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作 者:关丽[1] 许松涛[1] 聂凯[1] 张丹[2] 李鑫娜[1] 许文波[1] 马学军[1]
机构地区:[1]中国疾病预防控制中心病毒病预防控制所,北京102206 [2]广州医科大学基础医学院
出 处:《中华预防医学杂志》2015年第11期1009-1013,共5页Chinese Journal of Preventive Medicine
摘 要:目的建立基于羟基萘酚蓝(hydroxynaphtholblue,HNB)颜色变化的简单、灵敏和快速的环介导逆转录等温扩增技术(RT—LAMP)检测方法,应用于柯萨奇病毒A6型(coxsackievirusA6,CV-A6)的检测。方法针对CV-A6的VPl基因设计6条特异引物,在等温条件下(63℃)进行50min扩增反应。扩增前在反应体系中加入HNB,通过观察颜色变化进行检测结果判定。使用多种肠道病毒进行特异性验证,使用梯度稀释的体外转录CV-A6全VP1基因RNA进行灵敏度分析,同时与实时荧光定量逆转录PCR(rRT—PCR)检测结果进行比较,并对92份手足口病患者临床标本进行检测。结果本研究建立的RT-LAMP方法对除CV-A6外的23种肠道病毒的检测结果均为阴性,灵敏度为100拷贝/反应,与rRT—PCR方法相当。在对92份手足口病临床标本的检测中,检测结果与rRT-PCR方法相符,Kappa值为1,灵敏度和特异性均为100%。结论本研究建立的针对CV-A6的LAMP检测方法,特异度高,灵敏度与rRT—PCR相当,有望应用于CV—A6感染的快速筛选,具有在基层医疗卫生机构和现场推广与应用的潜力。Objective To develop a simple, rapid and sensitive colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of coxsackievirus A6 (CV-A6) based on the colour chang of hydroxy naphthol blue (HNB). Methods The method employed a set of six primers that recognized sequences of VP1 gene for amplification of nucleic acid under isothermal conditions at 63 ℃ for 50 min. The products were detected through visual inspection of color change by the pre-addition of HNB dye. The specificity was validated by detecting a collection of different human enteroviruses. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of CV-A6 VP1 gene, and compared with real-time RT-PCR (rRT-PCR) in parallel. This assay was evaluated with 92 clinical specimens from patients with hand-foot-mouth disease. Results A positive color (sky blue) was only observed in the preparation of CV-A6, whereas none of the other 23 kinds of human enteroviruses showed a color change. The HNB based RT-LAMP showed a sensitivity of 100 copies/reaction, which was at the same level as that of the rRT-PCR. The result of RT-LAMP in analysis of 92 clinical specimens was consistent with that of the rRT-PCR. The kappa correlation between the two methods was 1 and both of the sensitivity and specificity of the RT-LAMP assay were 100%. Conclusion The established RT-LAMP assay had good specificity and sensitivity and thus demonstrated to be a promising screening tool for CV-A6 infection. It also has the potential to be used in resource-limited clinical sites and field study.
关 键 词:聚合酶链反应 柯萨奇病毒感染 环介导逆转录等温扩增
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