Abstract
ObjectivesRickettsia rickettsii is the causative agent of Rocky Mountain spotted fever, which is the most severe spotted fever group (SFG) rickettsiosis. Developing a simple and reliable detection method is required.MethodsA detection method for R. rickettsii was established based on a recombinase polymerase amplification (RPA) assay and the lateral flow (LF) test. A specific target sequence was screened, and corresponding primers and probes were designed, synthesized, and screened for establishing an RPA assay with high amplification efficiency. Reagent concentrations, amplification time, and loading volume for strip development were optimized. The detection limit, analytic sensitivity and specificity were evaluated.ResultsA rapid, visual, sensitive and specific method for the detection of R. rickettsii based on RPA and the LF test was successfully established. The novel method had a limit of detection of 10 to 50 copies/reaction without recognizing other organisms. Analytical sensitivity and specificity were ≥90% and 100%, respectively, as evaluated by animal and simulative human samples.ConclusionsUsing the established method, detection could be completed in 30 min with visually detectable results by the naked eye, without requirement of any instrument except a constant temperature equipment. The technique shows superior detection performance and is promising for wide use in the field as well as resource-limited areas for R. rickettsii detection.
Highlights
A rapid, visual, sensitive and specific method for the detection of R. rickettsii based on recombinase polymerase amplification (RPA) and the lateral flow (LF) test was successfully established
Rickettsia rickettsii is the causative agent of Rocky Mountain spotted fever (RMSF), which is the most severe spotted fever group (SFG) rickettsiosis [1,2]
Doxycycline is available for the treatment of RMSF, fatal cases are still reported yearly, and case fatality rates approach 7% in some regions to which the disease is highly endemic in the United States [4,5,6]
Summary
Rickettsia rickettsii is the causative agent of Rocky Mountain spotted fever (RMSF), which is the most severe spotted fever group (SFG) rickettsiosis [1,2]. The diagnosis of RMSF relies on serological and molecular tests, with immunofluorescence assay (IFA) being the gold standard. Molecular tests include conventional polymerase chain reaction (PCR), nested PCR, and real-time quantitative PCR (RT-qPCR). PCR and nested PCR are not reliable for use in acute-phase blood samples, which may have very few organisms [6]. They may need another step of sequencing to confirm the species, which is time consuming. RT-qPCR has been proved to be more sensitive and useful [1,6] It requires a fluorescence quantitation instrument, which is expensive and not suitable for areas with limited instruments and infrastructure
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