Rapid Detection of Vibrio parahaemolyticus in Shellfish by Real-Time Recombinase Polymerase Amplification
- 8 Downloads
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.
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.
- Faye O, Soropogui B, Patel P, ElWahed AA, Loucoubar C, Fall G, Kiory D, Magassouba N, Keita S, Kondé MK, Diallo AA, Koivogui L, Karlberg H, Mirazimi A, Nentwich O, Piepenburg O, Niedrig M, Weidmann M and Sall AA (2015) Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015. Eurosurveillance: bulletin europeen sur les maladies transmissibles = European communicable disease bulletin 20Google Scholar
- Han HH, Li FQ, Yan WX, Guo YC, Li N, Liu XM, Zhu JH, Xu J, Chen Y, Li XG, Lv H, Zhang YQ, Cai T, Chen YZ (2015) Temporal and spatial variation in the abundance of total and pathogenic Vibrio parahaemolyticus in shellfish in China. PLoS One 10Google Scholar
- He PY, Zhu GY, Luo JY, Wang HH, Yan Y, Chen LX, Gao WJ, Chen ZW (2016) Development and application of a one-tube multiplex real-time PCR with melting curve analysis for simultaneous detection of five foodborne pathogens in food samples. J Food SafGoogle Scholar
- Kanungo S, Sur D, Ali M, You YA, Pal D, Manna B, Niyogi SK, Sarkar B, Bhattacharya SK, Clemens JD, Nair GB (2012) Clinical, epidemiological, and spatial characteristics of Vibrio parahaemolyticus diarrhea and cholera in the urban slums of Kolkata, India. BMC Public Health 12:1–9CrossRefGoogle Scholar
- Malcolm TTH, Cheah YK, Radzi CWJWM, Kasim FA, Kantilal HK, John TYH, Martinez-Urtaza J, Nakaguchi Y, Nishibuchi M, Son R (2015) Detection and quantification of pathogenic Vibrio parahaemolyticus in shellfish by using multiplex PCR and loop-mediated isothermal amplification assay. Food Control 47:664–671CrossRefGoogle Scholar
- Mccarter LL (1999) The multiple identities of Vibrio parahaemolyticus. J Mol Microbiol Biotechnol 1:51–57Google Scholar
- Mudoh MF, Parveen S, Schwarz J, Rippen T, Chaudhuri A (2014) The effects of storage temperature on the growth of Vibrio parahaemolyticus and organoleptic properties in oysters. Front Public Health 2Google Scholar
- Nishibuchi M, Kaper JB (1995) Thermostable direct hemolysin gene of Vibrio parahaemolyticus: a virulence gene acquired by a marine bacterium. Infect Immun 63:2093–2099Google Scholar
- Nordstrom JL, Vickery MCL, Blackstone GM, Murray SL, Depaola A (2007) Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters. Appl Environ Microbiol 73:5840–5847CrossRefGoogle Scholar
- Valiadi M, Kalsi S, Jones IGF, Turner C, Sutton JM and Morgan H (2016) Simple and rapid sample preparation system for the molecular detection of antibiotic resistant pathogens in human urine. Biomed Microdevices In Press:1–10Google Scholar
- Wang RZ, Zhong YF, Gu XS, Yuan J, Saeed AF, Wang SH (2015) The pathogenesis, detection, and prevention of Vibrio parahaemolyticus. Front Microbiol 6:144Google Scholar
- Yang MJ, Ke YH, Wang XS, Hang R, Wei L, Lu HJ, Zhang WY, Liu SW, Chang GH, Tian SG (2016) Development and evaluation of a rapid and sensitive EBOV-RPA test for rapid diagnosis of Ebola virus disease. Sci Rep 6Google Scholar