Rapid detection of H5 avian influenza virus by TaqMan‐MGB real‐time RT‐PCR

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 102 to 108 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.