A Novel One-step Real-time RTPCR Assay To Detect The 2009 Pandemic H1N1 Influenza Virus

Objective:Because of the wide spread of pandemic (H1N1) 2009 virus, it is necessary to develop a method that can detect this virus with high sensitivity and specificity. In April 2009, WHO published a protocol which use real time RTPCR to detect pandemic (H1N1) 2009 virus. This protocol has been revised in October 2009. However, when pandemic (H1N1) 2009 virus has already spread in the entire world, the genomic sequences will have changed. The detection limit of the protocol WHO recommended has declined. The task of this thesis is to develop a new real time RTPCR method that has higher sensitivity and specificity to detect pandemic (H1N1) 2009 virus.Method:We extracted the total RNA from clinical samples using QIAamp viral RNA Mini Kit. Then, the protocol WHO recommended detecting pandemic (H1N1) 2009 virus would be used to find positive samples. We propagated the pandemic (H1N1) 2009 virus from positive clinical samples in the allantoic fluid in SPF chicken eggs. The HA gene had been cloned from pandemic (H1N1) 2009 virus. Primers were designed on the basis of sequence information obtained from the Influenza Sequence Database (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html). All available HA,NA and MP sequences of pandemic (H1N1) 2009 virus which were reported in Chinese mainland from the database were aligned each, and five sets of primers and probes were designed specifically targeting the consensus section of these genes of pandemic (H1N1) 2009 virus each by using the beacon designer(version 7.6) program(Premier Biosoft). These five primers and probes sets were used to amplify total RNA extracted from pandemic (H1N1) 2009 virus, and then the best one would be selected. The sensitivity of the novel set would be determined by in vitro transcribed HA gene. The specificity of the novel set would be determined by total RNAs extracted from other types of influenza virus. Finally, the 83 clinical samples would be used to determine the sensitivity of the novel primers and probe set. The effectiveness of the novel primers and probe set would be evaluated compared to the method WHO recommended.Result:Among the five pre-designed primers and probes sets, the HA-3 set exhibit the best amplification resμLt. The detection limit of HA-3 set is 2-3 copies in-vitro transcribed HA gene, which is ten times higher than the primers and probe set WHO recommended. The specificity of HA-3 set is similar with the method WHO recommended. Among the 83 samples tested by the real-time RTPCR WHO recommended,26 were successfully characterized as 2009 pandemic H1N1 influenza virus and the other 57 samples were tested negative. However,32 samples were successfully characterized as 2009 pandemic H1N1 influenza virus by the novel real-time RTPCR method.Conclusion:The novel designed HA-3 primers and probe have great effectiveness in detecting pandemic (H1N1) 2009 virus. They can be used in respiratory specimens and viral cultures.