Development And Preliminary Application Of New Technology For The Detection Of Influenza Virus

Objective:To develop and evaluate two new multiplex RT-PCR assays for simultaneous detection of pandemic influenza A H1N1, seasonal influenza A H1N1 and H3N2 virus;To establish and evaluate one rapid and sensitive colorimetric reverse transcription loop mediated isothermal amplification (RT-LAMP) method to detect pandemic influenza A H1N virus.Methods:â‘ Multiplex RT-PCR for simultaneous detection of pandemic influenza A H1N1,seasonal influenza A H1N1 and H3N2 virus with a GeXP system.1) Eight pairs of specific primers were designed based on the conserved sequences of all influenza A virus, all influenza A H1N1 virus, seasonal influenza A H1N1 and H3N2 virus, pandemic influenza A H1N1 virus and the internal control-human RNase P by GeXP eXpress Profiler.2) To develop and evaluate multiplex RT-PCR assay with different influenza viruses and throat swab samplesâ‘¡Multiplex rRT-PCR assay for simultaneous detection of pandemic influenza A H1N1,seasonal influenza A H1N1 and H3N2 virus with CFX96 real-time system.1) Four pairs of specific primers targeting the conserved sequences of pandemic influenza A H1N1 virus,seasonal influenza A H1N1 and H3N2 virus and the internal control-human RNase P were designed by Primer Premier5.2) To develop and evaluate multiplex rRT-PCR assay by CFX96 system with different influenza viruses and throat swab samples.â‘¢RT-LAMP method for detection of pandemic influenza A H1N1.1) Three pairs of primers for RT-LAMP were designed based on the conserved sequences of pandemic influenza A H1N1 virus by Primer Explore 4.0.2) To develop and evaluate RT-LAMP method with different influenza viruses and throat swab samples.Results:â‘ The assays for different human influenza virus and serial dilutions of RNA from in vitro transcription of HA gene of pandemic Influenza A H1N1 virus indicated that GeXP method was highly specific with a detection limit (10 copies RNA) superior to currently accepted assays. Twenty nine throat swab samples from patients with influenza symptoms were analyzed. Pandemic influenza A H1N1 virus was detected in 28 samples (97%) by GeXP method compared with only in 22 samples (76%) by rRT-PCR recommended by WHO. Six inconsistent specimens were further confirmed positive using revised rRT-PCR developed by National Influenza Center.â‘¡The sensitivity of multiplex rRT-PCR for detection of pandemic Influenza A H1N1 virus was 20 copies of RNA and each primer set showed no cross reaction with other viruses.This multiplex rRT-PCR assay was validated with 29 throat swab samples..The positive rate(93%) using multiplex rRT-PCR was higher than that of rRT-PCR (76%) recommended by WHO. Five inconsistent specimens were further confirmed positive using revised rRT-PCR developed by National Influenza Center.â‘¢The assay for serial dilutions of RNA from in vitro transcription of the HA gene of pandemic Influenza A H1N1 virus and different influenza virus showed that the sensitivity of RT-LAMP assay was 60 copies of RNA with high specificity. The assay was validated with 29 throat swab samples.The detection results of 22 specimens agreed with those obtained using rRT-PCR recommended by WHO.Four inconsistent samples were further confirmed positive using revised rRT-PCR developed by National Influenza Center.Conclusion:We successfully developed two new multiplex RT-PCR assays for simultaneous detection of pandemic influenza A H1N1, seasonal influenza A H1N1 and H3N2 and one rapid and sensitive RT-LAMP method to detect pandemic influenza A H1N1 virus.