Establishment Of A Real-time Fluorescence Quantitative PCR Array For Detection Of Viral Encephalitis And Meningitis Syndrome Pathogens

Viral encephalitis and meningitis syndrome is an acute infectious disease caused by viral infection.The clinical manifestations are characterized by fever,headache,vomiting,accompanied by varying degrees of disturbance of consciousness or meningeal irritation.At present,many virus can cause viral encephalitis and meningitis.Among these viruses,herpes simplex virus,arboviruses and human enterovirus are the most common ones.Because of the risks and social burdens caused by such diseases,it is particularly important for early identification,diagnosis,control and treatment.In this study,clinical specimens(cerebrospinal fluid,serum and stool samples)collected during 2015 and 2016 from patients with viral encephalitis and meningitis syndrome in Shanxi Province,Hebei province and Hunan province were rapidly detected for 16 virus pathogens associated with viral encephalitis syndrome by the use of TaqMan real-time quantitative RT-PCR Array methodcombined with laboratory automatic workstation.The results showed that the total detectable rate of CSF in 608 specimens was 25.31%,the total detectable rate of blood was 3.92%,and the total detectable rate of stool was 6.25%.The main pathogens detected in cerebrospinal fluid specimen were enterovirus(23.77%),human herpes virus(0.61%)and Japanese encephalitis virus(0.61%).The main pathogen detected in serum specimens was enterovirus(2.94%)and that in stool specimens was enterovirus(6.25%).Combined with laboratory automatic workstation,the turn-aroundtime of TaqMan real-time fluorescence quantitative RT-PCR Array method established in this study can be shortened to 2 hours.The assay can be used to determine whether there is single or mixed infections of common virus in the samples.The proposed methods rapid,sensitive,high-throughput and easily standardized,and provides an efficient solution for rapid identification of common viral pathogens associated with meningitis encephalitis syndrome.Via the above detection of viral encephalitis and meningitis syndrome pathogens,we found that enterovirus(HEVs)was the most common pathogen present in the samples.In order to develop a more rapid and sensitive detection and serotyping assay,a one-step nested RT-PCR(real-time nested RT-PCR,RTN RT-PCR)method was then established in this study.By downloading the whole genome sequence of 33 enteroviruses collected by GenBank,we designed the internal and external primers and optimized the working condition.The results showed that it was less likely to form primer dimers when the concentration of the outer primer was 10nM and the concentration of inner primer was 200nM.The best amplification efficiency was achieved when the annealing temperature of outer primers was set at 64℃ and that of the inner primers was set at 52 ℃ and the cycle number of outer primers was adjusted to 17.A one-step nested RT-PCR was then used for the specific detection of cultured EV71,CVA16,CVA10 and CVA6 virus strains.The typical specific amplification curves with no cross-reaction among these four viruses were achieved.The sensitivities for the detection of EV71,CVA16,CVA10 and CVA6 were as low as 108 dilutions which is lower than that of real-time fluorescence quantitative PCR for the detection of pan-enterovirus.Finally,the one-step nested PCR,the two-steps nested PCR and the real-time fluorescence quantitative PCR method were used and compared for the detection of HEVs using 140 CSF and fecal samples,the positive rate of one-step nested RT-PCR,two-step nested PCR and the real time fluorescence quantitative PCR was 22.14%,33.57%and 17.14%,respectively.The main advantages of one-step nested RT-PCR method were simple,fast,specific and more sensitive than quantitative PCR.In addition,one-step nested RT-PCR was able to identify the serotypes of enterovirus by sequencing the purified PCR products or eliminate the electrophrosis step by using the melting curve analysis.Therefore,it isfairly suitable to be used in the remote disease control system with a limited budget.Although the sensitivity of one-step nested RT-PCR is slightly lower than that of the two-steps nested PCR,it can avoid the risk of cross contamination caused by the PCR products transfer brought by the two-steps nested PCR.The method established in this study provides a fast and reliable alternative for detecting HEVs with high sensitivity and is more suitable to be applied in local centers of disease control and prevention.