反转录环介导等温扩增技术对丙型肝炎病毒1b和2a基因型检测

A Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Detection of HCV Genotypes 1b and 2a in China

利用反转录环介导等温扩增技术(RT-LAMP)对中国主要的丙型肝炎病毒(HCV)1b和2a基因型进行扩增。首先,收集临床采样标本,对其进行定量和定性。第二,分别根据HCV 1b和HCV 2a序列的5’非转录区(5’UTR)设计相应的RT-LAMP引物。第三,利用与非特异模板的交叉反应以评价各引物的特异性,利用内切酶酶切产物以进一步保证扩增准确。第四,利用倍比稀释的模板以评价各引物的灵敏性,并用依赖钙黄绿素/Mn2+的可视化方法与电泳结果比较。最后,利用RT-LAMP对所有采样标本进行HCV 1b和HCV 2a两组平行反应,SPSS统计学软件对其实用性初步评价。结果显示,所设计的RT-LAMP引物特异性好,HCV 1b和HCV 2a型产物大小均与预期相同。最低可检测的模板量为100IU/mL,且可视化染色方法电泳结果相一致。统计学软件比较扩增结果表明RT-LAMP与实时荧光定量PCR之间没有统计学差异(P>0.05)。综上,RT-LAMP设备简单,操作简便,具有在基层医疗机构的应用前景。

Reverse transcription loop-mediated isothermal amplification （RT-LAMP） was used to detect hepatitis C virus （HCV） lb and 2a （the major genotypes in China）. First, clinical samples were collected, and the genotypes and viral load determined. Second, RT-LAMP primers of HCV lb and 2a were designed according to the 5r untranslated region of the HCV. Third, the specificity of RT-LAMP was tested by non-lb and non-2a HCV viruses, and the products analyzed by endonuclease. Fourth, the limit of detection was tested using diluted samples, and the results determined by calcein/Mn2＋-dependent visual methods. Finally, the consistency of RT-LAMP and real-time fluorescent polymerase chain reaction （PCR） was compared to evaluate the clinical practicality. Results showed that RT-LAMP primers had good specificity because there was no cross-reactivity and the target sequences were identified correctly by endonuclease. The limit of detection was 100 IU/mL and calcein/Mn2＋-dependent visual methods were easier to use compared with electrophoresis. After analyses of all amplification results by statistical software, we found no significant difference between RT-LAMP and real-time fluorescent PCR （P〉0.05）. In conclusion, RT LAMP requires only simple apparatus and simple operation, which is very helpful in primary-health-care institutions.