应用通用引物的实时 RT-PCR 同时检测地表水中的肠道病毒。

Simultaneous detection of enteroviruses from surface waters by real-time RT-PCR with universal primers.

机构信息

Key Laboratory of Northwest Water Resource, Ecology and Environment, Ministry of Education, School of Environmental and Municipal Engineering, Xi'an University of Architecture and Technology, Xi'an 710055, China.

出版信息

J Environ Sci (China). 2010;22(8):1261-6. doi: 10.1016/s1001-0742(09)60248-5.

DOI:10.1016/s1001-0742(09)60248-5

PMID:21179967

Abstract

In order to realize simultaneous quantitative detection of various enteroviruses from water samples, a real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) method was developed with universal primer pairs designed based on the highly conserved non-coding region sequences of genome targeting poliovirus, coxsackievirus and enterovirus 71. The recombinant plasmid was constructed as enterovirus DNA standard by cloning poliovirus cDNA into a pMD18-T vector. The real-time RT-PCR method utilizing SYBR Green I was optimized. As a result of a series of examinations, the detection limit of the method was found to be 2.31 genome equivalent copy (GEC)/microL, the intra- and inter-assay variations were lower than 2% and 5%, respectively, and enteroviruses were well distinguished from other microorganisms. There was a good linear relationship (r2 = 0.997) between the logarithm of viral density and cycle threshold in a wide range of 2.31 x 10(0) to 2.31 x 10(9) GEC/microL. The validity of the method was further proved by its application for the detection of enteroviruses from various practical water samples.

摘要

为了实现对水样中多种肠道病毒的同时定量检测，根据针对脊髓灰质炎病毒、柯萨奇病毒和肠道病毒 71 的基因组靶向高度保守的非编码区序列设计了通用引物对，建立了实时逆转录聚合酶链反应（real-time RT-PCR）方法。将脊髓灰质炎病毒 cDNA 克隆到 pMD18-T 载体中，构建了肠道病毒 DNA 标准品作为重组质粒。利用 SYBR Green I 对实时 RT-PCR 方法进行了优化。经过一系列检查，发现该方法的检测限为 2.31 基因组等效拷贝（GEC）/μL，内和间试验变异分别低于 2%和 5%，并且肠道病毒与其他微生物很好地区分。在 2.31 x 10(0) 到 2.31 x 10(9) GEC/μL 的宽范围内，病毒密度的对数与循环阈值之间存在良好的线性关系（r2 = 0.997）。该方法应用于各种实际水样中肠道病毒的检测，进一步证明了其有效性。