Rapid and Visual Detection of Vibrio parahaemolyticus in Aquatic Foods Using bla_CARB-17 Gene-Based Loop-Mediated Isothermal Amplification with Lateral Flow Dipstick (LAMP-LFD)

Abstract

Vibrio parahaemolyticus is recognized as one of the most important foodborne pathogens responsible for gastroenteritis in humans. The bla CARB-17 gene is an intrinsic β-lactamase gene and a novel species-specific genetic marker of V. parahaemolyticus. In this study, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) was developed targeting this bla CARB-17 gene. The specificity of LAMP-LFD was ascertained by detecting V. parahaemolyticus ATCC 17802 and seven other non-V. parahaemolyticus strains. Finally, the practicability of LAMP-LFD was confirmed by detection with V. parahaemolyticus-contaminated samples and natural food samples. The results showed that the optimized reaction parameters of LAMP are as follows: 2.4 mmol/l Mg 2+ , 0.96 mmol/l dNTPs, 4.8 U Bst DNA polymerase, and an 8:1 ratio of inner primer to outer primer, at 63 o C for 40 min. The optimized reaction time of the LFD assay is 60 min. Cross-reactivity analysis with the seven non-V. parahaemolyticus strains showed that LAMP-LFD was exclusively specific for V. parahaemolyticus. The detection limit of LAMP-LFD for V. parahaemolyticus genomic DNA was 2.1 × 10 -4 ng/μl, corresponding to 630 fg/reaction and displaying a sensitivity that is 100-fold higher than that of conventional PCR. LAMP-LFD in a spiking study revealed a detection limit of approximately 6 CFU/ml, which was similar with conventional PCR. The developed LAMP-LFD specifically identified the 10 V. parahaemolyticus isolates from 30 seafood samples, suggesting that this LAMP-LFD may be a suitable diagnostic method for detecting V. parahaemolyticus in aquatic foods.

Multiplex PCR-Lateral Flow Dipstick Method for Detection of Thermostable Direct Hemolysin (TDH) Producing V. parahaemolyticus

“…Data from previous published studies have demonstrated that the TLH gene is conserved in all V. parahaemolyticus strains and has been suggested as a good target for the detection of all V. parahaemolyticus strains [38][39][40]. For the identification of pathogenic V. parahaemolyticus strains, toxR [38], 16-23s rRNA [38], or bla CARB-17 [41] have been previously used. However, the TDH gene is commonly reported among pathogenic strains and has been recommended as a robust target for the identification of pathogenic V. parahaemolyticus strains.…”

LAMP-Based Point-of-Care Biosensors for Rapid Pathogen Detection

“…Non-hybridized FITC probes attach to gold-labelled anti-FITC antibodies, generating a biotin-free double complex that travels from the test to the control line. The detection sensitivity with extracted genomic DNA was 600 fg/reaction for Mycoplasma pneumoniae [ 82 ], 100 fg/reaction for methicillin-resistant Staphylococcus aureus (MRSA) [ 83 ], and Pseudomonas aeruginosa [ 84 ] 630 fg/reaction for V. parahaemolyticus [ 85 ] and 13.5 fg/µL for Salmonella [ 86 ]. For obtaining a higher sensitivity and accuracy, nanomaterials and enzymatic enhancements were investigated by various research groups.…”

Development of a Dual Mode UCNPs-MB Biosensor in Combination with PCR for Sensitive Detection of Salmonella

“…Normal PCR is limited by the utilization of hazardous chemicals and gel electrophoresis for qualitative purposes and is a time-consuming, and cumbersome procedure. Although qPCR and digital PCR have overcome the disadvantages of normal PCR, the requirement for high-cost reagents and trained operators has significantly limited their widespread application [ 9 , 10 ]. Additionally, several isothermal amplification assays, including loop-mediated isothermal amplification (LAMP), rolling loop amplification (RCA), and hybridization chain reaction (HCR), are currently being developed for bacterial assays and require only a thermostatic water bath or a metal bath to achieve amplification in a shorter time (less than 30 min) [ 11 ].…”