Rapid Detection of Vibrio parahaemolyticus in Shellfish by Real-Time Recombinase Polymerase Amplification
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Vibrio parahaemolyticus (V. parahaemolyticus) is a zoonotic pathogen generally found in seafood. To detect the foodborne pathogen rapidly and accurately for food safety measures, we developed a real-time recombinase polymerase amplification (RPA) method. An evaluation of the specificity and sensitivity of the method is discussed here. A set of primers and probe was specially designed to target the tlh gene, which is usually regarded as a marker of total V. parahaemolyticus strains. During the reaction, target DNA was amplified and tagged with specific fluorophore within 10 min and at an incubation temperature of 40 °C. In addition to fast amplification and low temperature, the fluorescence signal was synchronized with the amplification of products for the generation of real-time data. The detection limit of this assay was 0.4 pg/μL of DNA, which is comparable to assays that use the bacterial culture as template, 4 × 103 cfu/mL. The real-time RPA method had a stable performance when testing the spiking shellfish samples at the same level of contamination by the pathogen in different kinds of shellfish. Thus, the real-time RPA method shows great potential for on-site detection of V. parahaemolyticus, especially in low-resource settings.
KeywordsReal-time recombinase polymerase amplification tlh Vibrio parahaemolyticus Pathogen detection Shellfish Food safety
The authors would like to acknowledge the Ningbo Academy of Inspection and Quarantine for providing the bacterial strain for this study and allowing us to finish the assay of culturing strains in the laboratory there. Authors also appreciated the assistance and guidance of the staff there and the professionals at the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
Peng Zhu and Weifang Gao contributed equally to this work.
This study was funded by the Ningbo Science and Technology Innovation Team (2015C110018); Ningbo Marine Algae Biotechnology Team (2011B81007); Zhejiang Provincial Public Welfare Technology Program of China (2017C33133); K.C. Wang Magna Fund in Ningbo University (SS); LiDakSum Marine Biopharmaceutical Development Fund; National 111 Project of China; the Earmarked Fund for Modern Agro-industry Technology Research System, China (CARS-49); Zhejiang Xinmiao Talents Program (2015R405013); and Scientific Research Foundation of Graduate School of Ningbo University (G16091).
Compliance with Ethical Standards
Conflict of Interest
Peng Zhu declares that he has no conflict of interest. Weifang Gao declares that he has no conflict of interest. Hailong Huang declares that he has no conflict of interest. Jinpo Jiang declares that he has no conflict of interest. Xianfeng Chen declares that he has no conflict of interest. Jianzhong Fan declares that he has no conflict of interest. Xiaojun Yan 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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