Detection of main flaviviruses using polymerase chain reaction and microwell plate hybridization / 解放军医学杂志

ABSTRACT

Objective To establish a specific,sensitive and applied method for the detection and differentiation of dengue virus types Ⅰ-Ⅳ,Japanese encephalitis virus and yellow fever virus.Methods Based on the genomes sequence analysis,6 pairs of primers were designed.The special capture probes of dengue virus types Ⅰ-Ⅳ,Japanese encephalitis virus and yellow fever virus were amplified,cloned and sequenced.Then the microwell plates were precoated using these capture probes,and the forward primers were labeled using biotin.The samples were then amplified using the biotin labeled forward primers and reward primers.The microwell plate hybridization was processed for detecting and differentiating the virus.The precoated DNA concentration,precoated time,hybridization temperature and hybridization time were optimized carefully.Results The A value of positive samples were over 0.5,while the average A value of the negative samples was less than 0.1.The S/N value exceeded 10.0.Sensitivity experiment suggested the method of PCR-ELISA could detect the virus RNA in 107 times dilution,while RT-PCR could detect the virus RNA in only 106 times dilution.The stability experiment of PCR-ELISA using DVⅠ suggested that the within-batch coefficient of variation was 6.21%,the between-batch coefficient of variation was 9.92%;the within-batch coefficient of variation in negative control was 1.92%,and the between-batch coefficient of variation in negative control was 3.68%.No visible changes were found on the performance of the coated microwell plates when stored in 4℃for 6 monthes.Conclusion PCR-ELISA is a more sensitive and specific method than RT-PCR is in the early detection and type identification of dengue Ⅰ-Ⅳ types virus,Japanese encephalitis virus and yellow fever virus.