Detection of Vibrio parahaemolyticus in shellfish using polymerase chain reaction-enzyme-linked immunosorbent assay

Abstract

Aims: This study evaluated the application of polymerase chain reaction–enzyme‐linked immunosorbent assay (PCR‐ELISA) for the detection of Vibrio parahaemolyticus in shellfish. Methods and Results: The PCRs were selected to amplify a species‐specific sequence region. In particular, internal tl biotin‐labelled oligonucleotide probe was used to capture the DIG‐labelled PCR products. Next, the probe PCR product hybrids, immobilized on a streptavidin‐coated microtiter plate, were detected with peroxidase‐conjugated anti‐digoxigenin antibody (anti‐DIG‐POD) and the colorimetric peroxidase substrate ABTS [2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid)] using an ELISA plate reader. Conclusions: The PCR‐ELISA system described is a feasible, sensitive method for the direct and specific detection of V. parahaemolyticus in shellfish samples. Compared with gel‐based detection methods, PCR‐ELISA in this study increased sensitivity by 100‐fold for V. parahaemolyticus. Significance and Impact of the Study: The PCR‐ELISA described may be used for potential rapid detection in routine shellfish analysis for the seafood industry. The sector requires simultaneous large‐scale sample screenings to monitor contamination levels in processing plants and evaluate the performance of the hazard analysis and critical control point (HACCP) system. PCR‐ELISA also proved to be economical, with a cost of about 9 Euros per sample, and the quick assay taking 8 h to complete starting from DNA extraction.

Development of a Real-Time Resistance Measurement for Vibrio parahaemolyticus Detection by the Lecithin-Dependent Hemolysin Gene

“…The use of high annealing temperatures reduces the overall PCR duration by at least 1 h, permitting the rapid detection of V. parahaemolyticus in the fish tissues, which might be due to the presence of inhibitory materials. The primer detection limits were reported to be 100 pg and 1 ng for DNA purified from bacterial culture and infected oyster tissue, respectively [19], and a previous study reported that PCR-ELISA increased the detection sensitivity for V. parahaemolyticus by 100-fold in comparison to gel-based detection methods [20]. Electrochemical methods have attracted considerable interest because of its intrinsic advantages such as portability, low cost, high sensitivity, and low power requirement.…”

Application of PCR-ELISA in Molecular Diagnosis

“…As this detection uses gene-specific probes for detection, the specificity of the tool is very prominent [2, 6]. Not only can samples with that particular gene be detected, but also they can be quantified based on colour intensity.…”

“…Several studies compared the use of qPCR, PCR-ELISA, and conventional PCR for the detection of poultry virus, infectious bursal disease virus, and the summary of the results is listed in Table 1 [2, 10–14]. …”

“…There are also a number of publications using PCR-ELISA in the food industry such as the detection of harmful food-borne pathogens such as Campylobacter sp., Salmonella [6, 29–32], Listeria monocytogenes [33, 34], Escherichia coli [26, 35], Brucella melitensis [36], and Vibrio parahaemolyticus [2]. Not limiting the use of the method in the medical and food industries, the study of PCR-ELISA extends even to the veterinary industry.…”

“…From then on, numerous studies on immunodetection of DNA using enzyme linked immunosorbent assay techniques were published, which subsequently lead to the introduction of polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA). This method combines both PCR and ELISA into a single analytical technique and its application is very much similar to ELISA except that this method allows the detection of nucleic acid instead of protein [2]. …”