Development of EV71, CA16 and other enterovirus vrial real-time qualitative PCR diagnostic kit
10.3760/cma.j.issn.1003-9279.2013.03.022
- VernacularTitle:EV71、CA16及通用型肠道病毒实时荧光定量PCR诊断试剂盒的研发
- Author:
Li-Qin LI
1
;
Jing ZHONG
;
Lin-Fu ZHOU
;
Fu-Chu QIAN
;
Jia-Wei WANG
;
Li-Cheng DAI
Author Information
1. 湖州市中心医院
- Keywords:
Polymerase chain reaction;
Hand,foot and mouth disease;
Enterovirus
- From:
Chinese Journal of Experimental and Clinical Virology
2013;27(3):224-227
- CountryChina
- Language:Chinese
-
Abstract:
Objective A novel multiplex real-time RT-PCR kit was developed to detect EV71,CoxA16 and other human enteroviruses simultaneously with an internal amplification control to avoids false negatives,which used for hand,foot and mouth disease in the clinical diagnosis and epidemiological surveillance.Methods Design specific primers and probes of EV71,CA16,other intestinal virus and internal amplification control,improve the extraction method of virus nucleic acid.Optimization the detection system of real-time quantitative PCR.Research the products of the accuracy,stability,precision,amplification efficiency and detection of linear range.Results The primers and probes had high spicificity.The Viral RNA extraction effect of this Kit is as same as that of QIAamp Viral RNA mini Kit (QIAGEN company),but less reagent cost.The optimal concentrations of primers and probes are 0.2 μmol/L for all the upstream and downstream primers,0.06 μmol/L for probes of other human enteroviruse,0.08 μmol/Lfor probes of EV71 and CA16 respectively.The kit has good stability,accuracy and precision.The amplification efficiencies of EV71,CoxA16 and other human enteroviruses are 106％,101％ and 105％ and the detection of linear range is from 109 copies/μl-102 copies/μl.Conclusion The novel multiplex realtime RT-PCR kit for detecting EV71,CoxA16 and other human enteroviruses simultaneously with an internal amplification control has good stability,accuracy,precision and amplification efficiencies.So it has great value in clinical application.
