Kit for detecting genotypes of bovine herpesviruses 1.1 and 1.2

Abstract

The invention provides a primer group for detecting genotypes of bovine herpesvirus 1.1 and 1.2, and also provides a kit for detecting genotypes of bovine herpesvirus 1.1 and 1.2, the kit is high in detection sensitivity, good in specificity and high in repeatability, and the detection method provided by the invention can be used for qualitative detection and rapid typing, and is simple in process and easy to operate.

Description

Technical Field

[0001] The invention belongs to the field of molecular biological detection, and in particular relates to a kit for detecting bovine herpes virus 1.1 and 1.2 genotypes. Background Art

[0002] Bovine herpesvirus type 1 (BHV-1) belongs to the Herpesviridae family, Alphaherpesvirinae subfamily, Varicellavirus genus. It is a key pathogen causing infectious bovine respiratory disease or abortion. It can cause a series of disease syndromes in cattle, including bovine respiratory disease (BRD), reproductive tract disease vulvovaginitis and abortion, conjunctivitis and severe neonatal disease, posing a threat to animal husbandry and causing significant economic losses. Currently, inactivated or attenuated vaccines are used against bovine herpesvirus type 1 (BHV-1). However, they only partially protect against wild-type bovine herpesvirus type 1 (BHV-1) infection.

[0003] Bovine herpesvirus type 1 (BHV-1) is divided into type 1.1 and type 1.2. The symptoms of BHV-1 virus infection with different genotypes are similar, but epidemiological data show that the clinical effects of BHV-1 strain 1.1 and BHV-1 strain 1.2 are different. Therefore, it is particularly important to identify the BHV-1 virus genotype before vaccine immunization. In the prior art, commercial ELISA kits can be used to confirm BHV-1 infection, but it is impossible to distinguish which genotype it is infected with. The method of distinguishing by combining restriction enzyme cutting and monoclonal antibody (mAb) is cumbersome and costly. Therefore, it is urgent to develop a method for distinguishing the genotype of bovine herpesvirus type 1 that is simple to operate, fast and accurate. Summary of the invention

[0004] In view of this, the present invention aims to overcome the defects in the prior art and provide a dual PCR detection method for identifying bovine herpesvirus 1.1 and 1.2 genotypes.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] A primer set for detecting bovine herpesvirus genotypes 1.1 and 1.2, the primer set comprising the following primers:

[0007] The nucleotide sequence of primer IBRV-1.1-F is shown in SEQ ID NO: 1;

[0008] The nucleotide sequence of primer IBRV-1.1-R is shown in SEQ ID NO: 2;

[0009] The nucleotide sequence of primer IBRV-1.2-F is shown in SEQ ID NO: 3;

[0010] The nucleotide sequence of primer IBRV-1.2-R is shown in SEQ ID NO:4.

[0011] A kit for detecting bovine herpesvirus 1.1 and 1.2 genotypes, the kit comprising the following components:

[0012] The nucleotide sequence of primer IBRV-1.1-F is shown in SEQ ID NO: 1;

[0013] The nucleotide sequence of primer IBRV-1.1-R is shown in SEQ ID NO: 2;

[0014] The nucleotide sequence of primer IBRV-1.2-F is shown in SEQ ID NO: 3;

[0015] The nucleotide sequence of primer IBRV-1.2-R is shown in SEQ ID NO: 4;

[0016] PCR 10×Ex Taq Bufferr (Mg2+plus), dNTP Mixture, DNA template, ddH2O.

[0017] Furthermore, the PCR amplification conditions of the kit are: pre-denaturation at 95°C for 5 min; 95°C for 20 s, 56°C for 30 s, 72°C for 40 s, for a total of 35 cycles, and finally extension at 72°C for 10 min.

[0018] The kit for detecting bovine herpesvirus 1.1 and 1.2 genotypes is used in identifying bovine herpesvirus 1.1 and 1.2 genotypes.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] This kit has high detection sensitivity and good specificity. Through the detection of nucleic acid samples such as bovine respiratory syncytial virus, bovine viral diarrhea virus, bovine rotavirus, and bovine coronavirus, only the positive template of bovine herpes virus type 1 (BHV-1) amplified the expected band. This kit has good detection repeatability. After three repeated tests on three mixed and single templates, the bands all obtained consistent results.

[0021] The detection method provided by the present invention can perform qualitative detection and rapid typing, and the method flow is simple, easy to operate, and can be quickly mastered. The method of the present invention is used to amplify type 1.1 DNA of bovine herpesvirus type 1 (BHV-1), type 1.2 DNA of bovine herpesvirus type 1 (BHV-1), and mixed DNA thereof as templates to verify the accuracy of the method. At the same time, 60 nasal swabs and 10 bovine lungs collected clinically were tested using the system, and expected results were obtained.

[0022] This method can simultaneously identify and detect the 1.1 and 1.2 genotypes of bovine herpesvirus type 1 (BHV-1), and has practical reference significance for the research of related virus inspection and quarantine technology. Through this method, the genotype information of bovine herpesvirus type 1 in livestock herds can be understood, and at the same time, the molecular epidemiology of viruses in dairy cows raised in some areas can be studied to understand the prevalence of the disease and provide data and technical support for the prevention and control of bovine herpesvirus disease. This method can be widely used in entry-exit inspection and quarantine departments, animal husbandry and veterinary departments and breeding units, and is of great significance for the effective prevention and control of epidemics. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure is the result of single-plex PCR amplification of 1.1 and 1.2 genotypes of bovine herpesvirus type 1 (BHV-1), wherein 1 is the negative control of 1.1, 2 is the amplified fragment of 1.1 (405 bp), 3 is the negative control of 1.2, 4 is the amplified fragment of 1.2 (212 bp), and M is the DL2000 marker;

[0024] Figure 2 The figure is the result of double PCR of 1.1 and 1.2 genotypes of bovine herpesvirus type 1 (BHV-1), wherein 1 is the negative control, 2 and 3 are the amplified fragments of 1.2 and 1.1 (212bp and 405bp), 4 is the amplified fragment of 1.1+1.2 (405bp+212bp), and M is the DL2000 marker;

[0025] Figure 3 This is a graph showing the sensitivity test results of the 1.1 genotype PCR method for single-stranded bovine herpesvirus type 1 (BHV-1), where 1-10 is pMD18-T-1.1: 1010 copies / μL-101 copies / μL, and M is DL2000 marker;

[0026] Figure 4 This is a graph showing the sensitivity test results of the 1.2 genotype PCR method for single bovine herpes virus type 1 (BHV-1), where 1-10 is pMD18-T-1.2:10 10 copies / μL-10 1 copies / μL, M is DL2000 marker;

[0027] Figure 5 This is the result of the sensitivity test of the dual PCR method, where 1-10 is pMD18-T-1.1+pMD18-T-1.2:10 10 copies / μL-10 1 copies / μL; M is DL2000 marker;

[0028] Figure 6 The figure is the result of the double PCR method specificity test, where 1 is the negative control, 2 is the positive control, 3-6 are bovine respiratory syncytial virus, bovine viral diarrhea virus, bovine rotavirus, and bovine coronavirus, respectively, and M is the DL2000 marker;

[0029] Figure 7 This is a graph showing the results of a duplex PCR method repeatability test, where 1-3 are bovine herpesvirus type 1.1, 4-6 are bovine herpesvirus type 1.2, 7-9 are bovine herpesvirus type 1.1+1.2, and M is DL2000 marker;

[0030] Figure 8 This is a diagram showing the results of the detection of viral nucleic acid using the double PCR method, where 1 represents bovine herpesvirus type 1.1 + type 1.2, 2 represents bovine herpesvirus type 1.1, and 3 represents bovine herpesvirus type 1.2; M represents the DL2000 marker. DETAILED DESCRIPTION

[0031] The following specific descriptions are exemplary and are intended to provide further explanation of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the protection scope of the present invention.

[0032] The test methods used in the following experimental examples are conventional methods unless otherwise specified.

[0033] Example 1: Synthesis of primers for dual PCR detection of genotypes 1.1 and 1.2 of bovine herpesvirus type 1 (BHV-1):

[0034] According to the comparative analysis of the gC gene sequences of the bovine herpes virus type 1.1 isolate Cooper and type 1.2 isolate K22 published on Genbank, specific primers were designed using SnapGene software, and the primers were synthesized by Jinweizhi Biotechnology Co., Ltd., and the amplification lengths were 405bp and 212bp, respectively. The code sequence in the present invention is shown in Table 1, wherein the primers are designed with reference to the gC gene sequences of the isolates Cooper and K22 of the 1.1 and 1.2 genotypes of bovine herpes virus type 1 (BHV-1) numbered KU198480 and KM258880 in the gene library, and the full gene sequence length is 1527bp.

[0035] Table 1 Primer sequences for dual PCR amplification of genotypes 1.1 and 1.2 of bovine herpesvirus type 1 (BHV-1).

[0036]

[0037] Example 2: Preparation of positive clone plasmid template

[0038] Take 200 μL of venom and extract according to the instructions of the Norwegian virus DNA / RNA extraction kit. Use the bovine herpes virus type 1.1 and type 1.2 DNA for PCR reaction, and set up a negative control (ddH2O). 20 different systems should be used, including: 10 systems including: set negative control. Use bovine (Mg 2+ plus) (20mM) 20m, dNTP Mixture (2.5mM each) 2.5, upstream and downstream primers (10mM) 1.0M each, DNA template 1.0M primer, add ddH20 to 20HM. The cycle is: 95℃ pre-denaturation 5min; 95℃ 30s, 56℃ 30s, 72℃ 40s, a total of 35 cycles, and finally 72℃ extension for 10min; the target fragment lengths are 405bp and 212bp respectively. The PCR products are recovered and purified by the kit and connected to the pMD18-T vector, transformed into DH5a Escherichia coli, and the positive clone plasmid verified by PCR analysis is used as the standard positive.

[0039] 1. PCR amplification and identification of recombinant plasmids: 1 recombinant plasmid of bovine herpes virus type 1.1 and type 1.2 was used as template, 10 plates were taken, and the virus plasmid was used as standard (Mg 2+ plus) (20mM) 20m, dNTP Mixture (2.5mM each) 2.5, upstream and downstream primers (10mM) 1.0μL each, DNA template 1.0μL, add ddH2O to 20μL, and perform PCR amplification.

[0040] 1.5% agarose gel electrophoresis test, the results are shown in the attached Figure 1 .

[0041] 2. Sequencing and identification of recombinant plasmids: The bacterial solution identified as positive plasmids by PCR amplification was cultured overnight and sent to the company for sequencing. Agarose gel electrophoresis showed that single-plex PCR reactions were performed using bovine herpes virus type 1.1 and type 1.2 recombinant plasmids as templates, respectively, and fragments of 405bp and 212bp of the same size were amplified. The sequencing identification was correct, and the correctly identified recombinant plasmid was the positive template.

[0042] Example 3: Establishment of a dual PCR amplification system

[0043] Single template and mixed template were used, where the components of the mixed template were mixed at equal concentrations, and a negative control (ddH2O) was set. The reaction used a 20 μL system, including: 10×Ex Taq Buffer (Mg 2+plus) (20mM) 2μL, dNTPMixture (2.5mM each) 2μL, 1.1 type upstream and downstream primers (10mM) 1.0μL each, 1.2 type upstream and downstream primers (10mM) 1.0μL each, DNA template 1.0μL each, add ddH2O to 20μL. Reaction conditions: 95% pre-denaturation for 5min; 95min for, 56min for, 72min for, a total of 35 cycles, and finally 72 loop extension for 10min. The results show that the primers have good specificity for their respective templates. The gel electrophoresis results are shown in the attached Figure 2 .

[0044] Example 4: Sensitivity test, specificity test and repeatability test

[0045] Select pMD18-T-1.1 and pMD18-T1.2 at a concentration of 10 10 The positive plasmids with 10 copies / μL were diluted 10 times in sequence, and the 10 dilution gradients were used as templates for amplification. The prepared standard positive plasmids were used as positive controls, and negative controls were set up at the same time. 1.5% agarose gel electrophoresis was used for detection. The reaction results were obtained by the attached Figure 3-4 It can be seen that the primers have a high sensitivity to template amplification, and the minimum positive plasmid detection of pMD18-T-1.1 and pMD18-T1.2 is 10 4 copies / μL, 10 5 Select pMD18-T-1.1 and pMD18-T1.2 with a concentration of 10 10 The mixed positive plasmids with 10 copies / μL were diluted 10 times in sequence, and the prepared standard positive mixed plasmids were used as positive controls, and negative controls were set up at the same time. 1.5% agarose gel electrophoresis was used for detection. The reaction results were determined by the attached Figure 5 It can be seen that the mixed primers have a higher sensitivity to mixed template amplification, and the minimum positive plasmid detection of pMD18-T-1.1 and pMD18-T-1.2 is 10 4 copies / μL.

[0046] The specific test uses nucleic acid samples such as bovine respiratory syncytial virus, bovine viral diarrhea virus, bovine rotavirus, and bovine coronavirus to perform PCR reactions according to the optimized reaction system and reaction parameters. At the same time, negative and positive controls are set up and tested by 1.5% agarose gel electrophoresis. The reaction results are shown in the attached Figure 6It can be seen that except for the positive control with two target bands, no bands appeared in the other negative controls, indicating that this method has high specificity. pMD18-T-1.1, pMD18-T-1.2, pMD18-T-1.1+pMD18-T-HNa1.2 were used as templates, and three replicates of each type of template were amplified. Negative and positive controls were set up at the same time, and 1.5% agarose gel electrophoresis was used for detection. The reaction results were shown in the attached Figure 7 It can be seen that the amplified band is stable, indicating that this method has good repeatability.

[0047] Example 5: Detection results of viral nucleic acid and clinical samples by dual PCR method

[0048] The established double PCR method was used to amplify the target fragments of 405 bp, 212 bp, and 405 bp + 212 bp using bovine herpes virus type 1.1 DNA, bovine herpes virus type 1.2 DNA, and their mixed DNA as templates, respectively, confirming the accuracy of this method. The results are shown in the attached Figure 8 The 60 clinical nasal swab samples and 10 bovine lung samples tested by this method were all negative.

[0049] The specific embodiments described above are only used to explain the present invention, and are not used to limit the present invention. Obvious changes or modifications derived from the spirit of the present invention are still within the protection scope of the present invention.

Claims

1. A primer set for detecting bovine herpesvirus genotypes 1.1 and 1.2, characterized in that: The primer set comprises the following primers: The nucleotide sequence of primer IBRV-1.1-F is shown in SEQ ID NO: 1; The nucleotide sequence of primer IBRV-1.1-R is shown in SEQ ID NO: 2; The nucleotide sequence of primer IBRV-1.2-F is shown in SEQ ID NO: 3; The nucleotide sequence of primer IBRV-1.2-R is shown in SEQ ID NO:

4.

2. A kit for detecting bovine herpesvirus genotypes 1.1 and 1.2, characterized in that: The kit comprises the following components: The nucleotide sequence of primer IBRV-1.1-F is shown in SEQ ID NO: 1; The nucleotide sequence of primer IBRV-1.1-R is shown in SEQ ID NO: 2; The nucleotide sequence of primer IBRV-1.2-F is shown in SEQ ID NO: 3; The nucleotide sequence of primer IBRV-1.2-R is shown in SEQ ID NO: 4; PCR 10×Ex Taq Bufferr (Mg2+plus), dNTP Mixture, DNA template, ddH2O.

3. The kit for detecting bovine herpesvirus genotypes 1.1 and 1.2 according to claim 2, characterized in that: The PCR amplification conditions of the kit are: pre-denaturation at 95°C for 5 min; 95°C for 20 s, 56°C for 30 s, 72°C for 40 s, for a total of 35 cycles, and finally extension at 72°C for 10 min.

4. A kit for detecting bovine herpesvirus 1.1 and 1.2 genotypes as claimed in claim 2, and its use in distinguishing bovine herpesvirus 1.1 and 1.2 genotypes.

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