key: cord-0985950-f9zmztef authors: Zhou, Jingyu; Wang, Qiujing; Zhu, Lijun; Li, Shibo; Li, Wei; Fu, Yongfeng; Cheng, Xunjia title: Development and evaluation of a rapid detection assay for severe fever with thrombocytopenia syndrome virus based on reverse-transcription recombinase polymerase amplification date: 2020-04-21 journal: Mol Cell Probes DOI: 10.1016/j.mcp.2020.101580 sha: 0f384fde75a9f3efe7d71ee35cd748967cc12fcc doc_id: 985950 cord_uid: f9zmztef Abstract Rapid detection of severe fever with thrombocytopenia syndrome virus (SFTSV) is crucial for its control and surveillance. In this study, a rapid isothermal real-time reverse-transcription recombinase polymerase amplification (RT-RPA) assay was developed for the detection of SFTSV. The detection limit at 95% probability was 241 copies per reaction. A test of 120 serum samples of suspected severe fever with thrombocytopenia syndrome (SFTS) patients revealed that the sensitivity and specificity of the RT-RPA assay was approximately 96.00% (95%CI: 80.46%–99.79%) and 98.95% (95% CI: 94.28%–99.95%), respectively; the kappa value was 0.9495 (P＜0.001). The Bland-Altman analysis showed that 87.50% of the different data points were located within the 95% limits of agreement, indicating a good correlation between the results from RT-RPA assays and those of RT-qPCR assays. In conclusion, the rapid and efficient RT-RPA assay can be a promising candidate for point-of-care detection method of SFTSV. Severe fever with thrombocytopenia syndrome virus (SFTSV), belonging to the 19 family Phenuiviridae, the genus Banyangvirus, the species Huaiyangshan 28 Currently, detection methods of SFTSV comprise nucleic acid testing, serological 29 detection, and virus isolation [1, 8, 9] . Due to the long turnaround time of serological 30 detection and virus isolation, nucleic acid testing, such as real-time Table 1 . 94 assays, RT-qPCR assays and RT-PCR assays. 95 Primer/ Probe Sequence (5'-3') The candidate primer pairs were tested for detection of the SFTSV RNA standard 171 using RT-RAA basic kits and the amplicons were analyzed with 0.8% agarose gel. The primer pair with the best amplification efficiency and specificity was used for the Table 1 ). Besides, different incubation temperatures and 177 concentrations of primers and exo-probe were tested for the specificity and rapidity of 178 the assay. The optimized performance was achieved when incubated at 39 °C with a 179 primer/probe final concentration settled to 420 nM/ 60 nM. The sensitivity of the SFTSV RT-RPA assay was determined by testing with a serial 183 dilution of RNA standard from 10 6 -10 copies/µL. Robust fluorescence signals of the 184 10 6 copies/µL standards were rapidly observed within 1.85 -1.92 minutes. Positive 185 signals of 10 5 -10 2 copies/µL dilutions were successfully detected within 2.73 -9.41 186 minutes ( Figure 1A) . Results from semi-logarithmic regression showed a good RT-qPCR assay also illustrated that the detection time of RT-RPA assay was shorter 190 than that of RT-qPCR assay by an average of 10 minutes. The detection limit was further determined by applying probit regression analysis 192 using a data set of six RT-RPA runs on the standard plasmids, and the predicted LOD 193 was 241 copies per reaction at 95% probability ( Figure 1C ). The specificity of the SFTSV RT-RPA assay was determined using nucleic acid Bergeron 460 Influence of sequence mismatches on the specificity of recombinase polymerase Zhang 471 Nucleic acid detection with CRISPR-Cas13a/C2c2 microdevice for multiplex and real-time identification of food poisoning bacteria 476 Stetten 478 Review: a comprehensive summary of a decade development of the recombinase