Establishment And Application Of RAPD Combined With Real-time PCR For Detecting Herpes Virus

Background:viruses is one of the major pathogens of the central nervous system infection,According to epidemiological statistics,the annual incidence of viral neurological infections in the world is 3.5-7.4/100,000.Patients with viral infections of the central nervous system(CNS)may present with a variety of neurological symptoms,most commonly dominated by either encephalitis or meningitis,myelitis.Encephalitis is associated with high morbidity and mortality,especially when diagnosis and treatment is delayed.Commonly used laboratory technique for the detection of viruses infection are cell culture and Antibody detection.The former is time-consuming,the latter is less specific and can not distinguish between previous infections and active infections,and the sensitivity of both is limited.Molecular biology detection technology greatly improves the detection rate of viral nervous system infection,such as Fluorescence quantitative PCR,multiplex PCR,LAMP(loop-mediated isothermal amplification).But several studies confirmed that the DNA load can be low in CSF in case of encephalitis.Therefore,it is necessary to establish a more sensitive,fast and specific detection method.Objective:To develop a more sensitive method for quantitative detection of herpes simplex virus,human cytomegalovirus,varicella-zoster virus by random amplified polymorphic DNA combined with fluorescence quantitative polymerase chain reaction(RAPD-qPCR).Methods:According to the literature,dozens of random primers for RAPD were selected and randomly amplified three herpes viruses.The stable and clear bands were selected to isolate,purify and sequence from 2%agarose gel electrophoretic plate.Then more than 99%matched gene sequences were chosen as the target fragment compared with the genebank existing virus sequences using blast-nr and in internal,specific primers were designed with primer 3.0.We use the products of RAPD as the template to process qPCRand adjust the reaction conditions for establishing the new diagnosis method.Take the HSV?HCMV and VZV DNA as templates to analyze the sensitivity and specificity of qPCR and RAPD-qPCR.55 patients with clinical viral neurological infections and 21 patients with non-infectious neurological diseases were included in the study to assess the detection rateResults:After screening,a set of the best random primer and specific primer for each virus was chosen,and the RAPD-qPCR could detect 1:106HSV,1:105 HCMV and 1:105 VZVDNA respectively,while the single qPCR could detect only 1:103HSV,1:102HCMV and 1:103 VZVDNA.The sensitivity of RAPD-qPCR is higher 100-1000 times than qPCR.CT value was less than 22.96 ±0.81 when detecting more than 1:105diluted virus DNA by RAPD-qPCR,so that the results was easy to distinguish.Among the 55 cases,8 was positive for VZV,and the positive rate was 14.55%,HSV and HCMV not detected in this study.21 patients with non-infectious neurological diseases were negative for three viruses.Conclusion:RAPD-qPCR was a more sensitive and specific method to detect three herpesviruses than single qPCR.Preliminary clinical application showed that:8 cases were positive for VZV DNA in 55 patients diagnosed with viral neurological infection,the detection rate was 14.55%.