目的 建立一种基于CRISPR/Cas12a的侧流层析技术检测人乳头瘤病毒(HPV)的方法,以实现对HPV的简单、准确、快速筛查.方法 通过将CRISPR/Cas12a结合PCR技术与侧流层析技术对常见的10种高危型HPV质粒亚型进行检测验证其可行性;着重对HPV16、HPV52、HPV58、HPV59四种亚型进行灵敏性及特异性验证;并对31例HPV临床样本进行检测,验证基于CRISPR/Cas12a的侧流层析技术(Lateral Flaw Assay based CRISPR,LFAC)的临床检测效能.结果 通过应用LFAC检测,结果表明10种高危型HPV质粒均能产生明显的检测条带,表明该检测系统具有稳定性;对HPV16、HPV52、HPV58、HPV59四种高危型HPV质粒检测结果表明,该方法具有高特异性与高灵敏度,灵敏度检测下限为1.04×104copies/μL;31例临床样本检测结果总符合率为96.77％,根据LFAC检测结果绘制ROC曲线,灵敏度为0.920,特异度为0.626.结论 通过将PCR技术、CRISPR/Cas12a和侧流层析法结合建立一种基于CRISPR/Cas12a的侧流层析技术,实现对HPV的可视化检测,为HPV的快速筛查与诊断提供了技术思路.
Establishment of a lateral flow assay based on CRISPR/Cas12a for HPV detection
Objective The study aims to establish a lateral flow assay based on CRISPR/Cas 12a for HPV detection,so as to achieve simple,accurate and rapid screening of HPV.Methods Combined with PCR,the feasibility of the lateral flow assay based on CRISPR/Cas 12a was verified by testing 10 common high-risk HPV plasmid subtypes,focusing on the sensitivity and specificity veri-fication of four subtypes,HPV16,HPV52,HPV58,and HPV59,and testing 31 HPV clinical samples.In this way,the clinical detection efficiency of the lateral flow assay based on CRISPR/Cas 12a(LFAC)was verified.Results The results of LFAC detection showed that all 10 high-risk HPV plasmids could produce detection bands,indicating that the detection system was stable.The results of the four high-risk HPV plasmids,HPV16,HPV52,HPV58 and HPV59,showed that the method had high specificity and high sensitivity,and the lower limit of sensitivity detection was 1.04×104 copies/µL.96.77％of the 31 clinical samples was consistent.The ROC curve was drawn according to the LFAC test results,with a sensitivity of 0.920 and a specificity of 0.626.Conclusion By combining PCR technology,CRISPR/Cas 12a and lateral flow assay,a lateral flow assay based on CRISPR/Cas 12a was established to achieve visual de-tection of HPV,providing technical ideas for the rapid screening and diagnosis of HPV.
human papillomavirusCRISPR/Cas 12alateral flow technology
NETL
NSTL
万方数据
