Establishment and application of real-time fluorescent quantitative RT-PCR method for detection of severe fever with thrombocytopenia syndrome virus
LI Pengfei
LI Quan
LI Menglan
WU Ling
LI Zhifeng
Abstract：Objective To establish a highly sensitive one-step real-time fluorescent quantitative reverse transcription PCR(RT-PCR)method based on Taqman probe for detection of severe fever with thrombocytopenia syndrome virus(SFTSV).Methods Primers and Taqman probes were designed targeting the conserved region of the S segment of the severe fever with thrombocytopenia syndrome virus genome to establish a real-time fluorescent quantitative RT-PCR method.The sensitivity,specificity and repeatability of the method were evaluated.And the clinical samples were used to verify the method.Results The limit of detection of this method was 100 copies/ml,and its sensitivity was 1 000 times than that of conventional RT-PCR.Within the linear range,there was a strong correlation(R2＞0.999)between the cyclic threshold(Ct value)and the viral RNA standard.The coefficient of variation for intra-and inter batch repeatability was all less than 1.0%.This method also exhibited high specificity and did not yield cross-reacting signals from other viral nucleic acids.And this method was evaluated using serum samples from severe fever with thrombocytopenia syndrom confirmed patients and healthy volunteers.The results showed that the detection sensitivity and specificity of this method were 100.0%.Furthermore,this method was used to detect the samples from 288 suspected SFTSV infected patients.It demonstrated that 14.9%of the patients were infected with SFTSV.This method was fast and only took 2 hours,including the nucleic acid extraction step.Conclusion The real-time fluorescent quantitative RT-PCR assay established in our study is a reliable method for the early diagnosis of SFTSV infection.
Keywords：severe fever with thrombocytopenia syndrome virusreal-time fluorescent quantitative RT-PCRspecificitylimit of detection
Publication Date：2025-04-20
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：6( 149-153,209 )