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Internally controlled recombinase-aided amplification (IC-RAA) assays for the detection of human papillomavirus genotypes 16 and 18 using extracted DNA and samples treated with nucleic acid releasing agent.
Archives of Virology ( IF 2.5 ) Pub Date : 2020-07-17 , DOI: 10.1007/s00705-020-04722-3
Jinrong Wang 1, 2, 3 , Jianli Liu 4 , Guowei Song 3 , Zhi Cao 5 , Jing Pan 3 , Xinna Li 2 , Yuan Gao 1, 2 , Juju Qi 3 , Ziwei Chen 2 , Guohao Fan 2 , Xueding Bai 2 , Ruiqing Zhang 1, 2 , Ruihuan Wang 2 , Qingxia Duan 1, 2 , Lixin Li 1, 3 , Xinxin Shen 2 , Xuejun Ma 2
Affiliation  

Cervical cancer is primarily caused by persistent infection with high-risk human papillomavirus (HPV), and 70% of cases are associated with HPV16 and 18 infections. The objective of this study was to establish rapid, simple, and sensitive internally controlled recombinase-aided amplification (IC-RAA) assays for the detection of HPV16 and 18. The assays were performed at 39 ℃ and were completed within 30 min. A total of 277 clinical samples of exfoliated cervical cells were tested by IC-RAA assays and commercial HPV real-time fluorescent PCR kits using extracted DNA and samples treated with nucleic acid releasing agent. The analytical sensitivity of the IC-RAA assay was found to be 10 copies/μL for the detection of HPV16 and 18 when using recombinant plasmids as targets. The optimal concentration of the internal control (IC) plasmid and 18 was 1000 copies/μL for HPV16 and 100 copies/μL for HPV18. The clinical sensitivity of the IC-RAA assays for HPV16 using extracted DNA and samples treated with nucleic acid releasing agent was 98.73% and 97.47%, respectively, with kappa values of 0.977 (P < 0.01) and 0.955 (P < 0.01), respectively, and 100% The specificity in both cases. For HPV18, the sensitivity and specificity were 100%, and the kappa value was 1 for both samples (P < 0.01). The IC-RAA assay is a promising tool for the detection of HPV16 and HPV18, especially in resource-constrained settings.



中文翻译:

内部控制的重组酶辅助扩增(IC-RAA)分析,用于检测人乳头瘤病毒基因型16和18,方法是使用提取的DNA和经过核酸释放剂处理的样品。

宫颈癌主要是由持续感染高危型人乳头瘤病毒(HPV)引起的,其中70%的病例与HPV16和18感染有关。这项研究的目的是建立快速,简单,灵敏的内部控制的重组酶辅助扩增(IC-RAA)检测HPV16和18的检测。检测在39℃进行,并在30分钟内完成。使用提取的DNA和经过核酸释放剂处理的样品,通过IC-RAA分析和商业HPV实时荧光PCR试剂盒对总共277个脱落的宫颈细胞的临床样品进行了测试。当使用重组质粒作为靶标时,发现IC-RAA检测HPV16的分析灵敏度为10拷贝/μL,检测灵敏度为18个拷贝/μL。内部对照(IC)质粒和18的最佳浓度对于HPV16为1000拷贝/μL,对于HPV18为100拷贝/μL。使用提取的DNA和经核酸释放剂处理的样品对HPV16进行IC-RAA分析的临床敏感性分别为98.73%和97.47%,kappa值为0.977(P  <0.01)和0.955(P  <0.01),两种情况的特异性均为100%。对于HPV18,两个样品的敏感性和特异性均为100%,kappa值为1(P  <0.01)。IC-RAA分析是检测HPV16和HPV18的有前途的工具,尤其是在资源有限的环境中。

更新日期:2020-09-12
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