关键词: 发热伴血小板减少综合征布尼亚病毒；巢式PCR；引物优化

Abstract:

Objective   PCR primers for amplification of severe fever with thrombocytopenia syndrome virus(SFTSV) were optimized in order to improve the detection sensitivity and specificity in sera from patients suffering from severe fever with thrombocytopenia syndrome(SFTS). Methods   RT-PCR primers were designed from the sequences of L, M, and S segments of SFTSV genome by using Primer Premier 5.0 and Oligo 6.0 softwares (L1,M1,S1) or obtained from references(L2, M2, S2). 12 primers were divided into 6 groups to amplify these segments. SFTSV RNAs in sera from patients with SFTS and normal sera were amplified by PCR and sequenced. SFTSV RNA detection sensitivity and specificity between primers were compared. At the same time, the relationship between the sensitivity and serum acquisition time were also compared. Results   RTPCR results showed that SFTSV detection sensitivity was different between primers. S-segment was most frequently amplified with one pair of primer(S1), while L-segment was amplified occasionally and M segment was never amplified. Sensitivity for the detection of SFTSV RNA in sera was 64.3% when it was perfomed within 7 d, compared with 37.5% in 7~14d. The difference between them was not statistically significant［P＞0.05(=0.377)］. No segment of SFTSV was amplified in normal sera. Specificity of all primers was 100%. Conclusion   S1 primer pair is the most sensitive one among all primer pairs derived from S, M- and L-segments of SFTSV for RT-PCR amplification of S-segment.

Key words: SFTSV； Nested-PCR； Primers optimization