Jul 11, 2022
Detection of JEV and WNV by quantitative real-time reverse-transcription (qRT)-PCR
- Wenjing Liu1,
- Shihong Fu2
- 1Department of Basic Medicine, School of Medicine, Qingdao University, Qingdao, People’s Republic of China;
- 2Department of Arbovirus, NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People’s Republic of China
Protocol Citation: Wenjing Liu, Shihong Fu 2022. Detection of JEV and WNV by quantitative real-time reverse-transcription (qRT)-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bdayi2fw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 05, 2020
Last Modified: July 11, 2022
Protocol Integer ID: 33848
Abstract
you can use this protocol to detect the RNA of your sample
Materials
RNA of mosquito grinding supernatant , serum sample and CSF samples,AgPath-IDTM One-step RT-PCR Kit (AM1005, Thermo Fisher Scientific, USA) ,Stratagene real-time PCR instrument (model MX300; Thermo Fisher Scientific, Waltham, MA, USA) .
Before start
RNA was extracted from all collected samples (serum, CSF, and mosquito grinding supernatant) using a Tianlong Nucleic Acid Automatic Extractor with the Tianlong Nucleic Acid Extraction Kit(EX-RNA/DNA virus, Suzhou Tianlong Biotechnology Co., Ltd, Suzhou, China).
Detection of JEV and WNV by quantitative real-time reverse-transcription (qRT)-PCR
Detection of JEV and WNV by quantitative real-time reverse-transcription (qRT)-PCR
The instrument used was a Stratagene ™, real time PCR machine (manufacturer: Thermo Scientific, model: MX300) for quantitative fluorescence qRT-PCR detection of JE virus and West Nile virus. AgPath-IDTMOne-step RT-PCR Kit (REF: AM1005, manufacturer: Thermo Fisher Scientific.) was the detection kit used, the total volume of the detection system was 20 μl, containing 10 μl of 2Xbuffer4 μl of RNAse Free Water4 μl, 1 μl of primers, probes, and enzyme mixture (Mix), and 2 μl of template RNA.
The reaction program was: 45 ° C for 10 min, 95 ° C for 10 min for 1 cycle, 95 ° C for 15 s and 60 ° C for 1 min for 40 cycles. Using primers and probes specific for the JE virus gene and the West Nile virus gene.
The final result is checked on the real time PCR machine,the sample with CT value over 35 thought to be negetive, sample with CT value lower than 35 thought to be positive.
The positive sample will be sent to do PCR specific to JEV genes to be ferther tested.