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 hantaan virus prevalent in central China

Abstract

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 hantaan virus (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/μL, 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/μL for SFGR-ompA, 1.075 copies/μL for SFTSV-L and 1.006 copies/μL 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 single template and multiple templates (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.

Fig 1. Schematic illustration of the proposed all-in-one real-time PCR assay.

(a) Detection principle schematic diagram; (b) Schematic of the SFGR, SFTSV and HTNV genome and corresponding primer sequence design. (Created by Figdraw., export ID:UTYWAc96d8).

Fig 2. Primer and probe concentration optimization.

(a) SFGR’s amplification curve; (b) SFTSV’s amplification curve; (c) HTNV’s amplification curve; (d) Best primer and probe concentration of SFGR; (e) Best primer and probe concentration of SFTSV; (f) Best primer and probe concentration of HTNV.

Fig 3. Amplification curves and standard curve construction used about 10¹~10⁶ copies/μL plasmids.

(a) SFGR’s amplification curve; (b) SFTSV’s amplification curve; (c) HTNV’s amplification curve; (d) SFGR’s standard curve; (e) SFTSV’s standard curve; (f) HTNV’s standard curve; The all-in-one real-time PCR assay reaction is performed using the optimal primer concentration. 95°C for 3 min; denaturation at 95°C for 10 s; annealing/elongation at 60°C for 30 s. 45 cycles.

Fig 4. Results of specificity experiment.

Only the positive control well has an amplification curve; HBV, HCV, EBV and CMV show no reaction curves.