CN108048600B - Fluorescent quantitative PCR detection method for infectious bovine rhinotracheitis virus - Google Patents
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Abstract
The invention discloses a primer, a probe, a kit and a method for detecting gE and gB genes of Infectious Bovine Rhinotracheitis Virus (IBRV) by fluorescent quantitative PCR. The primer and the probe for detecting the Infectious Bovine Rhinotracheitis Virus (IBRV) have high specificity and sensitivity, can detect the Infectious Bovine Rhinotracheitis Virus (IBRV), can detect various clinical samples, can quickly monitor the infection condition of the Infectious Bovine Rhinotracheitis Virus (IBRV) in clinic, and have short detection time, high detection sensitivity and high stability.
Description
Technical Field
The invention belongs to the field of virus detection, particularly relates to a method for detecting viral infectious diseases, and particularly relates to a method for detecting infectious bovine rhinotracheitis virus and the application field.
Background
Infectious Bovine Rhinotracheitis (IBR) is an acute, hot and contact infectious disease of cattle, and the etiology thereof is Infectious Bovine Rhinotracheitis Virus (IBRV), also known as bovine herpes virus type I (BHV-I), which clinically presents multiple types of diseases, such as upper respiratory tract mucositis, pustular vulvovaginitis, balanitis, conjunctivitis, juvenile bovine meningoencephalitis, mastitis, abortion and the like. The disease was first discovered in the united states as early as the 50 s of the 20 th century and was first isolated from affected cattle by Madin in 1956. Kendrick isolated the virus from sick cattle with infectious pustular vulva-vaginitis in 1958, and it was shown by studies that the virus was IBRV. The virus is firstly detected and separated from imported dairy cows of New Zealand in 1980. The world animal health Organization (OIE) classifies IBR as a B-type animal epidemic disease, and the animal and plant quarantine method in China is also specified, so that IBR must be detected in import and export cattle so as to control the occurrence and prevalence of the disease. Serological investigation proves that IBRV infection exists in cattle flocks in most areas in China, and serious influence is caused to cattle raising industry in China.
The research establishes a real-time fluorescent quantitative PCR method for detecting IBRV and distinguishing gE gene deletion vaccine strains and wild strains by taking gE and gB genes of IBR as target sequences. Provides an effective and practical detection method for the disease inspection and quarantine.
Disclosure of Invention
According to IBRVgE and gB gene sequences registered in GenBank, 2 pairs of specific primers and Taqman probes are designed. A real-time fluorescent quantitative PCR method for detecting IBRV gE and gB genes is established, so that the IBRV can be sensitively, efficiently, quickly and accurately detected.
The invention aims to identify IBRV wild virus infection and gE gene deletion vaccine immune animals by a double real-time fluorescent PCR method, and can also identify and detect 2 genes or carry out gB and gE single-gene identification.
The invention aims to provide a primer for detecting Infectious Bovine Rhinotracheitis Virus (IBRV) by fluorescent quantitative PCR, wherein the primer is shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
The invention aims to provide a primer for detecting Infectious Bovine Rhinotracheitis Virus (IBRV) by fluorescent quantitative PCR, wherein the primer is shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
The invention aims to provide a probe for detecting Infectious Bovine Rhinotracheitis Virus (IBRV) by fluorescent quantitative PCR, wherein the probe is shown as SEQ ID NO: 5, respectively.
The invention aims to provide a probe for detecting Infectious Bovine Rhinotracheitis Virus (IBRV) by fluorescent quantitative PCR, wherein the probe is shown as SEQ ID NO: and 6.
The invention aims to provide a probe for detecting Infectious Bovine Rhinotracheitis Virus (IBRV) by fluorescent quantitative PCR, wherein the 5 'end of the probe is respectively marked with ROX and FAM, and the 3' end of the probe is respectively marked with BHQ2 and TAMRA.
The invention also aims to provide a primer or a probe for preparing a kit for detecting Infectious Bovine Rhinotracheitis Virus (IBRV).
The invention aims to provide a reagent kit for detecting Infectious Bovine Rhinotracheitis Virus (IBRV) by fluorescent quantitative PCR, which comprises a primer for detecting the Infectious Bovine Rhinotracheitis Virus (IBRV) and a probe for detecting the Infectious Bovine Rhinotracheitis Virus (IBRV).
The invention specifically provides a method for detecting infectious bovine rhinotracheitis virus, which specifically comprises the following steps:
1. design and synthesis of primers and probes:
homology analysis was performed with DNAstar based on the IBRVgE and gB gene sequences published in GenBank (accession No.: NC001847.1), and primers and probes were designed using Oligo6.24 and synthesized from Shanghai. The amplification lengths were: 112bp and 78 bp. The 5' end of the probe is respectively marked by fluorescent groups ROX and FAM; the 3' end is labeled with fluorescence quenching groups BHQ2 and TAMRA.
2. Preparation of target DNA template
200. mu.L of the venom was extracted according to the instructions of the Takara Virus DNA/RNA extraction kit.
Adding 12.5 mu LMix and 9.5 mu LddH into 25 mu L system2O, template, 1. mu.L of each of the upstream and downstream primers. The cycle parameters are: pre-denaturation at 94 ℃ for 5 min; at 94 ℃ for 45s, at 59.7 ℃ for 45s, at 72 ℃ for 30s, for 30 cycles, and finally at 72 ℃ for 8 min; the target fragment length is 112bp and 78bp respectively, PCR product is recovered, purified by a kit and then connected into a pMD19-T vector, transformed into DH5a Escherichia coli, and positive clone plasmid identified by double enzyme digestion and verified by sequence determination analysis is selected as a standardAnd (4) positive.
IBRV real-time fluorescent quantitative PCR reaction system and parameters:
using a 20. mu.L reaction system, Premix Ex TaqTM(Probe qPCR) 10. mu.L, DNA template 2. mu.L, TaqMan Probe (10. mu. mol/L) 0.5. mu.L, upstream and downstream primers 0.6. mu.L each, and the remainder was made up to 20. mu.L with ultrapure water. The double fluorescent quantitative PCR system is prepared according to the proportion of 1: 1. The reaction parameters are as follows: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5 s; annealing and extension at 60 ℃ for 30s for 45 cycles.
In the invention, primers and fluorescent probes are designed according to BHV-1gE and gB gene sequences numbered NC001847.1 in a gene library, and the length of the BHV-1 gene sequence is 135305bp, so that a person skilled in the art can know the primers and fluorescent probes through public channels.
In the invention, BHV-1 gene is used as a template, a fragment with the length of 112bp is amplified by using a primer gE, and the fragment is cloned to a pMD-19T vector to obtain a target template.
In the invention, BHV-1 gene is used as a template, a fragment with the length of 78bp is amplified by using a primer gB, and the fragment is cloned to a pMD-19T vector to obtain a target template.
Meanwhile, the invention fully considers that when two templates of gE and gB are amplified simultaneously, the gE and gB compete for enzyme, primer and probe, which affects the amplification, and optimizes and researches the reaction condition and detection lower limit of the co-amplification system. The double fluorescent quantitative PCR system is prepared according to the proportion of 1: 1. The reaction parameters are as follows: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5 s; annealing and extension at 60 ℃ for 30s for 45 cycles.
Further, the specificity and repeatability of the method for detecting the infectious bovine rhinotracheitis virus are confirmed, and a standard curve is established.
The amplification system provided by the invention is used for respectively amplifying nucleic acid samples such as mycoplasma bovis, bovine parainfluenza virus type 3, bovine viral diarrhea virus, pasteurella, bovine and brucella ovis, and the detected samples have no positive amplification curve, which indicates that the method has better specificity.
Selecting the dilution of 1X 107DNA template of copies/μ L, 10-fold dilution 3 timesTo release at 1X 104～ 1×106Three dilutions of copies/μ L were used as templates for 4 repeated amplifications, and an in-group reproducibility test was established. The coefficient of variation is less than 2%, which shows that the method has good repeatability.
Take 1X 100～1×109copies/. mu.L 10 dilutions were used as DNA templates for real-time fluorescent quantitative PCR, with 3 replicates per dilution. And observing the copy concentration of the lowest detected template of TaqMan real-time fluorescent quantitative PCR, and comparing and analyzing with the ordinary PCR.
By implementing the specific inventive content of the present invention, the following advantageous effects can be achieved.
The method has short detection time, the detection time of the traditional virus separation method for the IBRV is at least more than 7 days, the detection time of the method comprises the steps of sample pretreatment until the detection result is within 2 hours, and the detection time is at least 30 minutes shorter than that of the conventional PCR detection method. The detection sensitivity is high, the method can detect 2 copies of recombinant plasmids, and the sensitivity is 1000 times higher than that of the conventional 100-fold method. The specificity is good, and only the IBRV is detected to be positive by detecting nucleic acid samples such as IBRV, mycoplasma bovis, bovine parainfluenza virus type 3, bovine viral diarrhea virus, pasteurella, cattle, brucella melitensis and the like. The repeatability is good, and the Ct values and the coefficient of variation obtained by different tests are within 2% through three times of repeated detection on different concentrations. The stability is high, and consistent results are obtained through repeated detection of samples. The method can carry out real-time monitoring and quantitative detection, and realizes the real-time monitoring of the whole reaction process and the quantitative detection of the result by receiving the fluorescence signal generated in each amplification reaction through an instrument. The method has simple flow, strong operability and easy mastering, and can be well finished without special training as long as the method has basic knowledge of molecular biology. The method can be used for monitoring the test process, effectively solves the problem of false negative results existing in the traditional detection method, can be used for monitoring the quality of a laboratory, and ensures the accuracy of the detection result. The method of the invention is used for detecting 27 parts of clinically collected bovine lung and 5 parts of clinically collected sheep lung by using the system, and expected results are obtained.
The method can simultaneously identify and detect 2 genes or carry out gB and gE single gene identification. The establishment of the method provides a better reference meaning for the research of related virus inspection and quarantine technologies, and has a guidance reference meaning for the development of standard detection reagents to a standard level. The method can be widely applied to entry and exit inspection and quarantine departments, animal husbandry and veterinary departments and breeding units, and has important significance for effective prevention and control of epidemic diseases.
Drawings
FIG. 1: IBRV PCR amplification results: wherein 1 and 2 are amplification products (112bp and 78bp) of gE and gB, and M is DL500 marker.
FIG. 2: the result of PCR amplification of the recombinant plasmid: wherein 1 and 2 are amplification products (112bp and 78bp) of gE and gB, and M is DL500 Marker.
FIG. 3: IBRV gE gene real-time fluorescence quantitative PCR standard curve: wherein the gE correlation coefficient R2The amplification efficiency E is 106.955, and the expression of the logarithm of the initial copy number and Ct value is-3.166 x + 39.77.
FIG. 4: IBRV gB gene real-time fluorescence quantitative PCR standard curve: wherein the coefficient of correlation R2The amplification efficiency E is 92.658, and the expression of the logarithm of the initial copy number and Ct value is-3.511 x + 37.969.
FIG. 5: IBRVgE gene real-time fluorescent quantitative PCR sensitivity amplification curve: wherein the curves 1-10 are 2 × 10 respectively9～ 2×100Real-time fluorescent quantitative PCR amplification with copies/. mu.L as template, 11 as negative control.
FIG. 6: IBRVgB gene real-time fluorescent quantitative PCR sensitivity amplification curve: wherein the curves 1-10 are 2 × 10 respectively9～ 2×100Real-time fluorescent quantitative PCR amplification with copies/. mu.L as template, 11 as negative control.
FIG. 7: IBRV gB gene fluorescence quantitative PCR specific amplification curve: wherein the curves 1-8 are respectively 2 × 109～2× 102And (3) performing real-time fluorescent quantitative PCR amplification by using copies/mu L as a template, wherein 9-14 amplification curves of mycoplasma bovis, bovine parainfluenza virus type 3, bovine viral diarrhea virus, pasteurella and bovine and ovine brucella are obtained respectively, and 15 is a negative control.
FIG. 8: an IBRV gE gene fluorescent quantitative PCR specific amplification curve: wherein 1 to 6 are each 2X 109～2×1047-11 of real-time fluorescent quantitative PCR with copies/mu L as a template line are respectively mycoplasma bovis, bovine parainfluenza virus type 3, bovine viral diarrhea virus, pasteurella and brucella bovis and sheep, and 12 is a negative control.
FIG. 9: IBRV dual implementation fluorescent quantitative PCR standard curve: wherein the correlation coefficients of gE and gB are both 0.999, the efficiencies are 94.626 and 96.779 respectively, which are both 90-110% and have good linear relation. The regression equations are, gE: -3.402 x + 39.878; and gB: y-3.458 x + 42.319.
Detailed Description
Example one method for detecting infectious bovine rhinotracheitis virus
1. Design and synthesis of primers and probes:
homology analysis was performed with DNAstar based on the IBRVgE and gB gene sequences published in GenBank (accession No.: NC001847.1), and primers and probes were designed using Oligo6.24 and synthesized from Shanghai. The amplification lengths were: 112bp and 78 bp. The 5' end of the probe is respectively marked by fluorescent groups ROX and FAM; the 3' end is labeled with fluorescence quenching groups BHQ2 and TAMRA.
2. Preparation of target DNA template
200. mu.L of the venom was extracted according to the instructions of the Takara Virus DNA/RNA extraction kit.
Adding 12.5 mu LMix and 9.5 mu LddH into 25 mu L system2O, template, 1. mu.L of each of the upstream and downstream primers. The cycle parameters are: pre-denaturation at 94 ℃ for 5 min; at 94 ℃ for 45s, at 59.7 ℃ for 45s, at 72 ℃ for 30s, for 30 cycles, and finally at 72 ℃ for 8 min; the lengths of the target fragments are 112bp and 78bp respectively, the PCR product is recovered, purified by a kit and then connected into a pMD19-T vector, transformed into DH5a Escherichia coli, and a positive clone plasmid which is identified by double enzyme digestion and verified by sequence determination analysis is selected as a standard positive.
IBRV real-time fluorescent quantitative PCR reaction system and parameters:
using a 20. mu.L reaction system, Premix Ex TaqTM(Probe qPCR) 10. mu.L, DNA template 2. mu.L, TaqMan Probe0.5. mu.L of a needle (10. mu. mol/L), 0.6. mu.L of each of the primers on the upstream and downstream sides, and the balance to 20. mu.L with ultrapure water. The double fluorescent quantitative PCR system is prepared according to the proportion of 1: 1. The reaction parameters are as follows: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5 s; annealing and extension at 60 ℃ for 30s for 45 cycles.
The primer code sequence of the invention is shown in Table 1, wherein the BHV-1gE and gB gene sequences numbered NC001847.1 in a reference gene library are designed with primers and fluorescent probes, and the BHV-1 gene sequence is 135305bp in length, and can be known by persons skilled in the art through public channels.
Table 1: the primer code and sequence table I are shown as the sequences of gE and gB primers and probes.
Example two: obtaining a target template
200. mu.L of the venom was extracted according to the instructions of the Takara Virus DNA/RNA extraction kit.
Adding 12.5 mu LMix and 9.5 mu LddH into 25 mu L system2O, template, 1. mu.L of each of the upstream and downstream primers. The cycle parameters are: pre-denaturation at 94 ℃ for 5 min; at 94 ℃ for 45s, at 59.7 ℃ for 45s, at 72 ℃ for 30s, for 30 cycles, and finally at 72 ℃ for 8 min; the lengths of the target fragments are 112bp and 78bp respectively, the PCR product is recovered, purified by a kit and then connected into a pMD19-T vector, transformed into DH5a Escherichia coli, and a positive clone plasmid which is identified by double enzyme digestion and verified by sequence determination analysis is selected as a standard positive.
1. PCR amplification identification of recombinant plasmid:
mu.L of the template was added with Premixx 12.5. mu.L of each of the upstream and downstream primers (10pmoL), and then water was added thereto to a volume of 1. mu.L, followed by PCR amplification and detection by 2% agarose gel electrophoresis.
2. Enzyme digestion identification of the recombinant plasmid: add 8. mu.L plasmid, 2. mu. Lbuffer, 1. mu. LEcoRI, 1. mu. LHindiIII into 200. mu.L tube, supplement ultrapure to 20. mu.L, mix well by centrifugation, digest in 37 ℃ water bath for 2h, detect by 2% agarose gel electrophoresis.
3. Sequencing of the recombinant plasmid: and (4) overnight culturing the bacterial liquid identified as the positive plasmid by enzyme digestion, and sequencing.
Agarose gel electrophoresis shows that BHV-1 whole gene DNA is used as a template, gE and gB are used as primers, 112bp and 78bp fragments with the same size are amplified, the 112bp and 78bp fragments are obtained by PCR amplification and enzyme digestion identification of recombinant plasmids, the sequencing identification is correct, and the correctly identified recombinant plasmids are target templates (attached figures 1 and 2).
Example three: establishment of real-time fluorescent quantitative PCR amplification system
Respectively using gE and gB as templates to perform amplification, 25pL reaction system, wherein Premix Ex TaqTM(Probe qPCR) 10. mu.L, DNA template 2. mu.L, TaqMan Probe (10. mu. mol/L) 0.5. mu.L, upstream and downstream primers 0.6. mu.L each, and the remainder was made up to 20. mu.L with ultrapure water. The dual fluorescent quantitative PCR system was performed according to the following protocol 1: 1, preparing the mixture. The reaction parameters are as follows: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5 s; annealing and extension at 60 ℃ for 30s for 45 cycles. The results show that the probes have better specificity to the respective templates, and the amplification curves are shown in figures 7 and 8.
Example four: drawing of standard curve
Taking the prepared standard DNA template, diluting by 10 times, and obtaining 1 × 10 DNA template9～1×100copies/. mu.L 10 gradients, which were used as standards of known concentration to draw standard curves. And (4) setting a negative control (using ultrapure water to replace a template) according to the optimized reaction system and reaction parameters. Evaluation of the correlation coefficient R of the Standard Curve2Values, amplification efficiency E values, standard curve slope K values and construction of a standard curve regression equation (fig. 3, 4, 9).
Example five: repeatability test, sensitivity test and specificity test
Selecting the dilution of 1X 107The DNA template of copies/. mu.L was diluted 3 times by 10-fold and 1X 104～ 1×106Three dilutions of copies/μ L were used as templates for 4 replicates respectively to establish the in-group replicate test and negative controls (see table 2 and table 3 for results).
Table 2: results of gE Gene reproducibility test
Table 3: gB gene repeatability test results
Take 1X 100～1×109copies/. mu.L 10 dilutions were used as DNA templates for real-time fluorescent quantitative PCR, with 3 replicates per dilution. The lowest detected template copy concentration of TaqMan real-time fluorescent quantitative PCR was observed and analyzed in comparison with the ordinary PCR, and a negative control was set up (FIGS. 5 and 6).
And performing specificity test by using mycoplasma bovis, bovine parainfluenza virus type 3, bovine viral diarrhea virus, pasteurella, bovine and brucella melitensis according to the optimized reaction system and reaction parameters. 5 dilutions of the prepared standard DNA template are taken as positive controls, and negative controls are set simultaneously.
Example six: preparation of multiplex real-time fluorescent quantitative PCR standard curve
Diluting the prepared gE and gB standard positive plasmid template by 10 times, and performing real-time fluorescence quantitative PCR detection by using the established reaction system and reaction parameters of multiple real-time fluorescence quantitative PCR (template is 2 standard positive plasmid mixed templates)
Example seven: detection of a sample
Quantitative detection was performed on 27 parts of bovine lung and 5 parts of ovine lung. As can be seen, the amplification curve appeared in the positive control, and the amplification curve appeared in the negative control. 9 parts of the bovine lung may be positive reaction of gB gene, 4 parts of the bovine lung are positive reaction of gB and gE double genes, and the rest samples are negative reaction.
The foregoing examples further illustrate the present invention and are not to be construed as limiting thereof. Modifications and substitutions to methods, steps or conditions of the invention are within the scope of the invention without departing from the spirit and nature of the invention.
Claims (2)
1. The application of primers and probes for detecting viruses by fluorescent quantitative PCR (polymerase chain reaction) in preparing a kit for detecting Infectious Bovine Rhinotracheitis Virus (IBRV), wherein the primers are shown as SEQ ID NO: 1-4, and the probe is shown as SEQ ID NO: 5-6, the 5 'end of the probe is labeled with ROX and FAM, respectively, and the 3' end of the probe is labeled with BHQ2 and TAMRA, respectively.
2. The kit for detecting the Infectious Bovine Rhinotracheitis Virus (IBRV) by the fluorescent quantitative PCR is characterized by comprising a primer and a probe for detecting the Infectious Bovine Rhinotracheitis Virus (IBRV), wherein the primer is shown as SEQ ID NO: 1-4, and the probe is shown as SEQ ID NO: 5-6, the 5 'end of the probe is labeled with ROX and FAM, respectively, and the 3' end of the probe is labeled with BHQ2 and TAMRA, respectively.
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| CN101701265A (en) * | 2009-11-17 | 2010-05-05 | 新疆出入境检验检疫局检验检疫技术中心 | Method for detecting infectious bovine rhinotracheitis virus and application thereof |
| CN104726614A (en) * | 2015-03-31 | 2015-06-24 | 河南牧业经济学院 | Detection primer, kit and detection method of infectious bovine rhinotracheitis virus |
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| CN101701265A (en) * | 2009-11-17 | 2010-05-05 | 新疆出入境检验检疫局检验检疫技术中心 | Method for detecting infectious bovine rhinotracheitis virus and application thereof |
| CN104726614A (en) * | 2015-03-31 | 2015-06-24 | 河南牧业经济学院 | Detection primer, kit and detection method of infectious bovine rhinotracheitis virus |
| CN105132590A (en) * | 2015-10-15 | 2015-12-09 | 上海市农业科学院 | LAMP visual detection method of infectious bovine rhinotracheitis viruses |
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