ATUL KUMAR PATERIYA, SHANMUGA NAGARAJAN, REKHA KHANDIA, SANDEEP BHATIA, HARSHAD MURUGKAR, MANOJ KUMAR AND FATEH SINGH
Abstract
The development of molecular diagnostic for avian influenza virus (AIV) is a continuous process due to evolution of virus after each passing outbreak. The molecular tests need to be reviewed after each outbreak for obtaining consistent laboratory results. In the present study, the oligos sequences of validated H5 Eurasian taqman RT-qPCR for H5 subtyping have been improved matching to the currently circulating strains for laboratory diagnosis of AIV. All available influenza H5 hemagglutinin gene sequences of Eurasian strains from the public databases were aligned in-silico with the recommended RT-qPCR oligos and modifications were done according to the matching sequences of the recently evolved strains. The modified oligos were validated with clinical samples and reference AI H5N1 isolates belonging to different clades. Multiple sequence alignment suggested the modifications at two, three and one positions respectively in probe, forward and reverse primers. Out of 1295 clinical samples tested, 251 amples were found positive. Assay could identify all the 115 virus strains of AI H5 subtype belonging to clade 2.2, 2.2.2, 2.3.2.1C, 2.3.2.1a and 2.3.4.4b. On comparison to the virus isolation, the positive and negative agreement was found to be 0.925 ± 0.025 and 0.983 ± 0.002 respectively. Analytical specificity of the modified assay was confirmed specifically with AI H5 subtype only whereas analytical sensitivity was found to be 13-130 copies of IVT-RNA and 0.1 EID50 of AI H5N1 virus. We propose that the modified RT-qPCR assay can be a choice for routine diagnosis of Eurasian strains of AI H5 subtypes.