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High-sensitivity BHV-2 (bovine herpes virus 2) quantitative real-time PCR (polymerase chain reaction) detection method and kit

A real-time fluorescence quantitative, BHV-2-P technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. High-accuracy detection requirements, the sensitivity is not as good as real-time quantitative PCR and other problems, to achieve the effect of high accuracy, low cross-hybridization interference, and improved repeatability

Inactive Publication Date: 2017-03-22
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, nested PCR is far less sensitive than real-time fluorescent quantitative PCR technology
[0008] On the one hand, real-time fluorescent quantitative PCR or nested PCR technology has not been used in the current detection methods for BHV-2; High sensitivity, high specificity, high accuracy detection requirements

Method used

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  • High-sensitivity BHV-2 (bovine herpes virus 2) quantitative real-time PCR (polymerase chain reaction) detection method and kit
  • High-sensitivity BHV-2 (bovine herpes virus 2) quantitative real-time PCR (polymerase chain reaction) detection method and kit
  • High-sensitivity BHV-2 (bovine herpes virus 2) quantitative real-time PCR (polymerase chain reaction) detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1 A kind of kit for detecting bovine herpes virus type 2

[0061] The kit includes:

[0062] (1) Primer BHV-2-F1, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0063] (2) Primer BHV-2-R1, the nucleotide sequence of which is shown in SEQ ID NO: 2;

[0064] (3) Primer BHV-2-F2, the nucleotide sequence of which is shown in SEQ ID NO: 3;

[0065] (4) Primer BHV-2-R2, the nucleotide sequence of which is shown in SEQ ID NO: 4;

[0066] (5) Probe BHV-2-P, the nucleotide sequence of which is shown in SEQ ID NO: 5, the 5' end of the nucleotide sequence is connected to the fluorescent group FAM, and the 3' end is connected to the quenching group BHQ1;

[0067] (6) Positive quality control product: containing the genomic DNA of bovine herpesvirus type 2 as a PCR template, with primers BHV-2-F1 and BHV-2-R1 as the plasmid of the DNA fragment amplified by PCR primers, the plasmid concentration is 1.0×10 8 copy / μL;

[0068] (7) Negative quality control...

Embodiment 2

[0069] Embodiment 2 A kind of method for detecting bovine herpes virus type 2

[0070] The detection kit used is the kit described in Embodiment 1 of the present invention.

[0071] Samples to be tested: 30 bovine blood or skin tissues from different sources, known to be infected with BHV-2, collected from the Animal Disease Prevention and Control Center of Anlu City, Hubei Province, sample numbers 1-30.

[0072] (1) Extract viral DNA from the samples to be tested: use the viral genomic DNA / RNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd., article number is DP315) to extract viral genomic DNA from 30 samples as the DNA to be tested. For the extraction method, please refer to the instruction manual of the extraction kit;

[0073] (2) Preparation of positive quality control products:

[0074] 1) With the genomic DNA of BHV-2 as PCR template, carry out PCR amplification with primer BHV-2-F1-1 and BHV-2-R1-1 as PCR primers:

[0075] The PCR system is ...

experiment example 1

[0102] Experimental Example 1 Sensitivity Experiment

[0103] The positive quality control product described in the present invention was diluted to 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 , 1.0×10 3 , 1.0×10 2 , 1.0×10 1 Copy / mL, as the sample to be detected, the sensitivity experiment was carried out on the detection method of Example 2, and the sensitivity was characterized by the lowest detection concentration. figure 1 For the sensitivity test results, according to figure 1 It can be seen that the plasmid copy number is 10 8 ~10 2 In the copy / mL range, there are curves with obvious exponential growth period, and the plasmid copy number is 10 2 copies / mL, there is still a fluorescent signal through amplification, so the detection sensitivity of the method provided by the invention is: 1.0×10 2 copies / mL.

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Abstract

The invention provides a kit and a method for detecting BHV-2 (bovine herpes virus 2) and particularly discloses a high-sensitivity BHV-2 quantitative real-time PCR (polymerase chain reaction) detection method and a kit. The kit comprises a primer group and a fluorescent probe for detecting the BHV-2; the primer group comprises four primers for nested amplification of specific DNA fragments of the BHV-2. With the adoption of the detection method, nested amplification and quantitative real-time PCR can be completed in one tube through one-step amplification by the aid of the four primers and the fluorescent probe. With the adoption of the BHV-2 quantitative real-time PCR detection method and the kit with high sensitivity, specificity and accuracy, quick detection of the BHV-2 can be realized, the kit is very convenient to apply, and on the basis of the advantages, the kit is suitable to be popularized and applied in livestock breeding units, animal inspection and quarantine departments and the like and has wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of biological detection and relates to a BHV-2 detection method and kit, in particular to a high-sensitivity BHV-2 real-time fluorescent quantitative PCR detection kit and method. Background technique [0002] Bovine herpesvirus type 2 (BHV-2) causes teat ulcers or necrotic lesions, and infection of dairy cows with this virus can lead to reduced milk production and can lead to secondary mastitis and even culling of infected cows. Infection with this virus can be difficult to diagnose at present, especially when secondary bacterial infection alters the appearance of the lesion; even in areas where disease occurs rarely, it can be confused with other viral infections of the teats. In the past two decades, many countries have isolated strains of the virus one after another. With the increasing demand for dairy products and the increasing requirements for the quality of dairy products, we need to pay more atten...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6848C12Q1/705C12Q2549/119C12Q2547/101C12Q2561/101C12Q2563/107
Inventor 刘贵明疏义林王绪敏王丽
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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