End-point rapid detection of total and pathogenic Vibrio parahaemolyticus (tdh+ and/or trh1+ and/or trh2+) in raw seafood using a colorimetric loop-mediated isothermal amplification-xylenol orange technique

Abstract

Background. Vibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis in humans worldwide. To ensure seafood safety and to minimize the occurrence of seafood-borne diseases early detection of total V. parahaemolyticus (pathogenic and non-pathogenic strains) and pathogenic V. parahaemolyticus (tdh+ and/or trh1+ and/or trh2+) is required. This study further improved a loop-mediated isothermal amplification (LAMP) assay using xylenol orange (XO) a pH sensitive dye to transform conventional LAMP into a one-step colorimetric assay giving visible results to the naked eye. LAMP-XO targeted rpoD for species specificity and tdh trh1 and trh2 for pathogenic strains. Multiple hybrid inner primers (MHP) of LAMP primers for rpoD detection to complement the main primer set previously reported were designed by our group to maximize sensitivity and speed. Methods. Following the standard LAMP protocol LAMP reaction temperature for rpoD tdh trh1 and trh2 detection was first determined using a turbidimeter. The acquired optimal temperature was subjected to optimize six parameters including dNTP mix betaine MgSO4 Bst 2.0 WarmStart DNA polymerase reaction time and XO dye. The last parameter was done using a heat block. The color change of the LAMP-XO result from purple (negative) to yellow (positive) was monitored visually. The detection limits (DLs) of LAMP-XO using a 10-fold serial dilution of gDNA and spiked seafood samples were determined and compared with standard LAMP PCR and quantitative PCR (qPCR) assays. Subsequently the LAMP-XO assay was validated with 102 raw seafood samples and the results were compared with PCR and qPCR assays. Results. Under optimal conditions (65 ?C for 75 min) rpoD-LAMP-XO and tdhLAMP-XO showed detection sensitivity at 102 copies of gDNA/reaction or 10 folds greater than trh1-LAMP-XO and trh2-LAMP-XO. This level of sensitivity was similar to that of standard LAMP comparable to that of the gold standard qPCR and 10-100 times higher than that of PCR. In spiked samples rpoD-LAMP-XO tdh-LAMP-XO and trh2-LAMP-XO could detect V. parahaemolyticus at 1 CFU/2.5 g spiked shrimp. Of 102 seafood samples LAMP-XO was significantly more sensitive than PCR (P < 0.05) for tdh and trh2 detection and not significantly different from qPCR for all genes determined. The reliability of tdh-LAMP-XO and trh2-LAMP-XO to detect pathogenic V. parahaemolyticus was at 94.4% and 100% respectively. Conclusions. To detect total and pathogenic V. parahaemolyticus at least rpoD-LAMPXO and trh2-LAMP-XO should be used as both showed 100% sensitivity specificity and accuracy. With short turnaround time ease and reliability LAMP-XO serves as a better alternative to PCR and qPCR for routine detection of V. parahaemolyticus in seafood. The concept of using a one-step LAMP-XO and MHP-LAMP to enhance efficiency of diagnostic performance of LAMP-based assays can be generally applied for detecting any gene of interest. Copyright 2024 Lamalee et al.