Establishment And Application Of Real-time Fluorescence Quantitative PCR Methods For Detection Of Common Viral Pathologys Causing Acute Viral Encephalitis And Viral Meningitis

ObjectiveTo establish Real-time fluorescence quantitative PCR(FQ-PCR) methods fordetection of EV, ADV, EBV, MuV, HHV-6, CMV, HSV-2, MV, HPeV, VZV, JEVand HSV-1.MethodsAccording to the references, primers and probes for common viral pathologyscausing acute viral encephalitis and viral meningitis were designed. FQ-PCRmethods for detection of EV, ADV, EBV, MuV, HHV-6, CMV, HSV-2, MV, HPeV,VZV, JEV and HSV-1were established, and the specificity, sensitivity andreproducibility for the FQ-PCR were verified.Results1. Specific fragments of EV, ADV, EBV, MuV, HHV-6, CMV, HSV-2, MV,HPeV, JEV and HSV-1were obtained and their plasmids were constructed.2.The quantitative detection limit of these methods were respectively1.30×10~1copies/μl,6.74×10~0copies/μl,1.44×10~1copies/μl,3.24×10~1copies/μl,3.00×10~0copies/μl,5.72×10~0copies/μl,7.00×10~0copies/μl,5.39×10~1copies/μl,5.76×10~0copies/μl,1.89×10~1copies/μl,6.49×10~0copies/μl,7.43×10~0copies/μl. Thecorrelation coefficient were over0.99, Amplification efficiency were0.9~1.1, When the detection for the same sample was repeated for three times, coefficient ofvariation(CV) of intra-and inter-assay were calculated and were all less than1.46%and3.13%respectively. No specific amplification was detected for any of their virusexpected target virus.ConclusionThe FQ-PCR methods for common viral pathogens causing acute encephalitisand meningitis were established with a high specificity, sensitivity and repeatability,which can be used to quatitative detecting clincal samples. ObjectiveIn order to provide basic data and scientific evidence for disease prevention andcontrol, the study on epidemic characteristics of common viral pathogens in childrenhospitalized with actue viral encephalitis and viral meningitis were carried out inChangsha from July2010to October2011.Methods1. CSF specimens were collected from hospitallized children diagnosed with acuteviral encephalitis or viral meningitis at the pediatrics medical center of first affiliatedhospital of Hunan normal university in Changsha from July2010to October2011.2. EV, ADV, EBV, MuV, HHV-6, CMV, HSV-2, MV, HPeV, VZV, JEV andHSV-1were detected by FQ-PCR methods.3. Epidemiology and clinical manifestations in hospitalized children with acuteviral encephalitis and viral meningitis were analyzed.Results1. FQ-PCR methods established have used to detected common viral pathologyscause of acute viral encephalitis and viral meningitis.2. Among the93CSF samples, the total viruses positive detected rate was38.71%(36/93), EV was the most common virus. The detected rate of EV was24.73%(23/93), ADV was5.38%(5/93), EBV was4.3%(4/93), MuV was3.23%(3/93),HHV-6was2.15%(2/93), CMV was2.15%(2/93), HSV-2was2.15%(2/93), MV was1.08%(1/93), HPeVwas1.08%(1/93), VZV, JEV and HSV-1was not detected.3. The detection rates in season distribution, sex distribution and age distributionall have no significant difference.4. Clinical presentation, CSF Findings and EEG character in positive cases andnegative cases have no significant difference. Conclusions1. FQ-PCR methods established can be used to detecting common viral pathologyscause of acute viral encephalitis and viral meningitis.2. The total detected rate of viruses was38.71%in our present study, EV was themost common virus.3. There was no significant difference between positive cases and negative casesin season distribution, sex distribution and the age distribution.4. There was no significant difference between positive cases and negative cases inClinical Presentation and CSF Findings, the time of collecting CSF and EEG character.