The Establishment Of Detection Method For H1n1, EV71, HBOV And HMPV By Loop-Mediated Isothermal Amplification

LAMP is short for loop mediated isothermal amplification and refers to cyclical amplification with the assistance of specific primers and polymerase in isothermal condition. This mechanism does not need denaturation or renaturation of nucleic acid and has advantage of rapid, efficient, high specificity and so on. So this method is widely used in molecular diagnostic, especially in pathogen diagnosis. This study chose A H1N1 influenza virus, enterovirus 71, human bocavirus and human metapneumovirus as object and established the LAMP detection method of these four viruses. The results as follows:1. According to H1N1 SC strain to get the primers for RT-LAMP using Align X and PrimerExplorer software, then process the RT-LAMP reaction. Research result showed that digestion of restriction enzyme was in accordance with the theoretical, sequencing result got 100% homology with template and no cross reaction with other related pathogen, sensitivity achieved 10 copies, 2 same positive samples were detected in 76 clinical samples together with RT-PCR and sequencing results were identical. The laboratory LAMP detection method of A H1N1 influenza virus was preliminarily established.2. According to EV71 AH strain to get the primers for LAMP using Align X and PrimerExplorer software, then process the RT-LAMP reaction. Research result showed that digestion of restriction enzyme was in accordance with the theoretical, sequencing result got 98% homology with template and no cross reaction with other related pathogen, sensitivity achieved 10 copies, 52 same positive samples including 42 nasopharyngeal swabs and 10 stool swabs were detected in 252 clinical samples including 151 nasopharyngeal swabs and 101 stool swabs together with RT-PCR, in agreement with raw data. The laboratory RT-LAMP detection method of EV71 was preliminarily established.3. According to HBoV HN strain to get the primers for LAMP using Align X and PrimerExplorer software, then process the LAMP reaction. Research result showed that digestion of restriction enzyme was in accordance with the theoretical, sequencing result got 100% homology with template and no cross reaction with other related pathogen, sensitivity achieved 10 copies, 5 same positive samples were detected in 76 clinical samples together with RT-PCR and sequencing results were identical. The laboratory LAMP detection method of HBoV was preliminarily established.4. According to hMPV HN strain to get the primers for RT-LAMP using Align X and PrimerExplorer software, then process the RT-LAMP reaction. Research result showed that digestion of restriction enzyme was in accordance with the theoretical, sequencing result got 98% homology with template and no cross reaction with other related pathogen, sensitivity achieved 10 copies, 5 positive samples were detected by RT-PCR and 7 positive samples were detected by RT-LAMP in 76 clinical samples together with RT-PCR and sequencing results were identical. The laboratory LAMP detection method of hMPV was preliminarily established.This study established LAMP detection methods for H1N1, EV71, HBoV, hMPV. It had advantage in high throughout, efficiency, operability. Meanwhile, the result was macroscopic, background was clear, and the determination of result was objective and accurate.This study established simple, rapid, sensitive, specific LAMP detection methods for these four viruses that could be used as a pre-clinical diagnostic tool in laboratories with limited equipment, especially under field conditions, also can offer experiment experiences and technical support.