A rapid and sensitive recombinase aided amplification assay incorporating competitive internal control to detect Bordetella pertussis using the DNA obtained by boiling

Rui-Qing Zhang; Gui-Xia Li; Xin-Na Li; Xin-Xin Shen; Yuan Gao; Le Wang; Tao Fan; Qing-Xia Duan; Ya-Kun Wang; Ji Wang; Zhi-Shan Feng; Xue-Jun Ma

doi:10.1016/j.ijid.2019.06.028

A rapid and sensitive recombinase aided amplification assay incorporating competitive internal control to detect Bordetella pertussis using the DNA obtained by boiling

Int J Infect Dis. 2019 Sep:86:108-113. doi: 10.1016/j.ijid.2019.06.028. Epub 2019 Jul 6.

Authors

Rui-Qing Zhang¹, Gui-Xia Li², Xin-Na Li³, Xin-Xin Shen⁴, Yuan Gao⁵, Le Wang⁶, Tao Fan⁷, Qing-Xia Duan⁸, Ya-Kun Wang⁹, Ji Wang¹⁰, Zhi-Shan Feng¹¹, Xue-Jun Ma¹²

Affiliations

¹ Hebei Medical University, Shijiazhuang, 050031, Hebei, China; NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address: hebmuzhangruiqing@163.com.
² Children's Hospital of Hebei Province, Shijiazhuang, 050031, Hebei, China. Electronic address: 13832179762@139.com.
³ NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address: zhaoyanghefeng@126.com.
⁴ NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address: x616815@126.com.
⁵ National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, National State Key Laboratory for Infectious Disease Prevention and Control, Beijing, 102206, China. Electronic address: gaoyuan@icdc.cn.
⁶ Children's Hospital of Hebei Province, Shijiazhuang, 050031, Hebei, China. Electronic address: luka_wl@163.com.
⁷ Hebei Medical University, Shijiazhuang, 050031, Hebei, China; NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address: hugengkun@163.com.
⁸ Hebei Medical University, Shijiazhuang, 050031, Hebei, China; NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address: 15227821073@163.com.
⁹ Children's Hospital of Hebei Province, Shijiazhuang, 050031, Hebei, China. Electronic address: 462405430@qq.com.
¹⁰ NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address: wangji19871229@sina.com.
¹¹ Hebei General Hospital, Shijiazhuang, 050051, Hebei, China. Electronic address: 15131129999@139.com.
¹² NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address: maxj@ivdc.chinacdc.cn.

PMID: 31288091
DOI: 10.1016/j.ijid.2019.06.028

Abstract

Objectives: Pertussis is a highly transmissible acute respiratory infection caused by the bacterial pathogen Bordetella pertussis. The purpose of this study was to develop a rapid, simple and sensitive diagnostic test for detecting this pathogen.

Methods: Here we present a recombinase aided amplification (RAA) assay incorporating competitive internal amplification control (IAC) to detect Bordetella pertussis using the DNA obtained by boiling. This assay was performed in a single closed tube at 39°C within 30min. A total of 115 clinical samples suspected of pertussis were collected and tested by the internally controlled RAA assay using both extracted DNA with the commercial kit and the DNA obtained by boiling. For comparison, the real-time PCR (RT-PCR) was also performed with DNA extraction in parallel.

Results: The sensitivity of the internally controlled RAA assay was 10¹ copies or 10CFU/ml per reaction in detecting plasmid DNA or B. pertussis strain. The optimum concentration of the IAC plasmid was determined to be 100 copies, and the introduction of IAC effectively reduced the occurrence of false negatives. Compared to the RT-PCR, RAA results with DNA extraction obtained 100% sensitivity and specificity, and the RAA results with heat-treated DNA showed 85.96% sensitivity and 100% specificity.

Conclusion: With the advantages of 45min turn-around time and simple steps of DNA purification, this assay could become a useful diagnostic tool for Bordetella pertussis detection and is potentially suitable for point-of-care identification to guide prompt clinical treatment.

Keywords: Bordetella pertussis; Internal amplification control; Recombinase aided amplification.

MeSH terms

Bordetella pertussis / genetics
Bordetella pertussis / isolation & purification*
Child
Child, Preschool
DNA, Bacterial / isolation & purification
Female
Hot Temperature
Humans
Infant
Male
Nucleic Acid Amplification Techniques / methods*
Nucleic Acid Amplification Techniques / standards
Real-Time Polymerase Chain Reaction
Recombinases*
Reference Standards
Sensitivity and Specificity
Whooping Cough / diagnosis*

Substances

DNA, Bacterial
Recombinases