Comparative Analysis of Molecular Methods for Detection of Influenza Viruses

Aims: Influenza is a serious threat to human population worldwide therefore continuous surveillance is required to update influenza seasonal vaccines. A rapid, sensitive, specific and cost effective diagnostic method will be much helpful for patient management in the present scenario. Present study is conceptualized for detection of influenza viruses by molecular methods and compare with ‘gold standard’ virus isolation.

Study Design: Standard strains of Influenza virus were used to standardize the molecular diagnostic assays and results were then compared with virus isolation.

Place and Duration of Study: Centre for Biotechnology, Maharshi Dayanand University, Rohtak, Haryana, India, between December 2015 and April 2016.

Methodology: Standard strains of Influenza A and B virus were used for influenza virus isolation using virus culturing in MDCK (Madin-Darby Canine Kidney) cell line by following standard tissue culture procedure. Isolated viruses were detected by Hemagglutination assay (HA) and typed by Hemagglutination inhibition assay (HI). Conventional one step RT-PCR, Taqman real time RT-PCR and RT-LAMP (Reverse transcription loop mediated isothermal amplification) were standardized on RNA extracted from standard strains. Sensitivity and specificity of these molecular methods were compared with each other as well as with virus culture (gold standard).

Results: Both influenza A and B virus strains were cultured in MDCK cells and produced cytopathic effect during virus culture. Conventional RT-PCR and real time RT-PCR detected both type of Influenza viruses. RT-LAMP also successfully detected and typed influenza viruses. RT-LAMP proved to be more rapid than other two molecular assays.

Conclusion: Molecular diagnostic methods are useful in detection and typing of Influenza viruses and these methods provide results in short period of time when compared with traditional virus culture methods. RT-LAMP is rapid, sensitive, specific and cost effective method for influenza virus detection and subtyping.