OBJECTIVEA 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.

METHODSDesign 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.

RESULTSThe 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 micromol/L for all the upstream and downstream primers, 0.06 micromol/L for probes of other human enteroviruse, 0.08 micromol/L for 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 10(9) copies/microl-10(2) copies/microl.

CONCLUSIONThe novel multiplex real-time 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.