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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。
China’s major HCV genotypes 1b and 2a were amplified using RT-LAMP. First, collect clinical specimens and quantify and characterize them. Second, the corresponding RT-LAMP primers were designed based on the 5 ’untranslated region (5’UTR) of HCV 1b and HCV 2a sequences, respectively. Third, cross-reactions with non-specific templates were used to evaluate the specificity of each primer and the product was digested with endonucleases to further ensure accurate amplification. Fourth, the use of a prime dilution template to evaluate the sensitivity of each primer was compared with the electrophoretic results using a calcein / Mn2 + visualization method. Finally, RT-LAMP was used to detect the parallel reactions between HCV 1b and HCV 2a in all samples. SPSS statistical software was used to evaluate its practicability. The results showed that the designed RT-LAMP primers had good specificity and the sizes of HCV 1b and HCV 2a products were as expected. The minimum detectable template amount was 100 IU / mL, and the visualization staining method electrophoresis results consistent. Statistical software comparison amplification results showed that there was no significant difference between RT-LAMP and real-time fluorescence quantitative PCR (P> 0.05). In summary, RT-LAMP equipment is simple, easy to operate, with the prospect of application in primary health care institutions.