Open AccessDevelopment of a rapid and visual detection method for Rickettsia rickettsii combining recombinase polymerase assay with lateral flow test
Author(s) -
Yong Qi,
Yufeng Shao,
Jixian Rao,
Wanpeng Shen,
Qin Yin,
Xiaoling Li,
Hongxia Chen,
Jiameng Li,
Wenwen Zeng,
Shulong Zheng,
Suyun Liu,
Yuexi Li
Publication year - 2018
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0207811
Subject(s) - rickettsia rickettsii , recombinase polymerase amplification , detection limit , biology , virology , polymerase chain reaction , microbiology and biotechnology , spotted fever , chemistry , chromatography , rickettsia , gene , genetics , virus
Objectives Rickettsia 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. Methods A 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. Results A 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. Conclusions Using 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.