Source
Institute of Virology, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, China. luyiyuzjh@yahoo.com.cn
Abstract
AIMS:
Real-time reverse transcription-polymerase chain reaction (RT-PCR) assay based on a TaqMan-minor groove binder (MGB) probe was developed for the rapid detection of avian influenza virus subtype H5.
METHODS AND RESULTS:
Conserved regions in the haemagglutinin genes of avian influenza viruses subtype H5 served as targets for the primers and TaqMan-MGB probe design. Concentrations of primers and probe were optimized to improve the sensitivity and specificity of the reactions. A plasmid containing the haemagglutinin gene was constructed and in vitro transcribed for a quantitative assay of copy numbers of the target gene. The results revealed that the optimal concentration of primers and probe was 640 and 480 nmol l(-1), respectively. The threshold of 100 copies of target molecules could be detected. The linear range for detection was determined as 10(2) to 10(8) molecules in reaction.
CONCLUSIONS:
It took less than 3 h to complete the detection from viral RNA extraction, with good sensitivity and repeatability.
SIGNIFICANCE AND IMPACT OF THE STUDY:
Real-time RT-PCR assay with MGB probe was an effective means for quick and quantitative laboratory detection and monitoring of H5 avian influenza viruses.