Primer composition, kit and method for detecting vesicular stomatitis Indiana viruses and vesicular stomatitis New Jersey viruses

A technology of vesicular stomatitis and primer composition, which is applied in the field of primer composition for detecting vesicular stomatitis virus Indiana and New Jersey types, can solve the problems of large electrophoresis pollution, complex instruments, and low sensitivity, and achieve detection sensitivity Improvement, accurate results, and the effect of reducing pollution

Active Publication Date: 2020-03-13
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing multiple LAMP methods in China have certain limitations, and it is impossible to determine which positive reaction caused the result, which is not a differential diagnosis in the true sense.
Qunyi Hua et al. in "Real-time Fluorescent Quantitative TaqMan RT-PCR Differential Detection of Vesicular Stomatitis Indiana and New Jersey Viruses" (Proceedings of the 20th Anniversary Celebration and the 10th Academic Symposium of the Livestock Infectious Diseases Branch of the Chinese Society of Animal Husbandry and Veterinary Medicine , p1312-1318) although a kind of differential detection method is disclosed in the article, but the sensitivity of this detection method is low, the instrument used is also complicated, and it can only distinguish VSV, can not distinguish two types
Fan Qing et al. disclosed a differential detection method in the article "Double fluorescent RT-LAMP method for differential detection of foot-and-mouth disease virus and vesicular stomatitis virus" (Journal of Animal Husbandry and Veterinary Medicine, 2018, 49 (5): 996-1004), but The sensitivity of this method is low, and the two types of vesicular stomatitis virus cannot be distinguished. After the reaction, electrophoresis is required to judge the result. Electrophoresis pollutes a lot, so it can only be carried out in the laboratory, and quarantine cannot be carried out at the grassroots level.

Method used

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  • Primer composition, kit and method for detecting vesicular stomatitis Indiana viruses and vesicular stomatitis New Jersey viruses
  • Primer composition, kit and method for detecting vesicular stomatitis Indiana viruses and vesicular stomatitis New Jersey viruses
  • Primer composition, kit and method for detecting vesicular stomatitis Indiana viruses and vesicular stomatitis New Jersey viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, the design of primer composition

[0028] Download the registered VSV-NJ and VSV-IND NS genes (phosphoprotein) on Genebank, use MEGA5.0 for comparative analysis, and use primer 5.0 and Primer explore V5 to design 2 sets of LAMP-specific primers to form a primer composition. Each set includes 4 primers and 1 probe: outer primers F3 and B3, inner primers FIP (FIP=F1c+F2) and BIP (BIP=B1c+B2)), probe Probe. The probe Probe is designed between F1c and B1c, and the two probe Probes are respectively labeled with different fluorescent groups: IND-Probe is labeled with CY 5.5 at the 5' end of the nucleotide sequence shown in SEQ ID No: 5 in the sequence listing Fluorescence, the 3' end is labeled with the BHQ3 quencher group, and it is bright red at a wavelength of 694nm; NJ-Probe is labeled with FAM fluorescence at the 5' end of the nucleotide sequence shown in SEQ ID No: 10 in the sequence listing, and the 3' end Labeled BHQ1 quencher, yellow-green at 520nm wav...

Embodiment 2

[0031] Example 2, Verification test of vesicular stomatitis virus Indiana type and New Jersey type dual fluorescence RT-LAMP detection method

[0032] 1. Extraction of RNA / DNA template

[0033] Refer to the instructions of EasyPure RNA Kid, a full-type gold RNA extraction kit, to extract the RNA of the virus strains shown in Table 1, and store the RNA template at -70°C for later use.

[0034] 2. Dual fluorescent RT-LAMP reaction system and reaction conditions

[0035] Using VSV-NJ and VSV-IND RNA as experimental templates, the primer concentration, BstDNA 3.0 polymerase, reaction temperature and reaction time in the reaction system were optimized respectively. The optimal reaction system for dual fluorescence RT-LAMP is: 1 μL RNA template, 2×buffer 12.5 μL (composition is 40mM Tris–HCl, 20mM KCl, 16mM MgSO 4 , 20mM (NH 4 ) 2 SO 4 , 0.2% (v / v) Tween 20, 1.6M betaine, and 2.8mM dNTPs. ), Bst DNA 3.0 polymerase 15U, AMV reverse transcriptase 20U, inner primers (IND-FIP, IND...

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PUM

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Abstract

The invention discloses a primer composition, a kit and a method for detecting vesicular stomatitis Indiana viruses and vesicular stomatitis New Jersey viruses. The primer composition designed and screened in the invention comprises two groups of specific primers and probes. Under the condition of an optimized reaction system and under optimized reaction conditions, vesicular stomatitis Indiana viruses and vesicular stomatitis New Jersey viruses can be simultaneously identified and detected in the same reaction tube; and the primer composition, the kit and the method have the characteristics of good specificity, high sensitivity, interference resistance, no pollution, convenience and quickness in operation, low cost and the like.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a primer composition, kit and method for detecting vesicular stomatitis virus Indiana type and New Jersey type Background technique [0002] Vesicular stomatitis is a highly contagious disease of various mammals caused by Vesicular Stomatitis Virus (VSV) belonging to Rhabdoviridae family Vesiculovirus. VSV can naturally infect cattle, pigs, horses and other mammals. In addition, insects and plants are also potential reservoirs of VSV, and arthropod vectors can also transmit VSV. The disease can cause blisters and ulcers on the tongue, mouth, hoof and udder of infected animals, and cause the affected animals to lose productivity, and it is difficult to distinguish it from foot-and-mouth disease in clinical symptoms. It has been listed as a statutory report by the World Health Organization (OIE) The animal disease, which is an exotic disease in my country, is listed as a se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2600/16C12Q2521/101C12Q2531/119C12Q2563/107C12Q2537/143
Inventor 谢芝勋范晴谢志勤谢丽基熊文婕黄娇玲张艳芳曾婷婷王盛罗思思邓显文刘加波
Owner GUANGXI VETERINARY RES INST
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