Establishment of reverse transcriptase Loop-mediated isothermal amplification technique for detection of type A H1N1 influenza virus

Abstract

Objective To establish a rapid, simple and visualized nucleotide detection technique for type A influenza H1N1 virus using reverse transcriptase loop-mediated isothermal amplification(RT-LAMP) Methods Two pairs of RT-LAMP primers were designed in accordance with the HA gene of type A H1N1 influenza virus. Then, the specificity of the primers was evaluated by detection of different viruses, and the sensitivity was confirmed by testing multiple proportional diluent clinical samples. Hydroxynaphthol blue(HNB) was used for visualized detection, at the same time, gel electrophoresis was used for verification. At last, clinical samples were detected by RT-LAMP, the consistency of RT-LAMP and TaqMan PCR methods was compared by chi-square test. Results The 4 primers of RT-LAMP have good specificity, which can identify type A H1N1 influenza virus accurately. The sensitivity of RT-LAMP was 2.5×103copies/ml by detection of diluent samples. The products of RT-LAMP could be detected by HNB visually, and the results is consistent with gel electrophoresis methods. After evaluation of statistical difference of RT-LAMP and TaqMan PCR, we find no significant difference with the two methods(P>0.05). Conclusion Our study has realized visually detection of type A H1N1 influenza virus by RT-LAMP with better sensitivity, which has some practical value in the primary care hospitals. Key words: Type A influenza H1N1 virus; Reverse transcriptase loop-mediated isothermal amplification(RT-LAMP); Visualization