Rapid detection of multiple resistance genes to last-resort antibiotics in Enterobacteriaceae pathogens by recombinase polymerase amplification combined with lateral flow dipstick

Chenze Lu; Jingwen Wang; Leiming Pan; Xiuying Gu; Wenjing Lu; Di Chen; Cen Zhang; Qin Ye; Chaogeng Xiao; Pengpeng Liu; Yulong Tang; Biao Tang; Guangrong Huang; Jiehong Fang; Han Jiang

doi:10.3389/fmicb.2022.1062577

Rapid detection of multiple resistance genes to last-resort antibiotics in Enterobacteriaceae pathogens by recombinase polymerase amplification combined with lateral flow dipstick

Front Microbiol. 2023 Jan 5:13:1062577. doi: 10.3389/fmicb.2022.1062577. eCollection 2022.

Authors

Chenze Lu¹, Jingwen Wang¹, Leiming Pan², Xiuying Gu³, Wenjing Lu⁴, Di Chen⁴, Cen Zhang⁴, Qin Ye⁴, Chaogeng Xiao⁴, Pengpeng Liu⁵, Yulong Tang⁶, Biao Tang⁷, Guangrong Huang¹, Jiehong Fang¹, Han Jiang¹

Affiliations

¹ Key Laboratory of Specialty Agri-Products Quality and Hazard Controlling Technology of Zhejiang Province, College of Life Sciences, China Jiliang University, Hangzhou, Zhejiang, China.
² Zhejiang Hongzheng Testing Co., Ltd, Ningbo, Zhejiang, China.
³ Zhejiang Gongzheng Testing Center Co., Ltd, Hangzhou, Zhejiang, China.
⁴ Institute of Food Science, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang, China.
⁵ Key Laboratory of Biosafety Detection for Zhejiang Market Regulation, Zhejiang Fangyuan Testing Group LO.T, Hangzhou, Zhejiang, China.
⁶ Hangzhou Tiannie Technology Co., Ltd, Hangzhou, Zhejiang, China.
⁷ State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products and Institute of Agro-Product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang, China.

Abstract

The worrying emergence of multiple resistance genes to last-resort antibiotics in food animals and human populations throughout the food chain and relevant environments has been increasingly reported worldwide. Enterobacteriaceae pathogens are considered the most common reservoirs of such antibiotic resistance genes (ARGs). Thus, a rapid, efficient and accurate detection method to simultaneously screen and monitor such ARGs in Enterobacteriaceae pathogens has become an urgent need. Our study developed a recombinase polymerase amplification (RPA) assay combined with a lateral flow dipstick (LFD) for simultaneously detecting predominant resistance genes to last-resort antibiotics of Enterobacteriaceae pathogens, including mcr-1, bla_NDM-1 and tet(X4). It is allowed to complete the entire process, including crude DNA extraction, amplification as well as reading, within 40 min at 37°C, and the detection limit is 10¹ copies/μl for mcr-1, bla_NDM-1 and tet(X4). Sensitivity analysis showed obvious association of color signals with the template concentrations of mcr-1, bla_NDM-1 and tet(X4) genes in Enterobacteriaceae pathogens using a test strip reader (R ² = 0.9881, R ² = 0.9745, and R ² = 0.9807, respectively), allowing for quantitative detection using multiplex RPA-LFD assays. Therefore, the RPA-LFD assay can suitably help to detect multiple resistance genes to last-resort antibiotics in foodborne pathogens and has potential applications in the field.

Keywords: Enterobacteriaceae; lateral flow dipstick; rapid detection; recombinase polymerase amplification; resistance genes to last-resort antibiotics.