Development of Fluorescence PCR for Detection of Hand Foot Mouth Viruses with MGB Probe / 现代检验医学杂志

ABSTRACT

Objective A fluorescence PCR methods was developed to detect EV71 and CoxAl6 and other enteroviruses simultaneously,which used for hand,foot and mouth disease (HFMD) viruses in the clinical rapid diagnosis.Methods Designed specific primers and probes of the enteroviruses gene which represents one of the highly conserved regions of the virus gene,and optimized the detection system of real-time quantitative RT-PCR.The positive control template and the standard curve were constructed.Researched the limit of detection,repeatability and specificity of the products,and tested 26 positive samples and 10 negative samples which collected on June 2015.Results The results showed that this experiment obtained positive recombinant plasmid,the range of the linear relation was from 8 × 102 to 8 × 108 copies/μl,and the detection result within this range was fine.The optimal concentrations of EVUN upstream and downstream primers were 0.50 μmol/L and the MGB probes were 0.30 μmol/L,RT-PCR reaction conditions as follows42℃ 30 min,95℃ 3 min.95℃ 5 s,60℃ 35 s,45 cycle.The limit of detection reached to 800 copies/μl.The CV of the repeatability assay was no more than 5％.Specificity was good,and no cross reaction with other infectious viruses.26 clinical positive samples and 10 negative samples were detected in this experiment,detection rate of positive samples was 100 ％ (26/26) and negative samples was 100 ％ (10/10) by fluorescent quantitative RT-PCR detection method.Conclusion The experiment demonstrated that the detection method of the fluorescent quantitative PCR for hand-foot-mouth viruses could be used for the rapid diagnosis in clinical application.