Development and Application of a Simple Molecular Short RT-PCR Assay for Detection and Differentiation of Influenza A Viruses.

摘要

Influenza A viruses causes annual epidemics resulting in about 3 to 5 million cases of severe illness, and about 250,000 to 500,000 deaths, worldwide. They also have the potential to cause pandemics as occurred in 1918, 1957, 1968 and 2009.;Conventional methods for laboratory identification of human influenza virus infections are commonly performed using immunoassays, viral neuraminidase activity; virus isolation in cell culture; or detection of influenza-specific RNA e.g., by reverse transcriptase polymerase chain reaction (RT-PCR). These tests differ in their sensitivity and specificity in detecting influenza viruses as well as in their commercial availability, the amount of time needed from specimen collection until results are available, and the tests' ability to distinguish between different influenza virus types (A versus B) and influenza A subtypes (e.g., 2009 H1N1pdm versus seasonal H1N1 versus seasonal H3N2).;Early detection and differentiation of Influenza A viruses in clinical specimens will prevent further spread and allow early intervention of the illness. Hence, a simple molecular RT-PCR assay was developed based on the influenza A virus surface glycoproteins Hemagglutinin (HA) and Neuraminidase (NA) to rapidly detect and differentiate H3N2 and H1N1pdm viruses in clinical samples. Primers were designed based on the HA and NA of the H3N2 and H1N1pdm subtype specific conserved regions. Initially, we applied this assay to wild type (wt) viruses and the high yield (hy) reassortants available in our laboratory. The assay was highly specific and sensitive in differentiating HA and NA of H3N2 and H1N1pdm viruses. Further the assay was used to detect H3N2 and H1N1pdm viruses from the clinical samples received from the clinical virology laboratory at Westchester Medical Center. Although the assay was very sensitive with wild type and reassortants, the sensitivity of the assay was very low with clinical specimens. Hence, we are planning to improve the sensitivity and specificity of the assay by applying the primers to the real time RT-PCR technique.