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

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 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.