Study On Internal Control Of Influenza A (h1n1) Virus Inspection By Real-time Rt-pcr

ObjectiveThe study is establish internal control for real-time RT-PCR of the influenza A (H1N1) virus inspection.Methods:Envelope protein HA and NA gene segments of the influenza A (H1N1) virus were synthesized and construct to vector pCMV to obtain the envelope protein expression plasmid pCMV-HA and pVRC-NA; Secondly, four sets of primers and probes were designed basing on M, HA, NA and NP gene segments, respectively, which was constructed to the internal control (IC) gene and inserted to the reporter gene EGFP, obtaining the pEGFP-IC; thirdly, the envelope protein expression plasmids, reporter plasmid and package plasmid were co-transfected to 293T cell line to produce pseudotyped particle with IC gene.Result:Four sets of primers and probe for M, HA, NA and NP gene segments, were examinated in virus inspection by real-time RT-PCR, CT value of which were 12.32,12.67,12.73 and 12.94, respectively. IC pseudotyped particle could infect A549 cells with expression the EGFP intracellularly. The morphism of IC pseudotyped particle was observed by transmission electron microscopy. The primer and probe for HA gene segment was used to inspect 10-fold serial diluted RNA of IC pseudotyped particle, of which CT value for the non-diluted is CT 11.3+0.15, for the 10-fold diluted is CT 15.61±0.17, for the 100-fold diluted is CT 19.16+0.02, and for the 1000-fold diluted is CT 24.4+0.19.Conclusion:Internal control for real-time RT-PCR of the influenza A (H1N1) virus inspection was successfully established and confirmed its availability by real-time RT-PCR.