Development of a rapid and visual nucleotide detection method for a Chinese epidemic strain of Orientia tsutsugamushi based on recombinase polymerase amplification assay and lateral flow test.

OBJECTIVES: Orientia tsutsugamushi is an obligate intracellular pathogen that causes scrub typhus. Diagnosing scrub typhus remains a challenge, and a sensitive, specific, simple, and rapid diagnostic test is still needed. METHODS: A recombinase polymerase amplification (RPA) assay combined with a lateral flow (LF) test targeting the 56-kDa gene of a Karp-like strain of O. tsutsugamushi was developed and optimized. The detection limits, sensitivity, specificity, and simulative clinical performance were evaluated. RESULTS: Primers and probe were screened to establish the RPA assay, and the reaction conditions were optimized. The detection limit was 10 copies/reaction in detecting plasmid DNA and 12 copies/reaction in detecting genomic DNA. The RPA-LF method could differentiate O. tsutsugamushi from other phylogenetically related bacteria. The sensitivity was 100% and specificity was over 90%, when evaluated using infected animal samples or simulative clinical samples. Furthermore, the method was completed in 20min at 37°C followed by a 3-5min incubation at room temperature for the development of an immunochromatographic strip, and the results could be determined visually. CONCLUSIONS: This method is promising for wide-ranging use in basic medical units considering that it requires minimal instruments and infrastructure and is highly time-efficient, sensitive, and specific for diagnosing scrub typhus.