| Introduction:Virus infection has gradually become the most common central nervous system infections in the clinical disease. Multiple viral infections such as arbovirus and enterovirus can cause varying degrees of neurological symptoms. Meningoencephalitis are caused by many viruses, including Eastern equine encephalomyelitis virus, Western equine encephalomyelitis virus, Venezuelan equine encephalomyelitis virus, Tick-borne encephalitis virus, Japanese encephalitis virus and West Nile virus from the arboviruses class, Nipah virus and Hendra virus from Henonipah virus genus, the paramyxovirus family, Coxackievirus A, B and Poliovirus from enterovirus genus, the paramyxovirus family. The twelve viruses belong to RNA viruses and are all highly infectious diseases pathogens, which could transmit through variety of ways, including insect-borne, droplets, contact transmission and so on. The current study demonstrates that meningoencephalitis viral infection could cause complex clinical symptoms, including fever, headache, cough, sore throat, body pain, headache, chills and fatigue. Some patients also have diarrhea, vomiting and even a sudden high fever, pneumonia when their sickness are more worsen. Serious condition could cause unconscious, convulsions, neck stiffness, respiratory failure, multiple organ injury and even leading to death. At present, there are no effective vaccines or drugs for prevention and treatment of twelve meningoencephalitis viruses.At present, the detection of meningoencephalitis virus is mainly relied on traditional methods such as virus isolation and serological detection. Virus isolation requires relatively high experimental conditions, longer time and larger workload and can not handle the large number of samples during the same period. Influencing factors such as easily variable virus and multiple cross homology with viruses in the sample are all likely to affect antigen typing in the neutralization test.With the rapid development of molecular biology diagnostic techniques in recent years, Fluorescence quantitative polymerase chain reaction (FQ-PCR) technique has become more and more important method for rapid diagnosis of virus. In many quantitative PCR technologies, FQ-PCR is widely used at home and abroad. The principle of FQ-PCR is that the starting point quantitative and real-time monitoring to accumulate fluorescence intensity through fluorescence detection system. FQ-PCR labeled TaqMan probe technique is most widely used, which overcome the difficulty of detection for low content virus in samples by traditional detection techniques. Through designing specific primers and probes for target gene and optimizing reaction system and reaction condition, a rapid, sensitive and specific FQ-PCR detection method can be established successfully. It only takes3-4hours to do it from the extraction of nucleic acid to the quantitative PCR process and large samples can be detected at the same time, so the method is able to save a lot of test time and response cost. The method has the characteristics of high sensitivity, specificity, good linear relationship, simple operation, high degree of automation, pollution prevention, high-throughput and rapid. It is now widely used as the rapid detection and quantitative analysis for human and animal viral diseases and so on. With further research and development, although there are some uncertainties, the method still has a broad application value.If we want obtain reliable results in the process of detection and quantification of meningoencephalitis viruses by FQ-PCR, the standards and controls must be needed. Due to some meningoencephalitis viruses are infectious and pathogenic and some viral infections reports are rare in China, It brings the tremendous difficulties and dangers to study, under this background, it is a very safe and effective way to generate standard RNA by pseudotype virus technology. Pseudotype virus is that it has its own genetic material and capsule membrane is coated with the glycoprotein of other virus, which has the characteristics of another virus infection. Pseudotyped virus has true virus envelope protein, so it has the same infection to host cells. The inconsistent phenomenon of genotype and the phenotype called pseudotyped, virus with this feature called pseudotyped virus. Pseudotyped virus is a tool for people to study the relationship between virus and host cell. It not only has the target glycoprotein for study on the capsule membrane, without the interference of other proteins, compared to the real virus, but also nucleic acid molecules encoding capsule membrane protein gene is modified out, a replication-defective type, so the virus can only infect a cell cycle and is biosafety. In order to achieve the aim of the stable expression transient expression of exogenous gene, the exogenous gene to be packaged with marker gene are integrated into the host cell genome by transfection based on retroviral vector. Through the retroviral replication and packaging signal provided by retroviral vector, it can get replication-defective pseudotype virus particle, of which exogenous gene wrapped by retrovirus capsid.Pseudotype virus particle, containing the target gene used as a control and standard of RNA virus by real-time reverse transcription-polymerase chain reaction (RT-PCR), has the following advantages:(1) no biological risk of infection.(2) It has a very good simulation effect with pathogen RNA, therefore, it have a very good similarity with the true viral of specimens in the nucleic acid extraction process. Standard RNA has no bio-security risks, while avoiding the problem that the plasmid standard in the commercial kits developed in the past could not effectively control the reverse transcription process of samples. Pseudotypevirus RNA does not easily appear cross-contamination on experimental apparatuses and lead to false positive results in the environment.Therefore, this research intends to take Eastern equine encephalitis virus, Western equine encephalitis virus, Venezuelan equine encephalitis virus, Tick-borne encephalitis virus, Japanese encephalitis Virus, West Nile virus, Nipah virus, Hendra virus, Enterovirus71, Coxsackievirus A16, Coxsackievirus B and Poliovirus, a total of twelve viruses for the research object, through constructing retroviral expression vector containing twelve viruses gene and screening to establish cell line producing high titer virus, then taking the pseudotype virus particle from the cell line as a RNA standard for viruses detection, a TaqMan one-step real-time RT-PCR Array is established, which is able to one-time detect twelve meningoencephalitis viruses for rapid diagnosis.Methods:1. The generation of twelve meningoencephalitis pseudotype viruses used as RNA standard. Target genes of twelve meningoencephalitis viruses were selected according to domestic and international references and were downed from the GenBank. Specific primers and TaqMan probes were designed based on the highly conservative sequence of each target gene, respectively and were all in the order combined in a gene fragment. Then target gene fragment was synthesized and cloned into PLPCX retroviruses vector for constructing recombinant plasmid containing twelve encephalitis and meningitis viruses gene by corporation. After identification of sequencing, recombinant retroviruses vector and coated plasmid PVSVG were co-transfected into GP2-293cell by liposome method to package pseudotype virus. Then GP2-293packaging cell were screening by G418to select cell producing target virus. Cell supernatant was collected every three or four days, altogether collected fourteen times. One-step real-time RT-PCR had been to evaluate pseudotype virus titer after removing the DNA contamination in virus RNA by identification of real-time PCR.2. Development of a TaqMan one-step real-time RT-PCR method for detecting twelve meningoencephalitis viruses one time.The optimal reaction system and reaction conditions of TaqMan one-step real-time RT-PCR was established by using pseudotype virus as a RNA standard to detect and differentiate twelve meningoencephalitis viruses one time in96well. The sensitivity, reproducibility and specificity of this method were also evaluated.Results:1. Twelve meningoencephalitis viruses gene were confirmed to insert into retroviruses expression vector by sequencing. It indicated that recombinant retroviruses vector PLPCX containing twelve meningoencephalitis viruses gene was constructed, successfully. 2. Recombinant retroviruses vector PLPCX containing twelve meningoencephalitis viruses gene and coated plasmid PVSVG were co-transfected into GP2-293cell by liposome method. The result of real-time PCR and RT-PCR proved that pseudotype virus had been successfully packaged after removing the DNA contamination in virus RNA. GP2-293cell line producing pseudotype virus was constructed by G418screening and the pseudotype virus titer peaked at10’copies/μl by establishing standard curve method.3. Uniform reaction system of TaqMan one-step real-time RT-PCR Array using pseudotype virus as a RNA standard was determined.25μl of total volume were as follows,2×one-step RT-PCR buffer12.5μl, forward primer0.1μl, reverse primer0.1μl, probe0.1μl, DEPC water4.2μl, Hot-start Taq enzyme0.5μl, reverse transcriptase0.5μl, dNTPs0.4μl, RNase inhibitor0.5μl, one-step enzyme diluent, RNA template5μl. Uniform reaction condition was as follows, reverse transcription at50℃for15min, The pre-degeneration at95℃for15min, then40cycle for degeneration at95℃for5s and Annealing/extension at60℃for45s.4. The evaluation results of methodology(1) Quantitative standard curves using standard RNA as analysis indicators were drawn and the correlations between Ct value of standard curve and copy number of initiative template achieved to over0.98. Amplification efficiencies were between90-120%. Slopes were between-3.0～-3.5.(2) Sensitivity result revealed that the lowest detectable limits of twelve meningoencephalitis viruses by TaqMan one-step real-time RT-PCR Array ranged from10’copies/μl to102copies/μl, whereas traditional RT-PCR ranged from104copies/μlto105copies/μl.(3) Repeatability result indicated that the intra-assay coefficients of variation of standard RNA with high concentration ranged from0.411%to2.358%, whereas the inter-assay ranged0.585%to4.024%. The intra-assay coefficients of variation of standard RNA with median concentration ranged from0.242%to2.109%, whereas the inter-assay ranged0.821%to3.070%. The intra-assay coefficients of variation of standard RNA with low concentration ranged from0.412%to2.160%, whereas the inter-assay ranged0.602%to2.973%. Coefficients of variation between Ct values of each concentration were all lower than5%.(4) Specificity result showed that no cross-reactions were found between Eastern equine encephalomyelitis virus, Western equine encephalomyelitis virus, Venezuelan equine encephalomyelitis virus, Japanese encephalitis virus, West Nile virus, Nipah virus, Hevdra virus, Enterovirus71, Coxackievirus A16, Tick-borne encephalitis virus, Coxsackievirus B, Poliovirus and Enterovirus75, Denge virus, Vibrio cholerae01. Vibrio cholerae0139, E. coli0157.Conclusions:Pseudotype virus containing twelve meningoencephalitis viruses gene is successfully prepared through the packaging technology of pseudotype virus and is used as a RNA standard to establish the TaqMan one-step real-time RT-PCR Array method. The method established shows high sensitivity, good repeatability and stong specificity and is able to one time detect twelve meningoencephalitis viruses in a panel. |
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