Early diagnosis of novel SFTS bunyavirus infection by quantitative real-time RT-PCR assay.
BACKGROUND
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease recently identified to be caused by a novel bunyavirus (SFTSV). The clinical diagnosis is urgently needed to differentiate the disease from other infections.


OBJECTIVE
To develop a sensitive quantitative real-time RT-PCR assay for rapid detection of SFTSV viral RNA and evaluate potential use for clinical diagnosis of SFTS.


METHODS
Primers and probes were designed to target the L, M, and S segments of SFTSV, and standard curves were established based on serial dilutions of in vitro transcribed viral RNA or viral RNA extracts. The serum samples collected from 70 laboratory confirmed SFTS patients, 114 non-SFTS patients, and 400 healthy donors were analyzed.


RESULTS
Based on three optimized primer-probe sets to detect L, M, S genes of SFTSV, the quantitative real-time RT-PCR assay could discriminate SFTSV infection from other vector-borne viral diseases in human with potential detection limit of 10 viral RNA copies/μl or 10 TCID(50)/ml virus load. Strong linear correlations (r(2)>0.99) between the C(t) values and viral RNA standards over a liner range were obtained. The assay specificity was determined by sequence alignment and experimentally tested on various related viruses. Evaluation of the study method with clinical serum samples showed 98.6% clinical diagnostic sensitivity and over 99% specificity.


CONCLUSIONS
The quantitative real-time RT-PCR assay established in this study can be used as a reliable method for early diagnosis of SFTSV infection.
National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
Yulan
Y
Sun
Mifang
M
Liang
Jing
J
Qu
Cong
C
Jin
Quanfu
Q
Zhang
Jiandong
J
Li
Xiaolin
X
Jiang
Qin
Q
Wang
Jing
J
Lu
Wen
W
Gu
Shuo
S
Zhang
Chuan
C
Li
Xianjun
X
Wang
Faxian
F
Zhan
Wenqing
W
Yao
Zhenqiang
Z
Bi
Shiwen
S
Wang
Dexin
D
Li
D001483:Base Sequence; D002044:Bunyaviridae Infections / Q000097:blood / Q000175:diagnosis* / Q000821:virology; D016022:Case-Control Studies; D017931:DNA Primers / Q000235:genetics; D042241:Early Diagnosis; D005814:Genes, Viral; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D057230:Limit of Detection; D016856:Phlebovirus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D012367:RNA, Viral / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D019562:Viral Load
D017931:DNA Primers; D007074:Immunoglobulin G; D012367:RNA, Viral