Development of Recombinase Polymerase Amplification Assays for Detection of Orientia tsutsugamushi or Rickettsia typhi

Abstract

Sensitive, specific and rapid diagnostic tests for the detection of Orientia tsutsugamushi (O. tsutsugamushi) and Rickettsia typhi (R. typhi), the causative agents of scrub typhus and murine typhus, respectively, are necessary to accurately and promptly diagnose patients and ensure that they receive proper treatment. Recombinase polymerase amplification (RPA) assays using a lateral flow test (RPA-nfo) and real-time fluorescent detection (RPA-exo) were developed targeting the 47-kDa gene of O. tsutsugamushi or 17 kDa gene of R. typhi. The RPA assay was capable of detecting O. tsutsugamushi or R. typhi at levels comparable to that of the quantitative PCR method. Both the RPA-nfo and RPA-exo methods performed similarly with regards to sensitivity when detecting the 17 kDa gene of R. typhi. On the contrary, RPA-exo performed better than RPA-nfo in detecting the 47 kDa gene of O. tsutsugamushi. The clinical performance of the O. tsutsugamushi RPA assay was evaluated using either human patient samples or infected mouse samples. Eight out of ten PCR confirmed positives were determined positive by RPA, and all PCR confirmed negative samples were negative by RPA. Similar results were obtained for R. typhi spiked patient sera. The assays were able to differentiate O. tsutsugamushi and R. typhi from other phylogenetically related bacteria as well as mouse and human DNA. Furthermore, the RPA-nfo reaction was completed in 20 minutes at 37°C followed by a 10 minute incubation at room temperature for development of an immunochromatographic strip. The RPA-exo reaction was completed in 20 minutes at 39°C. The implementation of a cross contamination proof cassette to detect the RPA-nfo fluorescent amplicons provided an alternative to regular lateral flow detection strips, which are more prone to cross contamination. The RPA assays provide a highly time-efficient, sensitive and specific alternative to other methods for diagnosing scrub typhus or murine typhus.

Fig 1. Experimental design.

(A), the two TwistAmp kits used for detection of amplicons using a lateral flow strip, a cross contaminated proof (XCP) cassette, or a real-time monitoring of fluorescent signal. (B),a flowchart of the experimental design from evaluation of primers, optimization of assay conditions, determination of detection limit and evaluation of assay performance.

Fig 2. Representative results of RPA-nfo for the detection of O. tsutsugamushi or R. typhi genomic DNA.

(A), different copy # of pure O. tsutsugamushi Karp strain genomic DNA as determined by qPCR were added in the RPA reaction. At the end of reaction, amplicons were removed and diluted as described in Materials and Methods for lateral flow strip detection of amplicons. Lane N: negative control, 1–4 contained 600, 220, 53 and 10 copies/reaction of O. tsutsugamushi DNA, respectively. (B), different copy # of pure R. typhi Wilmington strain genomic DNA as determined by qPCR were added as described. Lane N: negative control, lanes 1–4 contained 130, 45, 20, and 6 copies/reaction of R. typhi DNA, respectively. Control band, test band and sample application area are indicated by blue, red and green arrow, respectively.

Fig 3. Representative results of RPA-exo for the detection of O. tsutsugamushi or R. typhi genomic DNA.

(A), different copy # of pure O. tsutsugamushi Karp strain genomic DNA as determined by qPCR were added in the RPA reaction as described in Materials and Methods for real-time monitoring of amplicons. Negative control: (black line), 1000 (blue line), 200 (purple line), 50 (green line), and 10 (red line) copies per reaction of O. tsutsugamushi DNA, respectively. (B), different copy # of pure R. typhi Wilmington strain genomic DNA as determined by qPCR were added as described. Negative control: (black line), 500 (blue line), 250 (purple line), 125 (green line), and 50 (red line) copies per reaction of R. typhi DNA, respectively.

Fig 4. RPA-exo for the specific detection of O. tsutsugamushi or R. typhi DNA.

The RPA-exo is specific for the detection of O. tsutsugamushi (A) or R. typhi (B) DNA even though non-target related DNA was present in 1000 folds more in copy # than the positive control DNA. Positive control (blue line) containing 200 or 350 copies per reaction of O. tsutsugamushi or R. typhi DNA, respectively, 5x10⁴ copies per reaction of Coxiella burnetii genomic DNA (green line), 1x10⁴ copies per reaction of Leptospira genomic DNA (purple line), and 6x10⁴ copies per reaction of Bartonella genomic DNA (red line), and negative control (black line).

Fig 5. Evaluation of RPA-nfo detection of O. tsutsugamushi DNA in PCR confirmed positives and negatives.

RPA-nfo reaction was performed as described in the Materials and Methods. (A), ten negative and (B), 10 positive samples (previously confirmed [19]) were evaluated along with positive (P) and negative (N) controls.

Fig 6. Evaluation of RPA-nfo assay using XCP cassette.

The RPA-nfo assay for both 17 kDa gene of R. typhi and 47 kDa gene of O. tsutsugamushi was performed as described in Materials and Methods. Upon completion of the assay, the reaction tube was transferred to the cassette and the cassette was closed. The appearance of a red line along the T mark indicated the positive detection of FAM labeled probe. Cassette 1 reaction mixture contained no DNA template and complete primer sets for 47 kDa gene and 17 kDa gene with FAM labeled probes. Cassette 2 was the same as cassette 1 with the addition of 350 copies of O. tsutsugamushi DNA. Cassette 3 was the same as cassette 1 with the addition of 350 copies of R. typhi DNA. *Note: As mentioned previously, no C line was expected because there are no DIG-labeled primers in the RPA reactions, as these cassettes were initially designed for the LAMP assay.