Recombinase Polymerase Amplification-Based Assay for Rapid Detection of Listeria monocytogenes in Food Samples
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Listeria monocytogenes (L. monocytogenes) is a Gram-positive microbe and is reported to be potentially pathogenic for both humans and animals, exhibiting a high mortality rate of up to 30%. The invention of a fast and sensitive method of point-of-care testing (POCT) is of great significance for the field of food safety of the rapid detection of harmful microbes and prevention of food contaminated by such microbes from reaching the markets. A novel isothermal molecular amplification technique, recombinase polymerase amplification (RPA), has been used to detect L. monocytogenes in food combined with a highly sensitive end determining method, lateral flow dipstick (LFD) technology. RPA-LFD was implemented at 37 °C, and amplification only took 20 min. By optimizing the primers and probes, the system had no cross-reaction with seven other pathogens, and it was proven to have a 100 times higher sensitivity (1360 cfu/mL) than a real-time PCR for L. monocytogenes testing. Also, RPA-LFD was applied without complex or expensive equipment, only a thermostatic heater such as the Thermomixer Comfort was required. Thus, RPA-LFD was found to be a solution for POCT for L. monocytogenes, especially for low-resource locations with little available laboratory equipment.
KeywordsRecombinase polymerase amplification Lateral flow dipsticks Pathogen Point of care testing Food safety
We would like to acknowledge the Ningbo Academy of Inspection and Quarantine for providing the bacterial strain in inactivation to extract genomic DNA for this study and allowing us to finish the spiking assay in the laboratory there. And the assistance and guidance of the staff there and the professionals at Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases were also appreciated. We thank LetPub (www.letpub.com) for their linguistic assistance during the preparation of this manuscript.
Weifang Gao and Peng Zhu contributed equally to this work.
Compliance with Ethical Standards
This study was funded by Ningbo Innovation Team (2015C110018), Ningbo Marine Algae Biotechnology Team (2011B81007), K.C. Wang Magna Fund in Ningbo University (SS), and Scientific Research Foundation of Graduate School of Ningbo University (G16091).
Conflict of Interest
Weifang Gao declares that he has no conflict of interest. Peng Zhu declares that she has no conflict of interest. Hailong Huang declares that he has no conflict of interest. Xiaojun Yan declares that he has no conflict of interest. Jianzhong Fan declares that he has no conflict of interest. Xianfeng Chen declares that he has no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Informed consent was obtained from all individual participants included in the study.
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