A New Strategy For The Detection Of Multiple Pathogens Of Viral Encephalitis

Viral encephalitis in the central nervous system infection is a common butdangerous disease. Its progress is extremely rapid, causing morbidity andbringing a lot of problems due to the lack of an immediate diagnosis ortreatment. Although many patients can be treated with antiviral medicines, butthe specific pathogens of viral encephalitis are still not known. In order todecrease mortality and increase survival, we need to develop an effective andaccurate detection method to identify the viral agents which cause the disease.Current testing methods (such as viral culturing, ELISA assay, biochemicaldetection, etc.) are labor-intensive, expensive time-consuming and can not detectmultiple pathogens simultaneously. A new molecular detection method based onLuminex200, has the advantages of sensitivity, relatively low cost, rapiddetection and the ability to discriminate between species and strains if suitableprimers are selected.Objective To establish a new multiplex PCR diagnostic platform based on the Luminex200suspension array system, which is rapid, sensitive and specificin detecting several pathogens simultaneously. This platform compared totraditional isolation and identification of etiology, will provide us with thedetection of more economical, more timely and more comprehensive etiologicalinformation. Method In this study, the most five common pathogens causingviral encephalitis (EV71, CA16virus, ECHO virus, Japanese encephalitis virus,and Epstein-Barr virus) are included. We searched pathogen-specific genes fromGENEBANK, then designed specific primers and probes with Oligo6.0. Allprimer sequences were facilitated by application of the BLAST program for thespecifcity prediction to ensure specificity and accuracy of primers and probes.Choose five unique color-coded microspheres that can be suspended in theliquid phase. Use direct hybridization of a labeled polymerase chain reaction(PCR)-amplified target DNA to microsphere sets bearing oligonucleotidecapture probes specific for each sequence and finally to evaluate the feasibilityof clinical specimens. Result The single-target PCR results suggest that the fivepairs of primers we designed are specific. The hybridization temperature of55℃for15min was found to be optimal for hybridization reaction. The annealingtemperature appropriate for multiplex PCR was carried out at58℃. Thereactions were cycled40times:94℃for30s,58℃for90s, and72℃for90s,with a fnal extension step of72℃for10min. Based on this assay, we cansimultaneously determine five kind of viral (EV71, EB, ECHO, JEV, CA16).The specificity was100%. Also, we examined cerebrospinal fluid clinicalspecimens from44cases of viral encephalitis, including two cases ofEpstein-Barr virus encephalitis, two cases of JEV encephalitis, thirty-five casesof HFMD encephalitis, three cases of other central nervous system infection, thetest results was consistent with the staining and PCR results. The coincidence was100%. Conclusion This new multiplex detection assay based on theLuminex200suspension system is rapid and accurate. Also, it was found to behighly sensitive and specifc in simultaneous detection of several pathogens.This method will provide us with a more convenient detection, and morecomprehensive etiological information, and help us apply specific antiviraldrugs reasonably in clinic.