Objective:To establish a sensitive and specific real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) method for rapid detection of Getah virus (GETV).Methods:All the gene sequences of GETV were downloaded from GenBank database. Clustal X was used for sequence alignment, and specific primers and probes were designed according to highly conserved regions; we established a standard curve using the nucleic acid of GETV as a standard, and the sensitivity, specificity and stability of this method were evaluated respectively.Results:This method could specifically detect GETV and has no cross-reactivity with multiple arboviruses; the sensitivity was 1.0×10 pfu/ml, and the intra-assay and inter-assay coefficients of variation were less than 1%. One case was GETV positive in 196 batches of mosquitoes collected from Hunan province, Hebei province, Fujian province and Chongqing city.Conclusions:We established a TaqMan probe real-time quantitative RT-PCR with high sensitivity and specificity which can be used for screening.

Objective Genotypes(G)1,3,and 5 of the Japanese encephalitis virus(JEV)have been isolated in China,but the dominant genotype circulating in Chinese coastal areas remains unknown.We searched for G5 JEV-infected cases and attempted to elucidate which JEV genotype was most closely related to human Japanese encephalitis(JE)in the coastal provinces of China. Methods In this study,we collected serum specimens from patients with JE in three coastal provinces of China(Guangdong,Zhejiang,and Shandong)from 2018 to 2020 and conducted JEV cross-neutralization tests against G1,G3,and G5. Results Acute serum specimens from clinically reported JE cases were obtained for laboratory confirmation from hospitals in Shandong(92 patients),Zhejiang(192 patients),and Guangdong(77 patients),China,from 2018 to 2020.Seventy of the 361 serum specimens were laboratory-confirmed to be infected with JEV.Two cases were confirmed to be infected with G1 JEV,32 with G3 JEV,and two with G5 JEV. Conclusion G3 was the primary infection genotype among JE cases with a definite infection genotype,and the infection caused by G5 JEV was confirmed serologically in China.

Objective:A highly sensitive and specific real-time quantitative TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for rapid and accurate detection of Tibet orbivirus (TIBOV).Methods:The TIBOV genomic sequences from GenBank were analyzed by Clustal X 2.1 and the specific primers and probe were designed in the conserved segment of VP4 gene. RNA standard was obtained from in vitro transcription and a TaqMan RT-PCR assay for TIBOV was established. The sensitivity, specificity and repeatability of this method were evaluated. Results:The assay showed a good amplification curve within the range of 1.0×10 2~8 copies/reaction template, the detection limit was 1.0×10 2 copies/reaction, the coefficients of variation of Ct values in repeat detections were all less than 1.5%. No cross-reaction was found in this assay. Variable mosquito samples were screened by this assay and the result showed TIBOV negative. The prepared TIBOV simulated positive samples were 100% detected. Conclusions:The assay developed in this study is specific and sensitive for detection of TIBOV and can be used for laboratory detection and routine surveillance.