Establishment and clinical application of triplex taqman probes real-time PCR for pathogenesis of hand foot mouth disease

Abstract

Objective To establish the triplex Taqman probes real-time RT-PCR method for simultaneously detecting of EV71,CA16 and EV. Methods Retrospective study.Specific primers and probes were designed based on conserved regions of EV71,CA16 and EV.The sensitivity,specificity and reproducibility were assessed by the optimized reaction system.A total of 176 throat swabs as the experimental group were collected from children with suspected hand foot mouth disease (HFMD),who admitted from April 2012 to July 2012 in Nanjing Children′s Hospital affiliated to Nanjing Medical University.During this time,10 cases of healthy children,10 cases of outpatients with flu-like symptoms and 90 cases of inpatients in pneumology department of our hospital were recruited as control group,whose throat swabs were also collected.All of 286 samples were tested by the triplex Taqman probes real-time RT-PCR for simultaneously detecting EV71,CA16 and EV.SPSS13.0 was used to analyze the results. Results The sensitivities of the triplex Taqman probes real-time RT-PCR was 1.0×103copies per milliliter for EV71,CA16 and EV.It showed 100% specificity for 9 enterovirus and 3 non-enterovirus.Analysis with 1.0×103-1.0×105copies per milliliter constructed plasmids demonstrated high reproducibility with coefficient of variation of 0.44%-1.04% for EV71,0.38%-0.73% for CA16,and 0.46%-0.90% for EV.More over 176 samples collected from children with suspected HFMD were detected by triplex Taqman probes real-time RT-PCR and real-time RTPCR.The results showed 97.2% (171/176) agreement and 0.94 Kappa value with high concordance. Conclusions The triplex Taqman probes real-time RT-PCR detecting EV71,CA16 and EV simultaneously has been established successfully.The assay,with high sensitivity and specificity,provide good basis for the rapid clinical diagnosis of EV71,CA16 and EV and open up broad prospects for clinical and relevant researches.(Chin J Lab Med,2013,36:845-849) Key words: Hand; foot and mouth disease; Enterovirus A; human; Polymerase chain reaction