Abstract:

Objective To establish a real-time fluorescence quantitative reverse transcription-polymerase chain reaction（RT-PCR） for influenza A H1N1 virus RNA,and to investigate its clinical application value. Methods Specific primers and TaqMan probes were designed according to nucleotide sequences of HA and NA genes of influenza A H1N1 virus and nucleotide sequences in GenBank database. The system and conditions in the experimental reaction were improved,and the RT-PCR was established for influenza A H1N1 virus RNA. Its sensitivity,specificity and repeatability were evaluated. Totally,21 samples were collected,all of which were nasopharyngeal swab samples. A comparative study was conducted with chicken embryo culture method.Results The results of RT-PCR were different among different viruses. The RNA of influenza A H1N1 virus was positive,and the RNA of H1-H16 influenza viruses,seasonal influenza virus and swine influenza virus was negative. The sensitivity was 103 copies/μL RNA molecule,and the standard curver ² was 0.998. The cycle threshold（Ct） value within-run and between-run coefficients of variation（CV） of the positive RNA standard were <10%,and the Ct value of the negative RNA standard [diethyl pyrocarbonate（DEPC）] was <0,and the repeatability was good. In the 21 samples,only 2 samples were positive,and the remaining samples were all negative. Compared with chicken embryo culture method,the consistency was 100%. Conclusions The established RT-PCR for influenza A H1N1 virus RNA is simple,time consuming,specific and sensitive,which can be widely used in clinical practice.

Key words: Influenza A H1N1 virus, Real-time fluorescence quantitative reverse transcription-polymerase chain reaction, Determination method, Clinical application