Study On The Visualized Detection Of Hepatitis B And C Virus By Loop-mediated Isothermal Amplification

Hepatitis B virus(HBV)and hepatitis C virus(HCV)infection is a global public health problem,particularly in developing countries where experiment instruments are limited.The HBV and HCV nucleic acid detection is the best way to diagnose and treatment monitoring,the fluorescence quantitative PCR(FQ-PCR)method is widely used in large and medium-sized hospitals in China,however,the operation process of FQ-PCR is trival,the reagent and instrument are both expensive,and require skilled personnel to perform,this method is not sutibile for places where healthcare instruments are limited.Our study set up a calcein and hydroxynaphthol blue(HNB)-dependent virus detection method by visualized loop-mediated isothermal amplification(LAMP)for HBV and HCV detection in instrument-limited environments.Part I Visualized detection of hepatitis B virus by loop-mediated isothermal amplificationObjective:To establish a LAMP method for visualized HBV detection.Methods:Firstly,the LAMP primers were designed according to the conservative region of S retion on the NCBI.Secondly,clinical serum samples were collected and DNA from which were extracted by both testing kit and serum boiling method,FQ-PCR was used for quantify.Thirdly,the reaction condition of LAMP was optimized.The amplification specificity was verified through nonspecific templates amplification assay and enzyme digestion assay;the sensitivity was evaluated through testing serial dilutions of templates after kit extraction;to evaluate the anti-interference of LAMP,amplification result of LAMP and PCR was compared after amplification with boiled serum.At the same time,the effect was compared by SYBR Green I-dependent visual method and HNB-dependent visual method according to electrophoresis.Finally,clinical samples were amplified by LAMP and PCR,respectively;the two methods were compared with FQ-PCR(gold standard)by SPSS v17.0 statistical software.Results:After a series of preliminary experiments,the optimal amplification condition of LAMP was established.LAMP had good specificity,there was no nonspecific amplification.The sensitivity of LAMP was only 10 copies/tube,regardless of which extraction method,we could come to the conclution that the anti-interference of LAMP was good.The effect of the HNB-dependent visual method was the same with that of SYBR Green I-dependent visual method and electrophoresis,and the HNB-dependent visual method was not easy to cause aerosol pollution like SYBR Green I.In addition,the results of LAMP and FQ-PCR had no statistic difference using boiled serum(P>0.05),and this two methods were in good consistency(Kappa=0.762).However,the results of PCR using boiled serum and FQ-PCR had statistic difference(P<0.05),and this two methods were not in good consistency(Kappa=0.186).Conclusion:We designed common LAMP primers targeting the HBV S region for detection of four main HBV genotypes in China,the efficiency of amplification were increased by designing loop primers.What was more,the operation process was simplified using boiled serum,thus,a HNB-dependent LAMP method for visualized HBV detection was established.Part II Visualized detection of hepatitis C virus by reverse transcription loop-mediated isothermal amplification(RT-LAMP)Objective:To establish a RT-LAMP method for visualized HCV detection.Methods:Firstly,75 clinical samples were collected and detected by FQ-PCR(gold standard).Common RT-LAMP primers were designed according to the HCV 5' untranslated region(5'UTR)of 9 HCV subgenotypes.Then,the traditional RT-LAMP system and system after adding Taq DNA polymerase were compared for optimization.The specificity was evaluated by nonspecific templates amplification assay,the sensitivity of RT-LAMP was evaluated by detection of serial dilutions of templates using RT-LAMP and RT-PCR.In the meantime,the results were judged with calcein-dependent and HNB-dependent visual method,and the effects were compared with that of electrophoresis.At last,clinical samples were detected by RT-LAMP and RT-PCR,the two methods were compared with FQ-PCR by SPSS v17.0 statistical software.Results:Results showed that the reaction efficiency could be increased by 20 minutes after adding Taq DNA polymerase to the traditional RT-LAMP system.RT-LAMP showed good specificity because no nonspecific targeting templates were amplified.After detection of diluted templates,the sensitivity of RT-LAMP was 10 IU/tube,which was 10 fold higher than that of RT-PCR.In addition,the effects of calcein-dependent and HNB-dependent visual methods were the same with that of electrophoresis.The 75 clinical sample detection results indicated that RT-LAMP showed good consistency with FQ-PCR(P>0.05,Kappa=0.762).Conclusion:We designed common RT-LAMP primers targeting the HCV 5 'UTR region of 9 HCV subgenotypes,thus,a calcein-dependent or HNB-dependent RT-LAMP method for visualized HCV detection was established.Part III Detection of HCV genotypes 1b and 2a by reverse transcription loop-mediated isothermal amplificationObjective:To establish a RT-LAMP method for HCV 1b and 2a genotyping.Methods:Firstly,74 positive clinical samples were collected,the genotypes and viral load were detected by FQ-PCR.Secondly,RT-LAMP genotyping primers were designed according to the 5? UTR of HCV 1b and 2a,respectively.Thirdly,the specificity of RT-LAMP was evaluated by nonspecific templates amplification assay and enzyme digestion assay.Fourth,the sensitivity of each set of primer was evaluated by amplification of serial dilutions of templates,and the results were determined by calcein-dependent visual methods.Finally,two parallel reactions for all clinical samples were operated by HCV 1b and 2a specific primers.The results were compared with FQ-PCR by SPSS v17.0 statistical software.Results:Results showed that RT-LAMP primers had good specificity because there was no cross reaction with nonspecific templates and the digesting fragments of HCV 1b or 2a were consistent with expectation.The detection limit was 100 IU/mL and the effect of calcein-dependent visual method was consistent with that of electrophoresis.After clinical sample detection by two parallel assays,the positive rate of RT-LAMP for HCV 1b detection was 97.37 %,and the positive rate of RT-LAMP for HCV 2a was 94.44 %.After analyses by SPSS 17.0 statistical software,we found no significant difference between RT-LAMP and FQ-PCR(P>0.05).Conclution:We designed two genotyping primers by comparing multiple sets of sequences,a calcein-dependent RT-LAMP method for visualized HCV genotyping was established.