Multiplex Reverse Transcription-nPCR: Application In The Study Of Causative Organisms Of Viral Encephalitis In Children

Objective Viral encephalitis(VE)is a kind of disease commonly occurred in childhood and constitutes complex etiology and pathogenesis. The present epidemic investigation indicates that herpes simplex virus (HSV) and enterovirus (EV) are mostly significant causes of acute VE. Conventional techniques for identifying both HSV and EV in VE have had limited success. So, we have devised a simple and robust PCR strategy—multiplex reverse transcription nest polymerase chain reaction (M-RT-nPCR) to detect HSV DNA(HSV-Ⅰand HSV-Ⅱ)and EV RNA(but not echovirus types 22 and 23)simultaneously to create a reliable,accurate,rapid and economic detection method. Methods Because EV is RNA virus, M-RT-nPCR was developed. The method involves a reverse transcription step followed by a multiplex nested PCR. In multiplex PCR more than one target sequence can be amplified by including more than one pair of primers in the reaction. It can effectively detect some kinds of viruses in a reaction. Subjects were 100 cerebrospinal fluid (CSF) specimens from VE diagnosed in our pediatrics during September 2001 and February 2003. We firstly detected 35 CSF specimens from VE that were positive for HSV or EV with Enzyme Linked Immunosorbent assay (ELISA) to test this method. Then HSV DNA and EV RNA in 65 CSF specimens from VE that were negative with ELISA and 73 specimens from control group were detected. Environment without RNase was created. Then we used a guanidium thiocyanate lysis buffer to extract viral RNA and DNA from CSF in one step. Primer design is the key to our success,the primer sequences have designed specifically according to the reference. PCR thermal cycling incubations used were as follows: reverse transcription was performed in a single reaction by incubation at 37℃ for 60 minutes and 95℃ for 5 minutes preceding 30 cycles of 45 seconds incubations at 95℃,50℃ and 72℃; Further amplification with internal (nested) primers was performed in a reaction mixture and under the reaction circumstance identical to that described above, except that 1ul of the initial reaction mixture was added as the template. All thermal cycling was performed using PE Applied Biosystems 9600 machines. Amplification products were identified by their molecular weight following electrophoresis of 10ul of the secondary reaction mixture through an ethidium bromide stained 1.5% agarose gel and UV light transillumination. To confirm the results, all PCR-positive specimens were tested at least twice and we compared the method with ELISA and typical nPCR.Results The average duration of tests was 5 hours. Compared the method with ELISA and typical nPCR, the concordance respectively was 96% and 100%. Of the 100 VE CSF specimens, 35 were found to be positive including that HSV was detected in 18 specimens (18%), EV in 17 specimens (17%), P>0.05, there were no differences in their incidence rates. Among 26 serious ill patients' CSF of the 100 specimens, HSV was detected in 14 specimens (53.85%), EV was detected in 7 specimens (26.92%), P<0.05,the difference is significant. The detection rates varied with age variation, among 0～, 3～, and 7～14 years old group HSV positive rates respectively were 7.4%, 11.9% and 35.5%; EV positive rates respectively were 25.9%, 19.05% and 6.5%, both P<0.05.Conclusion M-RT-nPCR assay is a reliable,economic and rapid method to detect HSV and EV simultaneously in the earlyphase of disease. Such a method is worth application extensively in clinical practice. HSV and EV are mostly causative organisms in VE. HSV is a main organism in severe VE. The older children are, the higher the incidence rate of herpes simplex encephalitis is; That of enteroviral encephalitis is in the opposite direction.