A seminested recombinase polymerase amplification assay to detect rickettsial pathogens in clinical samples

Ying Zhang; Yan Hai; Biao Duan; Hu Long; Xiaofei Xie; Zhongqiu Teng; Feifei Yin; Mingliu Wang; Yanwen Xiong; Zhujun Shao; Weidong Guo; Aiping Qin

doi:10.1016/j.diagmicrobio.2023.116067

A seminested recombinase polymerase amplification assay to detect rickettsial pathogens in clinical samples

Diagn Microbiol Infect Dis. 2023 Dec;107(4):116067. doi: 10.1016/j.diagmicrobio.2023.116067. Epub 2023 Sep 9.

Authors

Ying Zhang¹, Yan Hai², Biao Duan³, Hu Long⁴, Xiaofei Xie⁵, Zhongqiu Teng⁶, Feifei Yin⁷, Mingliu Wang⁸, Yanwen Xiong⁶, Zhujun Shao⁶, Weidong Guo⁹, Aiping Qin¹⁰

Affiliations

¹ Center for Disease Control and Prevention of Xilingol League, Xilinhaote, Inner Mongolia, China; State Key Laboratory of Infectious Diseases Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
² Center for Disease Control and Prevention of Inner Mongolia, Hohhot, Inner Mongolia, China.
³ Institute of Endemic Diseases Control and Prevention of Yunnan, Dali, Yunnan, China.
⁴ Center for Disease Control and Prevention of Guilin City, Guilin, Guangxi, China.
⁵ State Key Laboratory of Infectious Diseases Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China; Hainan Medical College, Haikou, Hainan, China.
⁶ State Key Laboratory of Infectious Diseases Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
⁷ Hainan Medical College, Haikou, Hainan, China.
⁸ Center for Disease Control and Prevention of Guangxi, Nanning, Guangxi, China.
⁹ Center for Disease Control and Prevention of Xilingol League, Xilinhaote, Inner Mongolia, China; Institute of Endemic Diseases Control and Prevention of Yunnan, Dali, Yunnan, China. Electronic address: gwd.com@126.com.
¹⁰ State Key Laboratory of Infectious Diseases Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China. Electronic address: qinaiping@icdc.cn.

PMID: 37751629
DOI: 10.1016/j.diagmicrobio.2023.116067

Abstract

Treatment at the early stage of onset is vital for the prognosis of rickettsioses. But the absence of specific clinical symptoms complicates the diagnosis of this condition. Herein we established a seminested recombinase polymerase amplification assay (snRPA-nfo) that enables quick detection and differentiation of rickettsial pathogens in clinical samples with high sensitivity and specificity. The conserved 17-kDa protein gene of Rickettsia sibirica and the 47-kDa protein gene of Orientia tsutsugamushi were targeted for the duplex RPA-nfo assay. The snRPA-nfo assay exhibited an increased LOD in spiked blood samples, up to 1000-fold in comparison to standard RPA-nfo, and a better detection rate (83.3%, 5/6) than TaqMan PCR (16.6%, 1/6, Ct ≤ 35) in clinically confirmed patient blood samples. Thus, snRPA-nfo assay represents a promising alternative to TaqMan PCR in the early diagnosis of rickettsioses for point-of-care testing as well as in resource-limited settings.

Keywords: Orientia tsutsugamushi; Recombinase polymerase amplification (RPA); Rickettsia spp.; Rickettsioses; Seminested RPA.

MeSH terms

Humans
Nucleic Acid Amplification Techniques
Orientia tsutsugamushi* / genetics
Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
Recombinases
Rickettsia Infections* / diagnosis
Sensitivity and Specificity

Substances

Recombinases