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SYBR Green I Real-time PCR method for quickly detecting and indentifying Hantaan virus infection

A fast technology for Hantaan virus, applied in the field of molecular biology, virology, immunology and immune application, SYBR GreenⅠReal-time PCR, can solve the problem of insufficient specificity and sensitivity, unsuitable rapid on-site detection and separation Time-consuming and other problems of the identification method, to achieve the effect of simple and fast operation, good detection effect and strong contrast

Inactive Publication Date: 2013-12-11
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the classic separation and identification method is time-consuming, and the IFA method is not suitable for rapid on-site detection. The detection of HTNV antibody is only an indirect indicator of one side, and there are problems such as insufficient specificity and sensitivity, and inaccurate quantification.

Method used

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  • SYBR Green I Real-time PCR method for quickly detecting and indentifying Hantaan virus infection
  • SYBR Green I Real-time PCR method for quickly detecting and indentifying Hantaan virus infection
  • SYBR Green I Real-time PCR method for quickly detecting and indentifying Hantaan virus infection

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Embodiment Construction

[0030] 1. Design and synthesize a pair of real-time fluorescent quantitative PCR primers for Hantaan virus nucleic acid, provided by Beijing Aokeding

[0031] Sheng Biotechnology Co., Ltd. (Address: B206, No. 7, Fengxian Middle Road, Yongfeng Industrial Base, Haidian District, Beijing, Zip Code: 10094).

[0032] According to the S gene sequence of HTNV 76-118 strain (the accession number in GenBank is M14626.1), a pair of real-time fluorescent quantitative PCR primers for Hantaan virus nucleic acid were designed by using Oligo6.0 software and NCBI Blast analysis (see Table 1); Named respectively: upstream primer HTNV-F-13; downstream primer HTNV-R-13, the length of the amplified fragment is 144 bp.

[0033] Table 1. Primers designed for Hantaan virus

[0034]

[0035] The Hantaan virus nucleic acid is amplified by the above primers to accurately quantify the virus copy number in the sample. Total RNA from Vero E6 cells infected with HTNV 76-118 was extracted and reve...

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Abstract

The invention discloses an SYBR Green I Real-time PCR (Real-time fluorescent quantitative polymerase chain reaction) method for quickly detecting and indentifying Hantaan virus infection. The method comprises the following steps: designing a pair of specific primers mainly according to a conserved sequence of an S gene in a GenBank database; using a positive plasmid subjected to 10-time gradient dilution as a standard product; optimizing a reaction condition; establishing the SYBR Green I Real-time PCR method used for detecting a Vero E6 cell, a mouse and a clinical patient, which are infected by Hantaan virus nucleic acids according to qRT-PCR specificity amplification, wherein a copy number of the detection sensitivity can reach 101, and sensitivity, repeatability and specificity of the Hantaan virus nucleic acids are detected. Compared with a conventional Hantaan virus detection method, the method has the advantages that types of detecting samples are rich, the application range is wide, the contrast is strong, operation steps are relatively few, convenience and quickness are realized, the sensitivity and the repeatability are high, the specificity is good, and a linear relationship is good, and can provide a strong experimental basis on clinical and laboratory detection of hemorrhagic fever with renal syndrome as well as studying of epidemiology.

Description

technical field [0001] The invention belongs to the field of virus detection and relates to related fields such as molecular biology, virology, immunology and immune application. The invention specifically relates to a SYBR GreenⅠReal-time PCR method for rapid detection and identification of Hantaan virus infection. The samples for detection and identification of Hantaan virus nucleic acid include infection of Vero E6 cells, infection of experimental mice and clinical patients. It includes designing a pair of primers for specifically amplifying Hantaan virus, preparing standards, researching and optimizing experimental conditions, and performing qRT-PCR specificity on total RNA extracted from infected Vero E6 cells, infected experimental mice and clinical patient serum Amplification detection. Background technique [0002] Research status of diagnosis and detection of hemorrhagic fever with renal syndrome: [0003] Hantaan virus belongs to the genus Hantavirus in the Buny...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 吴兴安刘梓谕王芳张晓晓张芳琳程林峰袁利娟徐志凯
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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