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Detection of human Enterovirus 71 reverse transcription loop-mediated isothermal amplification(RT-LAMP)

Detection of human Enterovirus 71 reverse transcription loop...

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2012深圳市预防医学会学术研讨会

作者： H.Yaqing Z.Wenping Y.Zhiyi W.Xionghu Y.Shouyi Y.Hong D.Yingchun H.Guifang Department of Epidemiology Southern Medical University Department of Microbiology Shenzhen Centerfor Disease Control and Prevention Department of Health Statistics Center for Medical Information of Shenzhen City

Aims:In this study,a one-step,single-tube reverse transcription loop-mediated isothermal amplification(RT-LAMP) assay was developed and validated for the detection of human Enterovirus 71(EV71),the major aetiological agent of hand,foot and mouth disease(HFMD). Methods and Results:reverse transcription loop-mediated isothermal amplification assay was optimized to amplify VP1 gene in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63℃for 1 *** products were evaluated by visual inspection and agarose gel *** detection limit of RT-LAMP assay was 105) 100 TCID50 or 160 copies in samples after RNA extraction,which was 10-fold higher in sensitivity than traditional reverse transcription polymerase chain reaction (RT-PCR).The specific positive amplification was only observed in EV71 strains,while no amplification was detected in other tested *** with a specific Escherichia coli restriction enzymes V(EcoR V) demonstrated that the amplified product was unique.A good correlation between RT-LAMP and real-time RT-PCR was observed on the basis of the analysis of 33 clinical samples. Conclusions:reverse transcription loop-mediated isothermal amplification is a novel,alternative microbiological approach for rapid,sensitive and specific detection of EV71 in HFMD. Significance and lmpact of the Study:reverse transcription loop-mediated isothermal amplification assay is suitable for the diagnosis of EV71 infection as a routine diagnostic tool for HFMD because of fewer requirements of experimental conditions such as private clinics,field applications as well as an epidemiological survey in epidemic ***-LAMP can also be used as an alternative method for EV71 detection.

关键词： hand foot and mouth disease human Enterovirus 71 reverse transcription loopmediated isothermal amplification

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