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 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.

Figure 1

Specific evaluation of TaqMan‐MGB method for the detection of H5 avian influenza virus. The line of H5N1 amplification was indicated (A). The other lines represented the amplifications using nucleic acids from respiratory viruses (B), including avian influenza H7 and H9 viruses, influenza viruses A1 (H1N1), A3 (H3N2) and B, SARS‐CoV and RSV. The control reaction using DEPC‐treated water as template was also included.

Figure 2

Amplification profile of detection for the HA nucleic acid of H5 avian influenza virus. From left to right, the amplification contained 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10² and 10¹ copies of in vitro transcribed RNA, respectively.

Figure 3

Standard curve created by the analysis of known copies of target nucleic acid. Reactions with copy numbers of target gene from 10² to 10⁸ were used for creating the standard curve. The standard curve was plotted by the log concentration of copy numbers against C _t values.

Figure 4

Reproducibility assay for the detection of HA gene using real‐time PCR. Each concentration of positive amplification contained five repeats. From left to right, 10⁷, 10⁶ and 10⁵ copies of target molecules were used to estimate the reproducibility.

Figure 5

Detection of avian influenza virus H5N1 from a clinical sample using TaqMan‐MGB real‐time RT‐PCR. Sample A was positive control; sample B represented the amplification from the clinical specimen.