[Detection of Hepatitis C Virus by Reverse Transcription-loop Mediated Isothermal Amplification]

Hepatitis C virus(HCV)do harm to people’s health.The present study aims to establish a simple HCV detection method by reverse transcription-loop mediated isothermal amplification technique(RT-LAMP).A total of75 clinical samples were collected and pre-detected by fluorescence quantitative-polymerase chain reaction(FQ-PCR),which was considered as the gold standard.Firstly,four common primers were designed according to the conservative 5’UTR region of HCV on the NCBI website to establish an integrated RT-LAMP reaction system.Then,the reaction efficiency was evaluated by adding Taq DNA polymerase to the conventional system.The specificity of RTLAMP was evaluated by observing the length of fragment after endonuclease digestion and by a templates exchange assay,the sensitivity of RT-LAMP was evaluated by detection of diluted clinical templates.The results were compared with that of reverse transcription polymerase chain reaction(RT-PCR).At the same time,the performance was judged using calcein and hydroxynaphthol blue(HNB)stain methods,The two results were compared with that of electrophoresis method.At last,75 clinical samples were detected by both RT-LAMP and RT-PCR methods.Results showed that the reaction efficiency was increased 20 minutes after adding Taq DNA polymerase to the normal RT-LAMP system.RT-LAMP showed good specificity,the digestion length was consistent with our expectation(216bp)after restriction endonuclease cleavage assay,and only the templates of HCV were amplified using the common RT-LAMP primers.After detection of diluted temples,the sensitivity of RT-LAMP was 10IU/tube,which was 10 fold higher than that of PCR.In addition,the results using calcein and HNB stain methods were the same with that of electrophoresis method.After detection of all 75 clinical samples,the results indicated that RT-LAMP had worse consistency with RT-PCR(P<0.05,Kappa=0.375).However,RT-LAMP,on the contrary,showed good consistency with FQ-PCR(P>0.05,Kappa=0.762).In conclusion,RT-LAMP has characteristics of simplicity,specificity and sensitivity,and this technique is suitable for the primary care hospitals.