title: Simultaneous detection of enteroviruses from surface waters by real-time RT-PCR with universal primers.
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.
authors.affiliation: 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. zhangchongmiao@yahoo.com.cn
authors.forename: Chongmiao
authors.initials: C
authors.lastname: Zhang
authors.forename: Xiaochang
authors.initials: X
authors.lastname: Wang
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Liu
authors.forename: Dangcong
authors.initials: D
authors.lastname: Peng
mesh_terms: D004770:Enterovirus / Q000302:isolation & purification*; D004784:Environmental Monitoring / Q000379:methods*; D010957:Plasmids; D012313:RNA; D012367:RNA, Viral; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014871:Water Microbiology
chemical_list: D012367:RNA, Viral; C039919:RNA, recombinant; D012313:RNA