A CRISPR-Cas12a-based universal rapid scrub typhus diagnostic method targeting 16S rRNA of Orientia tsutsugamushi

Abstract

Scrub typhus is caused by Orientia tsutsugamushi infection and occurs frequently in an area called the Tsutsugamushi Triangle. Currently, there is no vaccine for O. tsutsugamushi, and its infection is treated with antibiotics such as doxycycline. Scrub typhus responds to effective treatment, and early treatment shortens the course of the disease, reduces mortality, and accelerates recovery. Therefore, it is important to rapidly diagnose O. tsutsugamushi infection to ensure successful outcomes. Here, we developed a CRISPR-Cas12a-based diagnostic method targeting the bacterial 16S rRNA to detect O. tsutsugamushi infection of all known genotypes. To reduce the possibility of contamination and increase field applicability, we designed the one-pot assay system in addition to conventional two-pot assay system. Using this method, we successfully detected up to 100 copies of in vitro transcribed O. tsutsugamushi 16S rRNA within 1 hour under isothermal conditions. In blood samples from patients confirmed to be infected with O. tsutsugamushi by nested PCR, the developed method exhibited a clinical sensitivity of 98% and high specificity. These data demonstrate that the presented method is applicable for the rapid and universal diagnosis of scrub typhus to facilitate timely and appropriate treatment.

Fig 1. Schematic diagrams of OT DETECTRs.

(A) Conventional two-pot OT DETECTR. (B) One-pot OT DETECTR.

Fig 2. Comparative analysis of the sensitivity of the OT DETECTRs to O. tsutsugamushi DNA and RNA.

Various ICUs (10⁵ to 10¹) of O. tsutsugamushi were lysed with a column-based extraction kit, and nucleic acids (DNA and RNA) were amplified via RT-RPA with primer sets specific for the 16S rRNA gene. RT-RPA products were detected using two-pot OT DETECTR combined with a fluorescence assay. Shown are results obtained using gRNAs (A) OT1 and (B) OT2 (n = 6 replicates). RFU, relative fluorescence unit; NC, no template control; ICU, infected cell-counting unit.

Fig 3. LoD analyses of OT DETECTRs.

(A, B) To determine the LoD of each OT DETECTR, different copy numbers (10³ to 10⁰) of IVT O. tsutsugamushi 16S rRNA were used as templates for the (A) two-pot and (B) one-pot OT DETECTR combined with a fluorescence assay. Values are presented as means ± s.d. (error bars) (n = 3 replicates; ** P < 0.01, * P < 0.05 between samples, two-sample t-test). RFU, relative fluorescence unit; NC, no template control.

Fig 4. Specificity analyses of OT DETECTRs.

To determine the specificities of OT DETECTRs, 100 ng of RNA from each bacterial species and 10⁵ copies of RNA from Rickettsia spp. were amplified using RT-RPA. The Cas12a trans-cleavage activity assay was subsequently performed, and the results were analyzed through fluorescence measurements. Values are presented as means ± s.d. (error bars) (n = 3 replicates; ** P < 0.01, between samples, two-sample t-test). RFU, relative fluorescence unit; NC, no template control; SA, Staphylococcus aureus; KP, Klebsiella pneumoniae; SE, Salmonella enteritidis; RS, Rickettsia spp.; OT, Orientia tsutsugamushi.

Fig 5. Clinical applicability analysis of two-pot OT DETECTR.

Two-pot OT DETECTR was performed on clinical samples from (A) 50 patients confirmed positive or (B) 75 patients confirmed negative for O. tsutsugamushi infection by nested PCR. RNA was extracted from whole blood, RT-RPA was performed, the Cas12a trans-cleavage activity assay was performed, and the results were determined by a fluorescence assay or LFA. Genomic RNA of O. tsutsugamushi (100 ng) was used as a positive control. Values are presented as means ± s.d. (error bars) (n = 3 replicates; *** P < 0.001, ** P < 0.01, * P < 0.05 between samples, two-sample t-test). RFU, relative fluorescence unit; C-line, control line; T-line, test line; NC, no template control; PC, positive control.

Fig 6. Clinical applicability analysis of one-pot OT DETECTR.

One-pot OT DETECTR combined with a fluorescence assay was performed on clinical samples from (A) 50 patients confirmed positive or (B) 75 patients confirmed negative for O. tsutsugamushi infection by nested PCR. Genomic RNA of O. tsutsugamushi (100 ng) was used as a positive control. Values are presented as means ± s.d. (error bars) (n = 3 replicates; *** P < 0.001, ** P < 0.01, * P < 0.05 between samples, two-sample t-test). RFU, relative fluorescence unit; NC, no template control; PC, positive control.