides and is identical with the nucleotide sequence shown in the sequence table in SEQ ID No: 1, and the nucleotide sequence has the same function;
2) SEQ ID No. in the sequence Listing: 2, a nucleotide sequence shown in the specification; or the SEQ ID No: 2 through substitution and/or deletion and/or addition of one or more nucleotides and is identical to the nucleotide sequence shown in SEQ ID No: 2, the nucleotide sequence has the same function;
3) SEQ ID No. in the sequence Listing: 3, a nucleotide sequence shown in 3; or the SEQ ID No: 3 through substitution and/or deletion and/or addition of one or more nucleotides and is identical to the nucleotide sequence shown in SEQ ID No: 3, the nucleotide sequence has the same function;
4) SEQ ID No. in the sequence Listing: 4, a nucleotide sequence shown in figure 4; or the SEQ ID No: 4 and the nucleotide sequence is subjected to substitution and/or deletion and/or addition of one or more nucleotides and is identical with the nucleotide sequence shown in SEQ ID No: 4, the nucleotide sequence has the same function;
5) SEQ ID No. in the sequence Listing: 5, a nucleotide sequence shown in seq id no; or the SEQ ID No: 5, and the nucleotide sequence is subjected to substitution and/or deletion and/or addition of one or more nucleotides and is identical with the nucleotide sequence shown in SEQ ID No: 5, the nucleotide sequence has the same function;
6) SEQ ID No. in the sequence Listing: 6, a nucleotide sequence shown in the specification; or the SEQ ID No: 6, and the nucleotide sequence is subjected to substitution and/or deletion and/or addition of one or more nucleotides and is identical with the nucleotide sequence shown in SEQ ID No: 6, and the nucleotide sequence has the same function.
In particular, the same function is particularly useful for detecting infectious bovine rhinotracheitis virus IBRV and/or bovine viral diarrhea virus BVDV; or the same function is specifically a conserved nucleotide sequence which can be used for specifically identifying, combining or amplifying the non-coding region at the 5' end of bovine infectious rhinotracheitis virus gB gene sequence No. AY330349.1 or bovine viral diarrhea virus BVDV.
Specifically, when the composition comprises the sequence table of SEQ ID No: 3 and/or SEQ ID No.: 6, the nucleotide sequence shown in SEQ ID No: 3 and/or SEQ ID No.: 6, a fluorescent group is marked at the 5 'end of the nucleotide sequence shown in the formula 6, and a quenching group is marked at the 3' end.
Specifically, when the composition comprises the sequence table of SEQ ID No: 3 and/or SEQ ID No.: 6, the nucleotide sequence shown in SEQ ID No: 3 is marked with FAM fluorescent group at the 5 'end and quenching group ECLIPSE at the 3' end, and the nucleotide sequence shown in SEQ ID No: 6, a ROX fluorescent group is marked at the 5 'end of the nucleotide sequence shown in the formula 6, and a quenching group ECLIPSE is marked at the 3' end.
It is a further object of the present invention to provide a kit comprising a composition according to any of the present invention.
It is a further object of the present invention to provide a method for detecting infectious bovine rhinotracheitis virus and/or bovine viral diarrhea virus, said method comprising detecting using at least one primer and/or probe composition of 1) -6) as follows:
1) SEQ ID No. in the sequence Listing: 1, a nucleotide sequence shown in 1; or the SEQ ID No: 1 and the nucleotide sequence shown in the sequence table is subjected to substitution and/or deletion and/or addition of one or more nucleotides and is identical with the nucleotide sequence shown in the sequence table in SEQ ID No: 1, and the nucleotide sequence has the same function;
2) SEQ ID No. in the sequence Listing: 2, a nucleotide sequence shown in the specification; or the SEQ ID No: 2 through substitution and/or deletion and/or addition of one or more nucleotides and is identical to the nucleotide sequence shown in SEQ ID No: 2, the nucleotide sequence has the same function;
3) SEQ ID No. in the sequence Listing: 3, a nucleotide sequence shown in 3; or the SEQ ID No: 3 through substitution and/or deletion and/or addition of one or more nucleotides and is identical to the nucleotide sequence shown in SEQ ID No: 3, the nucleotide sequence has the same function;
4) SEQ ID No. in the sequence Listing: 4, a nucleotide sequence shown in figure 4; or the SEQ ID No: 4 and the nucleotide sequence is subjected to substitution and/or deletion and/or addition of one or more nucleotides and is identical with the nucleotide sequence shown in SEQ ID No: 4, the nucleotide sequence has the same function;
5) SEQ ID No. in the sequence Listing: 5, a nucleotide sequence shown in seq id no; or the SEQ ID No: 5, and the nucleotide sequence is subjected to substitution and/or deletion and/or addition of one or more nucleotides and is identical with the nucleotide sequence shown in SEQ ID No: 5, the nucleotide sequence has the same function;
6) SEQ ID No. in the sequence Listing: 6, a nucleotide sequence shown in the specification; or the SEQ ID No: 6, and the nucleotide sequence is subjected to substitution and/or deletion and/or addition of one or more nucleotides and is identical with the nucleotide sequence shown in SEQ ID No: 6, and the nucleotide sequence has the same function.
In particular, the same function is particularly useful for detecting infectious bovine rhinotracheitis virus IBRV and/or bovine viral diarrhea virus BVDV; or the same function is specifically a conserved nucleotide sequence which can be used for specifically identifying, combining or amplifying the non-coding region at the 5' end of bovine infectious rhinotracheitis virus gB gene sequence No. AY330349.1 or bovine viral diarrhea virus BVDV.
Specifically, when the composition comprises the sequence table of SEQ ID No: 3 and/or SEQ ID No.: 6, the nucleotide sequence shown in SEQ ID No: 3 and/or SEQ ID No.: 6, a fluorescent group is marked at the 5 'end of the nucleotide sequence shown in the formula 6, and a quenching group is marked at the 3' end.
Specifically, when the composition comprises the sequence table of SEQ ID No: 3 and/or SEQ ID No.: 6, the nucleotide sequence shown in SEQ ID No: 3 is marked with FAM fluorescent group at the 5 'end and quenching group ECLIPSE at the 3' end, and the nucleotide sequence shown in SEQ ID No: 6, a ROX fluorescent group is marked at the 5 'end of the nucleotide sequence shown in the formula 6, and a quenching group ECLIPSE is marked at the 3' end.
Specifically, the method further comprises performing a PCR reaction comprising at least one of the following 1) -8):
1) The reaction system of the PCR reaction comprises: when the composition comprises the sequence table SEQ ID No: 1-SEQ ID No.: 6, each 20 mu L of PCR reaction system contains the nucleotide sequence shown in SEQ ID No: 1-SEQ ID No.: 6 of the sequence of nucleotides shown in FIG. 6, 0.2. Mu. Mol/. Mu.L each;
2) The PCR reaction comprises Taqman double-fluorescence quantitative PCR reaction;
3) The PCR reaction comprises a Taqman double-fluorescence quantitative PCR reaction, wherein the reaction system of the Taqman double-fluorescence quantitative PCR reaction is as follows: every 20 mu L of reaction system contains 0.1ng-10ng of template DNA or cDNA extracted from the object to be detected; SEQ ID No.: 1-SEQ ID No.: 6 of the sequence of nucleotides shown in FIG. 6, 0.2. Mu. Mol/. Mu.L each; 50u Ex Taq HS;100mol dNTP Mixture,Mg 2+ The method comprises the steps of carrying out a first treatment on the surface of the The RNase Free pure water is added to 20 mu L;
4) The amplification procedure of the PCR reaction comprises: 95 ℃ for 10s; then, the mixture is circulated at 95 ℃ for 10s, 58 ℃ for 10s and 72 ℃ for 10s, and is subjected to more than 44 cycles, and the mixture is ended at 40 ℃ for 10 s.
Specifically, the temperature rising speed at 95 ℃ for 10S is 20 ℃/S;
the cycle is specifically 44 cycles;
the method does not include the method for diagnosing and treating a disease described in the twenty-fifth patent law.
The composition of any one of the invention, the kit of any one of the invention and the application of the method of any one of the invention.
Specifically, the applications include applications in at least one of the following 1) -6):
1) Identifying infectious bovine rhinotracheitis virus and/or bovine viral diarrhea virus;
2) Preparing a kit or related products for identifying infectious bovine rhinotracheitis virus and/or bovine viral diarrhea virus;
3) Detecting whether the pathogenic microorganism to be detected is infectious bovine rhinotracheitis virus and/or bovine viral diarrhea virus;
4) Preparing a kit or related products for detecting whether the pathogenic microorganism to be detected is a bovine infectious rhinotracheitis virus and/or a bovine viral diarrhea virus;
5) Detecting whether a sample to be detected contains infectious bovine rhinotracheitis virus and/or bovine viral diarrhea virus;
6) Preparing a kit or related products for detecting whether the sample to be detected contains the infectious bovine rhinotracheitis virus and/or the bovine viral diarrhea virus.
Any of the applications described does not include the method of diagnosing and treating a disease described in the twenty-fifth of the patent laws.
The beneficial effects of the invention include:
the real-time fluorescent quantitative PCR technology has the advantages of convenient operation, short time consumption and visual result, is widely applied to diagnosis of domestic animals and poultry, and can not only accurately and rapidly carry out qualitative diagnosis on the sample, but also determine the pathogen content in the sample. The first key technology is to design the primer and probe for fluorescence quantification, i.e. the requirement on the primer and probe is high. The primer design follows the principle that the parameters of the two pairs of primers are close, and when the fluorescent groups of the marker probe are selected, the two selected fluorescent groups are not interfered with each other and can be well distinguished. The Tm between primers is within 2 ℃, the Tm of a probe primer is within 1 ℃, and the primers have no cross interference and mismatch.
In a preferred embodiment, the probe provided by the invention is marked with a fluorescent group FAM (530 nM detection channel) or ROX (610 nM detection channel) at the 5 'end and a quenching group ECLIPSE at the 3' end, so that the marked probe greatly reduces the mutual interference and background value of the fluorescent signal of the probe and increases the color compensation, thereby increasing the success of the method.
In a preferred embodiment, the present invention optimizes primer concentration and probe concentration to give a better S-curve when the final concentration of the IBRV primer is 0.2. Mu. Mol/. Mu.L, the final concentration of the probe is 0.2. Mu. Mol/. Mu.L, the final concentration of the BVDV primer is 0.2. Mu. Mol/. Mu.L, and the final concentration of the probe is 0.2. Mu. Mol/. Mu.L, and a single peak appears in the dissolution curve.
In the method established by the test, through specificity, sensitivity and repeatability interference verification tests, in the specificity test of all the tested strains, only IBRV strains collect specific fluorescent signals (S curve) respectively at 530 mu m (FAM) and BVDV strains at 610 mu m (ROX), other strains do not collect fluorescent signals, and no cross signals appear among the strains, so that the two pairs of primers and probes used by the method have good specificity.
Sensitivity assays can detect 100 copies of IBRV and BVDV plasmid DNA, indicating established methods to detect IBRV and BVDV sensitivity.
The Ct value variation coefficients obtained by the repeatability test are all smaller than 4%, which shows that the repeatability is better.
The interference test is a key to test whether the established method of the invention can be used for identifying IBRV and BVDV clinically, because the two viruses which are mixed and infected clinically have different virus contents, and sometimes one virus has high content and the other virus has low content. Under the condition, the test design mixes the IBRV and BVDV templates with different concentrations, and then the method established by the test is applied to detection, so that good fluorescent signals can be obtained under the conditions of the high-concentration IBRV template, the low-concentration BVDV template, the low-concentration IBRV template and the high-concentration BVDV template, and the method established by the test can be used for detecting clinical samples.
The method established by the research is used for identifying and detecting the preserved clinical samples, and as a result, 2 parts of the samples are found to be mixed infection, and the sequences of the samples are analyzed to be 100% homologous with the sequences of IBRV and BVDV through sequence comparison. The method is used for indicating that bovine cases which are clinically infected with IBRV and BVDV simultaneously are easy to distinguish by using the method established by the research.
With the rapid development of the cattle raising industry, some old cattle diseases and new cattle diseases continuously appear, which brings difficulty to the prevention and control of the cattle diseases and restricts the development of the cattle raising industry. IBR and BVD are two bovine diseases that seriously affect the cattle industry, and the number of cases induced in recent years is increasing. Although the method for detecting IBR and BVD and the method for detecting IBR, BVD, FMD by triple fluorescence exist at present, the method established by the invention has higher sensibility than the existing double PCR detection method, can directly obtain the result without electrophoresis, and is quick and efficient; compared with triple fluorescence detection, the method has the advantages of low cost, less interference degree of the light source, accuracy and practicability.
Therefore, the method established by the research can rapidly, accurately and sensitively detect and distinguish IBRV and BVDV, and provides another better method for clinically distinguishing and diagnosing IBRV and BVDV infected cattle.
Drawings
FIG. 1 is a graph of a dual fluorescent quantitative PCR dissolution profile, wherein 1-6: 1.0X10 7 Copy number/. Mu.L-1.0X10 2 Copy number/. Mu.L; 7: 1.0X10 1 Copy number/. Mu.L, 8: negative control.
FIG. 2 is a graph of the results of a dual fluorescent quantitative PCR assay IBRV sensitivity assay (FAM channel), wherein, 1-6: 1.0X10 7 Copy number/. Mu.L-1.0X10 2 Copy number/. Mu.L; 7: 1.0X10 1 Copy number/. Mu.L, 8: negative control.
FIG. 3 is a graph of the results of a dual fluorescent quantitative PCR assay for BVDV sensitivity (ROX channel), wherein, 1-6: 1.0X10 7 Copy number/. Mu.L-1.0X10 2 Copy number/. Mu.L; 7: 1.0X10 1 Copy number/. Mu.L, 8: negative control.
FIG. 4 is a graph of the results of a dual fluorescent quantitative RT-PCR specific assay IBRV assay (FAM channel), wherein 1: IBRV;2: BVDV (Oregon CV 24); 3: BVDV (NADL); 4: BRV (014); 5: bacillus bovis;6: MB;7: CSFV;8: cattle mycoplasma;9: bovis pasteurella.
FIG. 5 is a graph of the results of a dual fluorescent quantitative RT-PCR specific assay BVDV (ROX channel), wherein 1: IBRV;2: BVDV (Oregon CV 24); 3: BVDV (NADL); 4: BRV (014); 5: bacillus bovis;6: MB;7: CSFV;8: cattle mycoplasma;9: bovis pasteurella.
FIG. 6 is a graph of the results of a dual fluorescent quantitative RT-PCR reproducibility assay IBRV assay (FAM channel), wherein, 1-3: 1.0X10 6 Copy number/. Mu.L; 4: negative control.
FIG. 7 is a graph of the results of a dual fluorescent quantitative RT-PCR reproducibility assay BVDV (ROX channel), wherein, 1-3: 1.0X10 6 Copy number/. Mu.L; 4: negative control.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
The molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The following examples and their detailed description are for the purpose of illustration and understanding of the invention and are not to be construed as unduly limiting the invention.
Example 1 establishment of Taqman double-fluorescence quantitative PCR method for simultaneously detecting IBRV and BVDV
Primer design
According to the conserved nucleotide sequences of the bovine infectious rhinotracheitis virus gB gene sequence (No. AY 330349.1) and the bovine viral diarrhea virus BVDV 5' non-coding region in GenBank, the ClustaV Method is applied to analyze the conserved sequences, then Primer design is carried out by applying Primer Express 2.0 software to the conserved sequence regions, 1 pair of bovine infectious rhinotracheitis virus specific primers and 1 Taqman probe are designed, 1 pair of bovine viral diarrhea virus primers and 1 Taqman probe are also designed, the designed Primer sequences are subjected to Blast comparison analysis through GeneBank, the Primer sequences selected through the comparison analysis are synthesized by Takara biological (Dalian) bioengineering limited company, and the Primer sequences are shown in Table 1.
In table 1, the nucleotide sequences of the primers IBRVF, IBRVR, IBRVPro, BVDVF, BVDVR, BVDVFPro are respectively shown in sequence table as SEQ ID No: 1-SEQ ID No.: 6, a nucleotide sequence shown in seq id no; wherein, the probe IBRVPro is SEQ ID No: 3, a fluorescent group FAM is marked at the 5 'end of the nucleotide sequence shown in the formula 3, and a fluorescence quenching group ECLIPSE is marked at the 3' end; the probe BVDVPro is shown in SEQ ID No: 6, a fluorescent group ROX is marked at the 5 'end of the nucleotide sequence shown in the formula 6, and a fluorescence quenching group ECLIPSE is marked at the 3' end.
TABLE 1
(II) template preparation
IBRV and BVDV strain proliferation: the IBRV and BVDV freeze-dried virus seed are respectively dissolved by 2mL aseptic operation of serum-free DMEM culture medium, then 0.5mL of the virus seed is inoculated to bovine kidney cell MDBK cells growing into a monolayer, the cell virus liquid with cytopathy appears after harvesting for 24-96 hours, the cell virus liquid is repeatedly and rapidly frozen and thawed for three times in a refrigerator at the temperature of minus 70 ℃, the collected cell liquid is centrifuged for 5min at the speed of 8 000rpm, and the supernatant after centrifugation is sucked and is placed at the temperature of minus 70 ℃ for standby or for extracting nucleic acid. Bovine kidney cells (MDBK) were purchased from the martial chinese collection of typical cultures.
Treating a bovine clinical sample to be tested: taking clinical samples stored at-70 ℃, centrifuging at 8 000r/min for 5min after dissolution, and taking the supernatant after centrifugation for standby or for extracting nucleic acid.
Extracting total RNA or DNA of the strain to be detected or the sample to be detected, wherein the extracted total RNA is used for reverse transcription into cDNA, and the reverse transcribed cDNA or the extracted DNA is directly used for double fluorescence PCR amplification or stored at the temperature of minus 30 ℃ for standby.
The nucleic acid extraction in this example was performed according to the instructions provided by the extraction kit Easy Pure Viral DNA/RNA. Easy Pure Viral DNA/RNA extraction kit and gel recovery purification kit were purchased from Beijing full gold biotechnology Co.
The cDNA transcription reaction system and process of this example include: cDNA transcription was performed on the extracted RNA in a 10. Mu.L transcription system comprising 5 XRT buffer 2. Mu.L, 50. Mu.M reverse transcription primer, random 9mer 0.5. Mu.L, AMV reverse transcriptase 0.5. Mu.L, RNA inhibitor 0.5. Mu.L, 10. Mu.M dNTPs 0.5. Mu.L, RNA template 2. Mu.L, dePC water 4. Mu.L, and the mixture was subjected to instantaneous centrifugation, followed by reverse transcription in a PCR apparatus at 42℃60 mm, 85℃5 mm, 12℃5 mm, and cDNA transcription was completed. The reagents of this example, 5 xRT-PCR buffer, AMV reverse transcriptase, reverse transcription primer (Random 9 mer), RNA inhibitor, dNTPs, etc., were purchased from Takara Bio (Dai) bioengineering Co., ltd.
(III) reaction System
Through an optimization experiment, the optimal reaction system selected in this embodiment is: each 20. Mu.L of the reaction system contains 1. Mu.L (0.1 ng-10 ng) of the template DNA or cDNA, 0.2. Mu.L (final concentration 0.2. Mu. Mol/. Mu.L) of each of the 20. Mu.mol/. Mu.L IBRVF and IBRVR primers, 0.2. Mu.L (final concentration 0.2. Mu. Mol/. Mu.L) of each of the 20. Mu.mol/. Mu.L IBRVpro probe, 0.2. Mu.L (final concentration 0.2. Mu. Mol/. Mu.L) of each of the 20. Mu.mol/. Mu.L BVDF and BVDR primers, and 0.2. Mu.L (final concentration 0.2. Mu. Mol/. Mu.L) of each of the 20. Mu.L BVDVP primer; in addition, the reaction system also comprises a buffer solution and water, wherein the buffer solution specifically selected in the embodiment is 2 XPremix Ex Taq (product No. RR390A of Takara Co., ltd., product No. RR) and 10. Mu.L of 5 u/. Mu.L of Ex Taq HS,10mM dNTP Mixture,Mg2 +) and the water specifically selected in the embodiment is RNase Free pure water and the water is added to 20. Mu.L.
(IV) reaction procedure and result detection
Uniformly mixing the components of the reaction system in the step (III), and performing amplification reaction in a fluorescent quantitative PCR instrument after instantaneous centrifugation, wherein the amplification procedure is 10S (20 ℃/S) at 95 ℃; then, the sample was subjected to 95℃for 10S (20 ℃ C./S), 58℃for 10S, and 72℃for 10S, FAM fluorescent signals were collected at 530. Mu.m, ROX fluorescent signals were collected at 610. Mu.m, and the sample was subjected to 44 cycles, ending at 40℃for 10S. The obtained detection Ct value is smaller.
The fluorescent quantitative PCR apparatus Lightcycler 2.0 used in this example was a product produced by Roche Inc. (Roche)
Example 2 Effect verification experiment of method
Establishment of standard curve and sensitivity test
Preparation of standard DNA: the target sequence of the primer IBRVF, IBRVR amplified IBRV described in example 1 was inserted into the pMD-18T plasmid, and the resulting positive recombinant plasmid was prepared and designated pMD-IBRV. The target sequence of the BVDV amplified by the primers BVDVF and BVDR described in example 1 was inserted into the pMD-18T plasmid, and the obtained positive recombinant plasmid was designated as pMD-BVDV. pMD-18T, etc. were purchased from Takara Bio (Dalian) bioengineering Co., ltd.
The extracted pMD-IBRV and pMD-BVDV positive recombinant plasmid DNA are used as standard DNA, its concentration is measured, mixed with identical concentration, then diluted into 1X 100-1X 10 according to 10-fold serial dilution 9 Copy/. Mu.L was added to the reaction system described in example 1, and double fluorescent quantitative PCR amplification was performed according to the amplification procedure described in example 1, repeated 3 times, and compared with single fluorescent quantitative PCR to establish a standard curve and test sensitivity.
The experimental results are shown in fig. 1-3. The results in FIG. 1 show that the primers, probes and methods of construction designed in example 1 showed a single peak for the amplification of IBRV and BVDV and a uniform Tm value for the product. The results of fig. 2 and 3 show that the method of the invention can detect 100 copies of both IBRV and BVDV, and has high sensitivity.
(II) specificity test
Double fluorescence quantitative PCR amplification was performed using the extracted IBRV, oregonCV, NADL nucleic acids as templates according to the reaction system and amplification procedure described in example 1, and specific fluorescence of 530 μm FAM (IBRV) and 610 μm ROX (BVDV) was collected and repeated 3 times. The same method is used, and nucleic acid templates of control strains bovine rotavirus Anhui isolate (014), bovine paratuberculosis (TB), bovine tuberculosis bacillus (MB), classical Swine Fever Virus (CSFV), mycoplasma bovis and bovine pasteurella are further added for verification, so as to verify the specificity of the method.
As shown in the experimental results in FIG. 4 and FIG. 5, when only IBRV or BVDV template is added into the reaction system, the primer and the probe for IBRV and BVDV are simultaneously added for double real-time fluorescence quantitative PCR detection, and the results show that specific fluorescence signal curves are respectively collected at the wavelengths of 530 μm FAM (IBRV) and 610 μm ROX (BVDV), no cross signal appears, and the two pairs of the primers and the probes have specificity. The nucleic acid templates of the control strains bovine rotavirus Anhui isolate (014), bovine paratuberculosis bacillus, bovine tuberculosis bacillus (MB), swine fever virus (CSFV), bovine mycoplasma and bovine Pasteurella are further detected and verified that no specific fluorescent signal curve is collected and no cross reaction exists with IBRV and BVDV strains, so that the method has strong specificity.
Bovine infectious rhinotracheitis virus (IBRV), bovine viral diarrhea isolate oreglon cv24 and NADL strain freeze-dried virus, control strain bovine rotavirus Anhui isolate (014) freeze-dried virus, bovine paratuberculosis, bovine tubercle bacillus (MB) dead bacteria were purchased from chinese veterinary drug monitoring institute, and swine fever virus (CSFV), mycoplasma bovis, and pasteurella bovis were all stored in this room.
(III) repeatability and interference test
Taking 1X 10 diluted as described in the above (one) 6 Performing double fluorescence quantitative PCR amplification according to the reaction system and the amplification procedure described in example 1, performing three-time repetition test, calculating Ct value and coefficient of variation (cv) of the three-time repetition test, and checking the in-batch repeatability; at the same time dilute 1X 10 6 The mixed DNA and reagent were placed at-20℃and repeated at 3d intervals, and the stability and batch-to-batch reproducibility of the reagents were examined. The IBRV and BVDV nucleic acid templates were combined at different concentrations and double fluorescent quantitative PCR amplification was performed according to the reaction system and amplification procedure described in example 1, respectively, to determine if interference occurred when the concentrations of IBRV and BVDV nucleic acids were greater and lower.
The results of the repeatability experiments are shown in FIGS. 6 and 7, for 1X 10 6 And (3) performing three repeated tests by taking the copy/mu L mixed DNA as a template, wherein the Ct values detected are 12.54, 12.66 and 12.70 respectively, and the variation coefficients are less than 4%. The samples stored at-20 ℃ are repeatedly detected, the Ct values detected are 12.22, 12.43 and 12.52 respectively, and the variation coefficients are smaller than 4 percent. The results show that the method hasHas good repeatability.
The method can also detect IBRV or BVDV simultaneously when the concentration of the IBRV template is higher and the concentration of the BVDV template is lower or when the concentration of the IBRV template is lower and the concentration of the BVDV template is higher by combining the standard DNA of the IBRV and the BVDV according to different concentrations, and the detection result has little variation with the Ct value detected by the IBRV or BVDV single fluorescent quantitative PCR, so that the established method has little interference to the detection of the IBRV and the BVDV.
(IV) detection of clinical samples
The method comprises the steps of collecting 127 parts of nasal mucus and anal mucosa cotton swab samples of cattle with diarrhea symptoms or nasal discharge symptoms in cattle farms in different places in Guangxi, respectively placing the cattle in 2mL of DMEM culture medium, repeatedly squeezing and eluting, and storing the liquid in a refrigerator at-70 ℃ for later use.
127 bovine clinical sample nucleic acids to which single-copy detection had been applied were tested for the presence of mixed infection using the reaction system and amplification procedure described in example 1 to evaluate their clinical utility.
The experimental result shows that 12 clinical samples of 127 cattle are proved to be positive for IBRV and 26 clinical samples of the cattle are proved to be positive for BVDV by applying single fluorescent detection; amplification assays were performed using the reaction system and amplification procedure described in example 1, wherein 2 parts of the confirmed IBRV and BVDV positive samples were mixed with infection and the copy numbers were 6.24X10, respectively 5 And 6.32X10 4 Copy/. Mu.L. 2 positive samples were analyzed by sequence alignment and were 100% homologous to the sequences of IBRV and BVDV. The detection result of clinical samples shows that bovine cases which are infected with IBRV and BVDV simultaneously clinically exist, and the method established by the research is easy to distinguish.
The above examples only show embodiments of the present invention, and the description thereof is more specific and detailed, but should not be construed as limiting the scope of the invention, but all technical solutions obtained by equivalent substitution or equivalent transformation shall fall within the scope of the invention.
Sequence listing
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Claims (5)
1. A primer and probe composition for detecting infectious bovine rhinotracheitis virusInfectious bovine rhino-tracheitis virus) The primer and probe composition of (1) -3) is shown in the specification, the amplification size of the target fragment is 120bp, and the primer and probe composition is used for detecting bovine viral diarrhea virusbovine viral diarrheamucosal disease virus) The primer and probe composition of (2) is 4) -6) below, and the amplification size of the target fragment is 88bp:
1) SEQ ID No. in the sequence Listing: 1, a nucleotide sequence shown in 1;
2) SEQ ID No. in the sequence Listing: 2, a nucleotide sequence shown in the specification;
3) SEQ ID No. in the sequence Listing: 3, a nucleotide sequence shown in 3;
4) SEQ ID No. in the sequence Listing: 4, a nucleotide sequence shown in figure 4;
5) SEQ ID No. in the sequence Listing: 5, a nucleotide sequence shown in seq id no;
6) SEQ ID No. in the sequence Listing: 6, a nucleotide sequence shown in the specification;
wherein, SEQ ID No.: 3 and the SEQ ID No.: 6 is FAM and ROX respectively, and the fluorescent groups marked at the 5' end of the nucleotide sequence shown in SEQ ID No: 3 and the SEQ ID No.: 6 are ECLIPSE, the 3' -end label quenching groups of the nucleotide sequence shown in the formula 6.
2. A kit comprising the composition of claim 1.
3. A method for detecting infectious bovine rhinotracheitis virus and/or bovine viral diarrhea virus, comprising detecting infectious bovine rhinotracheitis virus using the primer and probe composition of 1) -3) below and detecting bovine viral diarrhea virus using the primer and probe composition of 4) -6) below:
1) SEQ ID No. in the sequence Listing: 1, a nucleotide sequence shown in 1;
2) SEQ ID No. in the sequence Listing: 2, a nucleotide sequence shown in the specification;
3) SEQ ID No. in the sequence Listing: 3, a nucleotide sequence shown in 3;
4) SEQ ID No. in the sequence Listing: 4, a nucleotide sequence shown in figure 4;
5) SEQ ID No. in the sequence Listing: 5, a nucleotide sequence shown in seq id no;
6) SEQ ID No. in the sequence Listing: 6, a nucleotide sequence shown in the specification;
the amplification degree of the target fragment for detecting the bovine infectious rhinotracheitis virus is 120bp, the amplification degree of the target fragment for detecting the bovine viral diarrhea virus is 88bp, and the sequence of SEQ ID No: 3 and the SEQ ID No.: 6 is FAM and ROX respectively, and the fluorescent groups marked at the 5' end of the nucleotide sequence shown in SEQ ID No: 3 and the SEQ ID No.: 6, the 3' -end mark quenching groups of the nucleotide sequence are ECLIPSE;
the methods do not include diagnostic and therapeutic methods of disease.
4. The method of claim 3, further comprising performing a PCR reaction comprising at least one of the following 1) -4):
1) The reaction system of the PCR reaction comprises: when the composition comprises the sequence table SEQ ID No: 1-SEQ ID No.: 6, each 20 mu L of PCR reaction system contains the nucleotide sequence shown in SEQ ID No: 1-SEQ ID No.: 6 of the sequence of nucleotides shown in FIG. 6, 0.2. Mu. Mol/. Mu.L each;
2) The PCR reaction comprises Taqman double-fluorescence quantitative PCR reaction;
3) The PCR reaction comprises a Taqman double-fluorescence quantitative PCR reaction, wherein the reaction system of the Taqman double-fluorescence quantitative PCR reaction is as follows: every 20 mu L of reaction system contains 0.1ng-10ng of template DNA or cDNA extracted from the object to be detected; SEQ ID No.: 1-SEQ ID No.: 6 of the sequence of nucleotides shown in FIG. 6, 0.2. Mu. Mol/. Mu.L each; 50u Ex Taq HS;100mol dNTPMixture,Mg 2+; the RNase Free pure water is added to 20 mu L;
4) The amplification procedure of the PCR reaction comprises: 95 ℃ for 10s; then, the mixture is circulated at 95 ℃ for 10s, 58 ℃ for 10s and 72 ℃ for 10s, and is subjected to more than 44 cycles, and the mixture is ended at 40 ℃ for 10 s.
5. Use of the composition of claim 1, the kit of claim 2, the method of any one of claims 3-4, said use comprising use in at least one of the following 1) -6):
1) Identifying infectious bovine rhinotracheitis virus and/or bovine viral diarrhea virus;
2) Preparing a kit or related products for identifying infectious bovine rhinotracheitis virus and/or bovine viral diarrhea virus;
3) Detecting whether the pathogenic microorganism to be detected is infectious bovine rhinotracheitis virus and/or bovine viral diarrhea virus;
4) Preparing a kit or related products for detecting whether the pathogenic microorganism to be detected is a bovine infectious rhinotracheitis virus and/or a bovine viral diarrhea virus;
5) Detecting whether a sample to be detected contains infectious bovine rhinotracheitis virus and/or bovine viral diarrhea virus;
6) Preparing a kit or related products for detecting whether a sample to be detected contains bovine infectious rhinotracheitis virus and/or bovine viral diarrhea virus;
such applications do not include diagnostic and therapeutic methods of disease.
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| 范晴等.牛病毒性腹泻病毒和牛轮状病毒TaqMan二重实时荧光RT-PCR检测方法的建立.《中国兽医学报》.2011,(第10期),摘要，参见第1.1、1.3节，表1. * |
| 陈超等编著.《新技术与精准医学》.上海交通大学出版社,2017,第85页第2段. * |
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