Real-time multiplex PCR assay for detection and differentiation of rickettsiae and orientiae

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Abstract
The high incidence of rickettsial diseases in Southeast Asia necessitates rapid and accurate diagnostic tools for a broad range of rickettsial agents, including Orientia tsutsugamushi (scrub typhus) and Rickettsia typhi (murine typhus), but also spotted fever group infections, which are increasingly reported. We present an SYBR-Green-based, real-time multiplex PCR assay for rapid identification and differentiation of scrub typhus group, typhus group and spotted fever group rickettsiae using 47 kDa, gltA and ompB gene targets. Detection limits for amplification of these genes in reference strains ranged from 24 copies/μl, 5 copies/μl and 1 copy/μl in multiplex and 2 copies/μl, 1 copy/μl and 1 copy/μl in single template format, respectively. Differentiation by melt-curve analysis led to distinct melt temperatures for each group-specific amplicon. The assay was subjected to 54 samples, of which all cell-culture and 75% of characterised clinical buffy coat samples were correctly identified. Real-time PCR has the advantage of reliably detecting and differentiating rickettsial and oriential cell-culture isolates in a single-template assay, compared with the more time-consuming and laborious immunofluorescence assay. However, further optimisation and validation on samples taken directly from patients to assess its clinical diagnostic utility is required.