Establishment And Evaluation Of Real-time RT-PCR Method For Detection Of Enterovirus 71

Background and ObjectiveEnterovirus 71 (EV71)is the main etiological agent of Foot and Mouth Disease (HFMD)a childhood disease characterized by fever, multiple ulcers in the mouth and vesicles on the limbs and buttocks.It is associated with neurological complications such as aseptic meningitis,brainstem encephalitis and poliomyelitis-like paralysis which have led to fatalities.The classical "gold standard" diagnosis for EV71 is cell culture.However.this kind of diagnosis requires several weeks.Besides,it could be hindered by low viral titre or antigenie drifts in the specimens.Molecular methods such as PCR have been developed to detect EV71 specifically and proved to be more sensitive than the cell culture method.In recent years,real-time PCR was gained wider acceptance for viral diagnosis in laboratories because of higher sensitivity and specificity,and faster rate of detection,and it provides real-time monitoring of the amplification process through fluorescence emission.With the increased concerns of the fatal HFMD caused by EV71.there is a need for a rapid and specific method to distinguish this virus from other HFMD-causing enteroviruses such as Coxsackievirus A16 and Echovirus.Here,we have developed a rapid and more sensitive real-time RT-PCR assay for detecting the DNA of EV71 specifically from clinical specimens. Methods:Specific primers and probe were designed based on the EV71 nucleoprotein gene sequences published in Gene bank. A 226bp fragment was amplified by polymerase chain reaction (PCR) technique.After purification, the gene fragment was linked with the PGM-T vector to construct the recombinant plasmid (TA clone).By PCR identification and DNA sequencing analysis confirmed that the construction of recombinant plasmid was successful.With the recombinant plasmid as templates, we established a real-time PCR method and optimized the reaction system and condition. Then, the sensitivity, specificity and reproducibility of the system was tested.We conducted experiment to compare the real-time PCR with conventional RT-PCR and virus culture and isolation. Samples from HFMD were collected in Henan Province for EV71 isolation using RD cells and EV71 DNA detection using conventional RT-PCR.Results:1.Recombinant plasmid was conducted by TA clone.2.With the recombinant plasmid as templates.The optimal reaction condition was as follows:each 20μl PCR mixture contained 2.0μl of EV71 RNA,0.8μl concentration of each primer.and 0.4μl probe, RT-PCR Buffer 10μl;EX Taq-HS 0.2μl and 2ul RT Enzyme Mix.After 5 min at 42℃for predenaturation,the DNA templet was subjected to 40 cycles of PCR. Each cycle was 95℃for 5 sec(denaturation)and 60℃for 30 sec (annealing and extension).3.The constructed new real-time PCR method was evaluated.Quantification standard curve based on the geneomic copy was drawn.The linear equation was expressed as Y=-3.370X+49.790.The range of linear assay was obtained between 102 copies/μl and 108 copies/μl.The results showed the sensitivity was 102 copies/μl,but the sensitivity of conventional RT-PCR was 104 copies/μl.Real-time PCR method could distinguish EV71 from other closely related pathogenic microorganism.eg CA16,Echo25,Echo30,Rotavirus,Japanese Encephalitis.Reproducibility test showed that coefficient variables were all less than 11% in 5 different systems. 4.We conducted experiment to compare the real-time PCR with qualitative RT-PCR and virus culture and isolation.The positive rate of the real-time PCR is 61.2%,and the positive rate of conventional RT-PCR method, virus culture is 16.4% and 52.4%.Conclusions:One sensitive, specific and quick-performing method was established for EV71 based on TaqMan PCR, which could applied for HFMD diagnosis.