环介导等温扩增对人乳头瘤病毒可视化分型检测

Loop-Mediated Isothermal Amplification for visual typing of human papillomavirus

目的应用环介导等温扩增(LAMP)可视化检测人乳头瘤病毒(HPV)16、18、52、58亚型,探索LAMP技术在基层或现场的使用前景。方法首先,收集临床疑似HPV感染患者的宫颈脱落细胞标本。然后分别根据HPV16、18、52、58亚型E6、E7区域设计LAMP引物并优化反应体系,通过扩增不同亚型的模板验证这引物的特异性,并通过检测经过一系列稀释后的模板分析其灵敏度,同时在体系中加入钙黄绿素并将显色结果与电泳图对比以验证可视化结果的准确性。最后对临床标本分别用LAMP及PCR进行分型检测,将两种方式的结果进行一致性分析,比较两种方式有无统计学差异。结果优化好的LAMP体系特异性较好,对HPV16、18、52、58亚型的检测限分别为100、10^(3)、100、10^(3)IU/μL。另外,加入钙黄绿素后肉眼判定的效果和电泳效果相同。通过对临床样本分型检测的得出,LAMP与PCR的统计学差异不明显(Kappa值=0.854)。结论LAMP具有较好的灵敏度及特异性,可检测未经纯化的HPV病毒,适合条件简陋的基层或现场检测。

Objective The subtypes of human papillomavirus(HPV)16,18,52 and 58 were detected by annular mediated isothermal amplification(LAMP),and the application prospect of visual LAMP technology in grassroots or field was explored.Methods First,specimens of cervical exfoliated cells from patients clinically suspected of HPV infection were collected.Then respectively according to the E6,E7 area of HPV16,18,52,58 design LAMP primers and optimize the reaction system,through swap template experiment shows the specificity of the four sets of primers,through the analysis of the template detection gradient diluted its sensitivity,at the same time in the system to join the calcein color and the result was compared with electrophoresis figure to verify the accuracy of the visual results.Finally,LAMP and PCR were used for the classification of clinical specimens,and the results of the two methods were analyzed for consistency,and the statistical differences between the two methods were compared.Results the optimized LAMP system had good specificity,and the detection limits of HPV16,18,52 and 58 were 100、10^(3)、100、10^(3) IU/μL.In addition,the result of adding calcium chlorophyllin was similar to that of electrophoresis.Through the classification and detection of clinical samples,the results showed that there was no significant statistical difference between LAMP and PCR(Kappa value=0.854,P>0.05).Conclusion LAMP has good sensitivity and specificity,can detect unpurified HPV virus,and is suitable for basic or field detection with poor conditions.