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Kit for detecting eastern equine encephalitis virus and west equine encephalitis virus by real-time fluorescence quantitative RT-PCR

A western equine encephalitis, real-time quantitative technology, used in fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of time-consuming experiments and inability to quantify results.

Inactive Publication Date: 2011-02-09
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, laboratory research applications include ordinary multiplex RT-PCR and real-time quantitative RT-PCR, etc. The ordinary RT-PCR method requires gel electrophoresis, which is time-consuming and the results cannot be quantified

Method used

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  • Kit for detecting eastern equine encephalitis virus and west equine encephalitis virus by real-time fluorescence quantitative RT-PCR
  • Kit for detecting eastern equine encephalitis virus and west equine encephalitis virus by real-time fluorescence quantitative RT-PCR
  • Kit for detecting eastern equine encephalitis virus and west equine encephalitis virus by real-time fluorescence quantitative RT-PCR

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Embodiment 1

[0052] Example 1. A kit for detecting eastern equine encephalitis virus and western equine encephalitis virus by real-time fluorescent quantitative RT-PCR and its application

[0053] 1. Design of primers and probes

[0054] Design using SDS2.0 software. The real-time quantitative detection primers and probes of Western Equine Encephalitis Virus (WEEV) are selected from the E3 region of the viral structural protein, and the real-time quantitative detection primers and probes of Eastern Equine Encephalitis Virus (EEEV) are selected from the NSP3 region of the viral non-structural protein, by sequence Comparison analysis showed that the sequence homology with other viruses was less than 50%. The detection primers and probe sequences of WEEV and EEEV are shown in Table 1.

[0055] Table 1 Detection primers and probe sequences of WEEV and EEEV

[0056]

[0057] 2. Establishment and optimization of reaction system

[0058] 1. Sample preparation

[0059] The western equine encephalitis viru...

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Abstract

The invention discloses a kit for detecting eastern equine encephalitis virus and west equine encephalitis virus by real-time fluorescence quantitative RT-PCR. The kit provided by the invention comprises a primer pair as shown in the following (1) and (2): (1) EEEV forward primer: TGTGCGTACCTCCTCATCGTT, EEEV reverse primer: GACTGGCGTGAATCTCTGCT; (2) WEEV forward primer: AGGGATACCCCCGAAGGTT, WEEV reverse primer: GTGAATAGCACACGGGTGGTT. The kit can be applied to detecting EEEV and WEEV and has good sensitivity and specificity; in addition, the lowest EEEV virus titer that the kit can detect is 0.2TCID50 / ml and the WEEV virus titer that the kit can detect is 1TCID50 / ml.

Description

Technical field [0001] The invention relates to a kit for detecting eastern equine encephalitis virus and western equine encephalitis virus in real-time fluorescent quantitative RT-PCR in the field of biotechnology. Background technique [0002] Eastern equine encephalitis virus (EEEV) and western equine encephalitis virus (WEEV) are two viruses with similar antigenicity in the Togavirus family Alphavirus, which are mainly prevalent in the Americas. They are spread by mosquitoes, can infect horses, birds and humans, and can cause severe encephalitis symptoms. The fatality rate of EEEV infection can reach 36%, which is one of the most fatal encephalitis viruses. WEEV is less toxic to adults, but infections in children can cause severe encephalitis, with a mortality rate of about 10%. But they all have serious neurological sequelae. Although there are no reports of Eastern equine encephalitis virus and Western equine encephalitis virus infection in China, once they are imported ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/70C12N15/11G01N21/64
Inventor 康晓平杨银辉李裕昌刘洪林方蔡绪禹户义李靖常国辉祝庆余
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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