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Reagent and method for identifying rabies virus vaccine strain and wild type virus strain

A technology of rabies virus vaccine and wild-type virus, applied in the field of reagents for identifying rabies virus vaccine strains and wild-type strains, can solve the risk of rabies virus vaccine use, inability to distinguish wild-type virus strains and vaccine strains, and vaccine safety evaluation Insufficient application of clinical diagnosis and other problems, to achieve high specificity and sensitivity, good sensitivity effect

Inactive Publication Date: 2018-04-20
谭理琦
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods cannot distinguish between wild-type strains and vaccine strains, especially the lack of identification methods for rabies virus vaccine strains, and the application of vaccine safety evaluation and clinical diagnosis is insufficient, resulting in certain risks in the use of rabies virus vaccines

Method used

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  • Reagent and method for identifying rabies virus vaccine strain and wild type virus strain
  • Reagent and method for identifying rabies virus vaccine strain and wild type virus strain
  • Reagent and method for identifying rabies virus vaccine strain and wild type virus strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: reagent of the present invention

[0042] RVGF (upstream primer): TGACCACMAAGTCCGTGAGTTT

[0043] RVGF (downstream primer): AGCATCAGCCTCCATCAAGGT

[0044] RVGVaP (vaccine strain probe): HEX-GGAAAAGCATATACCATATTC–MGB

[0045] RVGViP (wild-type strain probe): FAM-GGAAAGGCGTATACCATATT-MGB

Embodiment 2

[0046] Embodiment 2: reagent specificity test of the present invention

[0047] Materials: rabies virus CVS-11 vaccine strain, JX-08-45 vaccine strain and BD06 wild strain, canine distemper virus nucleic acid, canine coronavirus nucleic acid, canine rotavirus nucleic acid and canine parvovirus nucleic acid, throat swabs of healthy dogs , set a blank water control. The primers and probes used the reagents in Example 1, which were synthesized by Shanghai Sangon Biotechnology Co., Ltd., and other reagents were biologically pure reagents.

[0048] Nucleic acid extraction: Take 200 μl of culture or tissue homogenate after centrifugation at 2000g, and use QIAGEN's RNeasy mini kit to extract RNA from various materials according to the product instructions.

[0049] Reaction system and reaction conditions: amplification using One Step PrimeScript TM RT-PCR Kit (Perfect Real Time) (TaKaRa) kit is used for fluorescence amplification test in 20μl reaction system. First, add 10μl of 2×O...

Embodiment 3

[0054] Embodiment 3: Reagent sensitivity test of the present invention

[0055] Preparation of rabies virus template by in vitro transcription:

[0056] Rabies virus BD06 wild strain RNA was extracted, RT-PCR amplification was performed with primers TGACCACMAAGTCCGTGAGTTT (SEQ ID NO: 1) and AGCATCAGCCTCCATCAAGGT (SEQ ID NO: 2), and the PCR product was purified and connected to the pGEM-T vector, After the direction of the recombinant plasmid was identified, the extracted plasmid was cut into linear lines with restriction enzymes, and then transcribed in vitro according to the instructions of Invitro Transcription T7Kit (Takara). The product was digested with DNase I, precipitated with 3M sodium acetate ethanol, and dissolved in In the water, calculate the corresponding copy number after measuring the concentration, sequentially from 10 6 copies / μl was diluted to 10 by 10 times 0 copy / μl, test according to the reaction system and reaction conditions in Example 2, to test the ...

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Abstract

The invention relates to the technical field of microbiological detection of veterinary pathogens and discloses a reagent and method for identifying a rabies virus vaccine strain and a wild type virusstrain. The reagent disclosed by the invention consists of an upstream primer of which the nucleotide sequence is shown as SEQ ID NO:1, a downstream primer of which the nucleotide sequence is shown as SEQ ID NO:2, a rabies virus vaccine strain probe of which the nucleotide sequence is shown as SEQ ID NO:3 and is modified with a fluorescent group 1 and a quenching group, and a rabies virus wild type virus strain of which the nucleotide sequence is shown as SEQ ID NO:4, and is modified with a fluorescent group 2 and a quenching group. The invention provides one group of primers and probe reagents with higher specificity and sensitivity; by means of the reagent, the aims of accurately identifying the rabies virus vaccine strain and the wild type virus strain are achieved, and the defects that the wild virus strain is difficulty distinguished from the vaccine strain by existing general methods such as virus separation, serological tests and an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method are made up; the reagent and the method can be comprehensively applied to safety evaluation and clinical diagnosis of a rabies virus vaccine.

Description

technical field [0001] The invention relates to the technical field of detection of veterinary pathogenic microorganisms, in particular to a reagent and method for identifying rabies virus vaccine strains and wild-type strains. Background technique [0002] Rabies virus (RV) belongs to the genus Lyssavirus of the family Rhabdoviridae. The shape is elastic, the nucleocapsid is helically symmetrical, the surface has an envelope, and it contains single-stranded RNA, which is the pathogen that causes rabies. [0003] Rabies virus G protein is the only viral structural protein that can induce the body to produce neutralizing antibodies for humoral immunity. Previous studies have identified several spatial and linear epitopes of G protein. The G gene encodes 524 amino acids (AA). At the amino terminal of the peptide chain, the first 19 residues are the signal peptide. During the maturation process, the signal peptide is excised to become a mature G protein containing 505 AA. It ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2561/113C12Q2563/107C12Q2545/114C12Q2521/107
Inventor 谭理琦郑晓聪秦智锋李汶松蔡良语卢奕良王津津孙洁林彦星马岚钟松清刘建利刘荭兰文升
Owner 谭理琦