Detection of JEV and WNV by quantitative real-time reverse-transcription (qRT)-PCR
Wenjing Liu, Shihong Fu
Abstract
you can use this protocol to detect the RNA of your sample
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).
Steps
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.
