Primer combination and GeXP detection method for simultaneously identifying 5 bovine viral dermatitis viruses

A primer combination, a technology for bovine viral diarrhea, applied in the directions of microorganism-based methods, biochemical equipment and methods, and the determination/inspection of microorganisms, which can solve problems such as indistinguishability

Inactive Publication Date: 2016-09-28
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the clinical symptoms of these five viruses are very similar and difficult to distinguish, it is necessa

Method used

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  • Primer combination and GeXP detection method for simultaneously identifying 5 bovine viral dermatitis viruses
  • Primer combination and GeXP detection method for simultaneously identifying 5 bovine viral dermatitis viruses
  • Primer combination and GeXP detection method for simultaneously identifying 5 bovine viral dermatitis viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Embodiment 1, design and preparation of primer combination

[0116] A large number of sequence analyzes and comparisons were carried out to obtain several primers for the identification of five bovine viral dermatitis viruses: FMDV, BTV, VSV, BVDV and IBRV. Preliminary experiments were carried out on each primer to compare performances such as sensitivity and specificity, and finally 5 pairs of specific primers for identifying 5 kinds of bovine viral dermatitis viruses were obtained. Each specific primer pair, forward primer and reverse primer, consists of a targeting segment and a universal primer segment, with the universal primer segment located 5' to the targeting segment.

[0117] The primer pair used to identify FMDV consists of the following two primers (5'→3'):

[0118] FMDV-F (Sequence 1 of the Sequence Listing): AGGTGACACTATAGAATA GCCGTGGGACCATACAGG;

[0119] FMDV-R (Sequence 2 of the Sequence Listing): GTACGACTCACTATAGGGA AAGTGATCTGTAGCTTGGAATCTC.

[0...

Embodiment 2

[0143] Embodiment 2, specificity

[0144] 1. Single template experiment

[0145] 1. Extract the total RNA of the sample to be tested and reverse transcribe it into cDNA. The samples to be tested are: FMDV type O inactivated virus, BTV type 4 inactivated virus, VSV NJ type inactivated virus, BVDV reference strain Oregon CV24 strain (BVDV-1 type), IBRV virus.

[0146] 2. Using the cDNA obtained in step 1 as a template, GeXP multiplex PCR was carried out using the primer combination in Example 1.

[0147] Multiplex PCR reaction system (20 μL): template 1 μL (about 100 ng), Genome Lab GeXP StarterKit 5×buffer 4 μL (the buffer contains universal primers, and the universal primers are primer A shown in sequence 16 of the sequence listing and sequence 17 of the sequence listing The composition of primer B shown, wherein the 5' end of primer A is labeled with CY5 fluorescent group, the working concentration of primer A and primer B are both 0.25μM), MgCl 2 (25 μM) 4 μL, primer mix ...

Embodiment 3

[0162] Example 3, universality

[0163]1. Extract the total RNA of the sample to be tested and reverse transcribe it into cDNA. The samples to be tested are: FMDV type O inactivated virus, FMDV type A inactivated virus, FMDV Asia type I inactivated virus, BTV type 4 inactivated virus, BTV type 8 inactivated virus, BTV type 9 inactivated virus, BTV 15 inactivated virus, BTV 17 inactivated virus, BTV 18 inactivated virus, VSV NJ inactivated virus, VSV IND inactivated virus, BVDV reference strain Oregon CV24 strain (BVDV-1 type), BVDV Reference strain NADL strain (BVDV-1 type), BVDV reference strain Yak strain (BVDV-1 type), BVDV strain GX-BVDV1, BVDV strain GX-BVDV2, BVDV strain GX-BVDV3, BVDV strain GX -BVDV4, BVDV strain GX-BVDV5, BVDV strain GX-BVDV6, BVDV strain GX-BVDV7, BVDV strain GX-BVDV8, BVDV strain GX-BVDV9, BVDV strain GX-BVDV10, BVDV strain GX- BVDV11, BVDV strain GX-BVDV12, BVDV strain GX-BVDV13, BVDV strain GX-041, IBRV virus.

[0164] 2. Using the cDNA obtaine...

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PUM

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Abstract

The invention discloses a primer combination and GeXP detection method for simultaneously identifying 5 bovine viral dermatitis viruses. The primer combination is composed of a primer pair I, a primer pair II, a primer pair III, a primer pair IV and a primer pair V. The invention also discloses a GeXP detection method for simultaneously identifying foot-and-mouth disease virus, bluetongue virus, vesicular stomatitis virus, bovine viral diarrhoea virus and infectious bovine rhinotracheitis virus. The GeXP detection method can simultaneously identify the 5 bovine viral dermatitis viruses. The method has the characteristics of higher flux, higher specificity and higher sensitivity, can be used for monitoring of bovine disease epidemiology and differential diagnosis of unexpected epidemic situations, and ensures the healthy development of cattle raising industry.

Description

technical field [0001] The invention relates to a combination of primers and a GeXP detection method for simultaneously identifying five kinds of bovine viral dermatitis viruses. Background technique [0002] Foot and Mouth Disease Virus (FMDV), Bluetongue Virus (BTV), Vesicular Stomatitis Virus (VSV), Bovine Viral Diarrheal Virus (BVDV), and Bovine Infection Infectious Bovine Rhinotracheitis Virus (IBRV) is the five main viruses that cause bovine viral dermatitis. Cows infected with the virus showed lesions in the mouth, between the mouth and nose, hoof, vulva and udder clinically, with blisters, erythema, cracking, necrosis and ulcers. Bovine foot-and-mouth disease caused by FMDV, bovine vesicular stomatitis caused by VSV and bovine bluetongue caused by BTV are highly acute infectious diseases of cattle, generally showing explosive epidemics and high mortality rates, and are recognized by the World Organization for Animal Health (OIE ) is listed as a Class A infectious d...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 谢芝勋范晴谢志勤邓显文谢丽基黄莉罗思思黄娇玲张艳芳曾婷婷王盛刘加波庞耀珊
Owner GUANGXI VETERINARY RES INST
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