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

发布时间：2018-05-29 05:01

本文选题：丙型肝炎病毒(HCV) + 中国　；参考：《病毒学报》2017年01期

【摘要】：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(P0.05)。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。
[Abstract]:Reverse transcriptional loop mediated isothermal amplification (RT-LAMP) was used to amplify the major hepatitis C virus (HCV) 1b and 2A genotypes in China. First, the clinical samples were collected to carry out quantitative and qualitative analysis. Second, the corresponding RT-LAMP primers were designed according to the 5 'non transcriptional region (5' UTR) of the HCV 1b and HCV 2A sequences. Third. The specificity of the specific template was evaluated to evaluate the specificity of the primers. Fourth, the sensitivity of the primers was evaluated by using the dilution template to evaluate the sensitivity of the primers, and the results were compared with the electrophoretic results using the visualization method dependent on calcein /Mn2+. Finally, all the samples were HC with RT-LAMP. V 1b and HCV 2A two groups were reacted in parallel, and SPSS statistical software was used to evaluate its usability. The results showed that the specificity of RT-LAMP primers was good, the size of HCV 1b and HCV 2 A was the same as expected. The minimum detectable template amount was 100IU/mL, and the visual staining method was the same. Statistics software compared the result of amplification. It shows that there is no statistical difference between RT-LAMP and real time fluorescence quantitative PCR (P0.05). To sum up, the RT-LAMP equipment is simple and easy to operate, and it has the prospect of application in the medical institutions of the grass-roots level.
【作者单位】：承德医学院病原生物学教研室;中国人民解放军白求恩国际和平医院全军肝病诊治中心;
【基金】：全军医药卫生科研基金项目(项目号:CBJ14C010);题目:一种便携式、简便、快速检测病原体核酸新装置的研制及其在呼吸道传染病的初步应用
【分类号】：R373.21

【相似文献】