Establishment Of Detecting Method For The Main Pathogens Of Hand-foot-mouth Disease And Its Preliminary Evaluation In Clinical

Text:Hand-foot-mouth disease (HFMD) is a viral disease caused by the enterovirus. The main pathogens are Enterovirus 71(EV71)and Coxsackie A16(Cox A16). They are common infections in infants and preschool children. A small portion of HFMD cases are complicated with more serious neural diseases, including meningitis, viral encephalitis, limb paralysis, myocarditis, even leading to fatality. So it is very important to establish a method for Early diagnosis. We want to develop rapid laboratory detection methods for viral nucleic acid and sera antibody for HFMD in this research and preliminary evaluate the methods in clinical.Objective:1.To establish the real-time fluorescent PCR method for detecting enterovirus, enterovirus 71 and coxsackievirus A 16 nucleic acid.2. To clone, express and purify the recombinant protein of enterovirus 71 VP1 and establish detecting method of IgM antibody.3. To evaluate the detection of IgM antibodies for human enterovirus 71(EV 71) in early diagnosis for the hand-foot-mouth disease (HFMD).Method:1. Primers and MGB probe were chosen for virus gene. The samples of 38 HFMD patients were analyzed by TaqMan-MGB PCR technique on a fluorescence real-time PCR instrument, and the results were compared with those by conventional RT-PCR.2. EV-71 VP1 gene fragment was amplified by RT-PCR. Then the gene fragment was inserted into the multiple cloning site of pRSET-A?? and expressed in E. coli,. which was purified as coating antigen to establish ELISA to detect EV71 VP1 IgM antibody. Then the sera of 31 hand-foot-mouth disease (HFMD) and 36 health children were detected.3.The sera and throat swabs from 38 patients which were clinically confirmed as HFMD,, were consecutively collected in our hospital on daily basis in 2010. These specimens were detected by EV 71 IgM antibodies assay, real time RT-PCR methods for EV 71 and Enterovirus.Results:1.The real-time fluorescent PCR positive rates of EV, EV71 and Cox A16 were 73.7%,61.0%,13.2%, and the conventional RT-PCR were 71.1%,55.3%,13.2% respectively. There were no significant differences between the two methods.2. The cloned EV71 VP1 gene and expressed proteins were about 891bp and 33KD as predicted. Of 31 specimens from the HFMD group tested by ELISA,7 samples showed positive reaction. No samples showed positive reaction in the group of healthy children. There were significant differences between the children with HFMD and healthy children.3. Among 38 HFMD patients, the cumulative positive rates of EV 71 IgM antibodies were:60.5% on day 1,71.1% on day 2,81.5% in the first 3-4 days,92.1% on day 5,92.1% on day 6, and the positive rate of nucleic acid detected by the real time RT-PCR for EV 71 and Enterovirus were 60.5%,73.6%.Conclusion:1.The real-time fluorescent PCR detecting method of EV, EV71 and Cox A16 nucleic acid have been established successfully.2. The detecting method of EV71 IgM antibody has been established successfully.3. The EV 71 IgM antibody would be positive in the hand, foot and mouth disease as early as day 1, and reach to peak on day 5,which can be used as one of indicators of early diagnosis of hand, foot and mouth disease.