Development and application of dual real time RT-PCR for avian influenza H5N6 virus
10.3760/cma.j.issn.1003-9279.2017.01.013
- VernacularTitle: H5N6禽流感病毒双重实时荧光RT-PCR的建立与应用
- Author:
Hanqing TAN
1
;
Jieping CHENG
;
Yingmei ZHU
;
Haifang TAN
;
Qiang HUANG
;
Lebin SU
;
Feng LIN
;
Tingguo DENG
;
Bijian LI
Author Information
1. Zhaoqing Center for Disease Control and Prevention, Zhaoqing 526060, China
- Publication Type:Journal Article
- Keywords:
Avian influenza H5N6 virus;
Real-time RT-PCR;
TaqMan-MGB probe;
Hemagglutinin gene;
Neuraminidase gene
- From:
Chinese Journal of Experimental and Clinical Virology
2017;31(1):62-65
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a TaqMan-MGB probe-based real-time fluorescence RT-PCR assay for avian influenza H5N6 virus used in rapid diagnosis for suspected cases and surveillance for outer environment of live poultry markets.
Methods:Based on the conservative sequences of avian influenza H5N6 virus for HA and NA gene published on GenBank, specific primers and TaqMan-MGB probes were designed to develop and optimize for the dual real-time RT-PCR assay. Specificity, sensitivity, repeatability and comparison tests were carried out.
Results:This dual real-time RT-PCR detection can be completed within 80 minutes. There was no cross-reaction with other subtypes of influenza virus and common respiratory pathogens. The minimum detection limit could be up to 10 copies/reaction. The correlation coefficient of standard curve for the gene of H5 and N6 were 0.999 and 0.993, and the coefficients of variation for cycle threshold were range from 0.151%-0.549%and 0.213%-0.575%, respectively. The positive and negative coincidence rates of the validation test were 100%.
Conclusions:This TaqMan-MGB probe-based dual real-time RT-PCR for avian influenza H5N6 virus was rapid, specific and sensitive. It will have a good use in early emergency detection of suspected cases and continuous monitoring of external environment in live poultry trade market.
