Development of an all-in-one real-time PCR assay for simultaneous detection of spotted fever group rickettsiae, severe fever with thrombocytopenia syndrome virus and orthohantavirus hantanense prevalent in central China

Central China has been reported to be one of the most important endemic areas of zoonotic infection by spotted fever group rickettsiae(SFGR), severe fever with thrombocytopenia syndrome virus (SFTSV) and orthohantavirus hantanense(HTNV). Due to similar clinical symptoms, it is challenging to make a definite diagnosis rapidly and accurately in the absence of microbiological tests. In the present study, an all-in-one real-time PCR assay was developed for the simultaneous detection of nucleic acids from SFGR, SFTSV and HTNV. Three linear standard curves for determining SFGR-ompA, SFTSV-L and HTNV-L were obtained within the range of 101-106 copies/L, with the PCR amplification efficiencies ranging from 93.46% to 96.88% and the regression coefficients R2 of >0.99. The detection limit was 1.108 copies/L for SFGR-ompA, 1.075 copies/L for SFTSV-L and 1.006 copies/L for HTNV-L, respectively. Both the within-run and within-laboratory coefficients of variation on the cycle threshold (Ct) values were within the range of 0.53%-2.15%. It was also found there was no statistical difference in the Ct values between with and without other non-target bloodborne virus nucleic acids (PSFGR-ompA =0.186, PSFTSV-L=0.612, PHTNV-L=0.298). The sensitivity, specificity, positive and negative predictive value were all 100% for determining SFGR-ompA and SFTSV-L, 97%, 100%, 100% and 99.6% for HTNV-L, respectively. Therefore, the all-in-one real-time PCR assay appears to be a reliable, sensitive, rapid, high-throughput and low cost-effective method to diagnose the zoonotic infection by SFGR, SFTSV and HTNV. Author SummarySpotted fever, severe fever with thrombocytopenia syndrome (SFTS), and hemorrhagic fever with renal syndrome (HFRS) sporadically have outbreaks in central China. Due to the similarities in clinical symptoms and the absence of reliable diagnostic methods, clinical diagnosis and treatment frequently result in misdiagnosis or missed diagnosis. Thus, the development of a fast and accurate diagnostic method is crucial for prevention and precise treatment. In this study, we designed an all-in-one real-time PCR assay to differentiate spotted fever group rickettsiae(SFGR), severe fever with thrombocytopenia syndrome virus (SFTSV) and orthohantavirus hantanense(HTNV). The gene ompA of SFGR, as well as the gene segment L of SFTSV and HTNV, were used as targets to design primers and probes for amplification. Through the verification of nucleic acid and clinical sample detection, the sensitivity of this detection method exceeded 97%, and its specificity was 100%. This new assay could be applied in epidemiology and clinical diagnosis, to control new outbreaks, reduce diagnostic and identification time, and improve test efficiency.