Development of a Rapid and Efficient RPA-CRISPR/Cas12a Assay for Mycoplasma pneumoniae Detection

Feina Li; Jing Xiao; Haiming Yang; Yao Yao; Jieqiong Li; Huiwen Zheng; Qian Guo; Xiaotong Wang; Yuying Chen; Yajie Guo; Yonghong Wang; Chen Shen

doi:10.3389/fmicb.2022.858806

Frontiers in Microbiology (Mar 2022)

Development of a Rapid and Efficient RPA-CRISPR/Cas12a Assay for Mycoplasma pneumoniae Detection

Feina Li,
Jing Xiao,
Haiming Yang,
Yao Yao,
Jieqiong Li,
Huiwen Zheng,
Qian Guo,
Xiaotong Wang,
Yuying Chen,
Yajie Guo,
Yonghong Wang,
Chen Shen

Affiliations

Feina Li: Laboratory of Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Center for Children’s Health, Beijing, China
Jing Xiao: Laboratory of Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Center for Children’s Health, Beijing, China
Haiming Yang: Department of Respiratory Diseases II, Beijing Children’s Hospital, National Clinical Research Center for Respiratory Diseases, National Center for Children’s Health, Capital Medical University, Beijing, China
Yao Yao: Department of Respiratory Diseases I, Beijing Children’s Hospital, National Clinical Research Center for Respiratory Diseases, National Center for Children’s Health, Capital Medical University, Beijing, China
Jieqiong Li: Laboratory of Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Center for Children’s Health, Beijing, China
Huiwen Zheng: Laboratory of Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Center for Children’s Health, Beijing, China
Qian Guo: Laboratory of Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Center for Children’s Health, Beijing, China
Xiaotong Wang: Laboratory of Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Center for Children’s Health, Beijing, China
Yuying Chen: Laboratory of Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Center for Children’s Health, Beijing, China
Yajie Guo: Laboratory of Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Center for Children’s Health, Beijing, China
Yonghong Wang: Laboratory of Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Center for Children’s Health, Beijing, China
Chen Shen: Laboratory of Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Center for Children’s Health, Beijing, China

DOI: https://doi.org/10.3389/fmicb.2022.858806
Journal volume & issue: Vol. 13

Abstract

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Mycoplasma pneumoniae (MP) is a one of most common pathogen in causing respiratory infection in children and adolescents. Rapid and efficient diagnostic methods are crucial for control and treatment of MP infections. Herein, we present an operationally simple, rapid and efficient molecular method for MP identification, which eliminates expensive instruments and specialized personnel. The method combines recombinase polymerase amplification (RPA) with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) 12a-based detection, with an optimal procedure less than 1 h from sample to result including DNA extraction (25 min), RPA reaction (39°C for 15-20 min), CRISPR/Cas12a detection (37°C for 10 min) and visual detection by naked eyes (2 min). This diagnostic method shows high sensitivity (two copies per reaction) and no cross-reactivity against other common pathogenic bacteria. Preliminary evaluation using 201 clinical samples shows sensitivity of 99.1% (107/108), specificity of 100% (93/93) and consistency of 99.5% (200/201), compared with real-time PCR method. The above data demonstrate that our developed method is reliable for rapid diagnosis of MP. In conclusion, the RPA-CRISPR/Cas12a has a great potential to be as a useful tool for reliable and quick diagnosis of MP infection, especially in primary hospitals with limited conditions.

Published in Frontiers in Microbiology

ISSN: 1664-302X (Online)
Publisher: Frontiers Media S.A.
Country of publisher: Switzerland
LCC subjects: Science: Microbiology
Website: http://www.frontiersin.org/journals/microbiology

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