|
Hand, foot and mouth disease is a viral infectious disease caused by enterovirus major pathogens enterovirus 71 (EV71) and coxsackievirus A16 (Cox A16). Infants and preschool children susceptible. A small part of the hand, foot and mouth cases may be complicated by severe neurological diseases, including meningitis, viral encephalitis, paralysis, even death. Therefore, early diagnosis is very important. This paper is to develop a hand, foot and mouth disease virus nucleic acid and serum antibody detection methods and preliminary clinical evaluation. Objective: To establish a real-time PCR method a simultaneous detection of enteroviruses, enterovirus 71 and Coxsackie virus type 16 and other hand-foot-mouth disease pathogens nucleic acid; enterovirus 71 gene was cloned in the prokaryotic expression system Expression, purification, serum IgM antibody detection; enterovirus 71 IgM antibody value in the early diagnosis of hand, foot and mouth disease and further assessment. Main methods: 1, according to the gene sequence of the nucleic acid of enteroviruses, enterovirus 71 and Coxsackie virus type 16 and other hand-foot-mouth disease pathogens, design, synthetic primers and probes, 38 cases of hand, foot and mouth disease in the real-time fluorescence PCR instrument amplification, detection and analysis of results, and to compare the test results with conventional RT-PCR method. 2, using the RT-PCR method, separation EV71 VPl gene; This fragment was inserted into an expression vector pRSET-A, to be expressed in E. coli, after purification of the expression product, detecting EV71 IgM antibody ELISA The clinical diagnosis of 31 cases of hand, foot and mouth in children and 36 healthy children serum detection. 3, since the onset of continuous daily collection the children serum and throat swab specimens in our hospital in 2010, 38 cases of hand, foot and mouth disease, EV 71 IgM antibodies were detected enterovirus nucleic acid EV 71 virus nucleic acid. Results: 1,38 patients with intestinal virus, enterovirus 71 and Coxsackie virus 16 real-time fluorescence PCR method positive rate: 73.7%, 61.0%, 13.2%, respectively, the positive rate of conventional RT-PCR method as follows: 71.1%, 55.3% and 13.2%. Statistical analysis showed that the real-time PCR method with conventional RT-PCR test results, the difference was not statistically significant. PCR product and recombinant protein size of approximately 910bp and 33 kD expected VP1 gene and protein fragments of the same size. ELISA showed that children with hand, foot and mouth seven cases positive, positive, statistically significant difference was not found in the healthy group. 3,38 cases of hand, foot and mouth disease children IgM antibody to accumulate positive rate, according to the number of days of onset were: 60.5% in one day, two days 71.1%, 81.5% in 3-4 days, 92.1% in the first five days, six days 92.1%; intestinal virus nucleic acid positive rate was 73.6%; the EV 71 specific nucleic acid positive rate was 60.5%. Conclusion: 1, to establish a real-time PCR for the detection HFMD pathogens EV 71, the expression of COX A16, EV method, and the method with conventional RT-PCR anastomosis degrees higher sensitivity and specificity of viral nucleic acid detection, two methods have consistency. Successfully established EV71 IgM antibody detection methods. 3, EV 71 IgM antibodies can occur in the first day of the hand, foot and mouth disease, the positive rate of up to five days to reach peak can be used as one of the early diagnosis of HFMD.
|