Nan Shao1, Fan Li1, Kai Nie2, Shi Hong Fu1, Wei Jia Zhang3, Ying He1, Wen Wen Lei1, Qian Ying Wang1, Guo Dong Liang1, Yu Xi Cao4, Huan Yu Wang1. 1. Department of Viral Encephalitis, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; State Key Laboratory of Infectious Disease Prevention and Control, Beijing 102206, China. 2. State key laboratory for genetic engineering and molecular virology, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China. 3. Department of Viral Encephalitis, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; State Key Laboratory of Infectious Disease Prevention and Control, Beijing 102206, China; School of Public Health, Shandong University, Jinan 250012, Shandong, China. 4. Office of laboratory management, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Abstract
OBJECTIVE: To detect Japanese encephalitis virus (JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction (RT-PCR) detection system was developed. METHODS: By aligning the full-length sequences of JEV (G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay. RESULTS: With the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/µL. The coefficients of variation of this real-time RT-PCR were all < 2.8%. The amplification efficiency of this method was between 90% and 103%. CONCLUSION: A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.
OBJECTIVE: To detect Japanese encephalitis virus (JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction (RT-PCR) detection system was developed. METHODS: By aligning the full-length sequences of JEV (G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay. RESULTS: With the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/µL. The coefficients of variation of this real-time RT-PCR were all < 2.8%. The amplification efficiency of this method was between 90% and 103%. CONCLUSION: A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.
Authors: Annaleise R Howard-Jones; David Pham; Neisha Jeoffreys; John-Sebastian Eden; Linda Hueston; Alison M Kesson; Vanathi Nagendra; Harsha Samarasekara; Peter Newton; Sharon C-A Chen; Matthew V O'Sullivan; Susan Maddocks; Dominic E Dwyer; Jen Kok Journal: Viruses Date: 2022-08-24 Impact factor: 5.818