A one-step reverse-transcription recombinase aided PCR assay for the rapid and sensitive detection of human enteroviruses

IF 3.5 Q1 PUBLIC, ENVIRONMENTAL & OCCUPATIONAL HEALTH
Xiuli Sun , Huanhuan Lu , Yanqing Tie , Mengchuan Zhao , Ruiqing Zhang , Zhenlu Sun , Guohao Fan , Fengyu Li , Fengyu Tian , Yaxin Hu , Mengyi Zhang , Xinxin Shen , Xuejun Ma , Zhishan Feng
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引用次数: 0

Abstract

Human enteroviruses (HEVs) include many different types that cause a wide range of diseases, and an effective method of genus-level identification has therefore significant clinical implications. However, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), the gold-standard method, still has shortfalls in diagnostic sensitivity and timeliness. Here we established a one-step real-time reverse-transcription recombinase-aided PCR assay (RT-RAP) to detect HEV fragment within an hour. The RT-RAP assay showed a detection limit of 5 copies/μL using recombinant plasmids and was extensively verified using 15 HEV strains. Among 15 types of HEV (species A-C), the sensitivity of RT-RAP was approximately 2–8 folds lower than that of the qRT-PCR in 9 types, and no-cross reaction with other viruses was observed. RT-RAP was further applied to analyze CSF and fecal specimens; the clinical performance demonstrated that the RT-RAP and the commercial qRT-PCR kit provided consistent results. These results indicated that RT-RAP assay may be a promising approach for rapid and sensitive detection of HEV.

一步逆转录重组酶辅助聚合酶链式反应快速灵敏检测人肠道病毒
人类肠道病毒(hev)包括许多不同类型,可引起广泛的疾病,因此有效的属水平鉴定方法具有重要的临床意义。然而,作为金标准的定量实时逆转录聚合酶链反应(qRT-PCR)在诊断敏感性和时效性方面仍存在不足。在这里,我们建立了一种一步实时逆转录重组酶辅助PCR (RT-RAP)检测方法,可以在一小时内检测出HEV片段。RT-RAP实验显示,重组质粒的检测限为5拷贝/μL,并在15株HEV菌株中得到了广泛验证。在15种HEV病毒(A-C种)中,有9种RT-RAP的灵敏度比qRT-PCR低约2-8倍,且与其他病毒无交叉反应。进一步应用RT-RAP分析脑脊液和粪便标本;临床表现表明,RT-RAP与商用qRT-PCR试剂盒的结果一致。这些结果表明,RT-RAP法可能是一种快速、灵敏检测HEV的方法。
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来源期刊
Biosafety and Health
Biosafety and Health Medicine-Infectious Diseases
CiteScore
7.60
自引率
0.00%
发文量
116
审稿时长
66 days
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