Design of a duplex Loop-mediated isothermal amplification assay suitable for real-time, and end-point, colorimetric detection of Vibrio parahaemolyticus, and a universal Internal Amplification Control, in mussel samples
Year: 2024
Type: article
Abstract: Vibrio parahaemolyticus remains as one of the most important foodborne pathogens worldwide, causing gastrointestinal disease in humans due to the consumption of raw, or undercooked, seafood. Even though molecular techniques, like PCR and qPCR, have allowed to overcome most of the limitations associated to culture-based methods, some persist such as the need for highly trained personnel, or the need to acquire expensive equipment. Loop-mediated isothermal amplification, LAMP, has emerged as a promising tool to solve these issues. The present study reports the development of a novel LAMP-based method for the detection of V. parahaemolyticus in mussels. This assay targets the gene toxR , and implements a novel, universal, freely available Internal Amplification Control (IAC) which was detected in a multiplex real-time LAMP format, where it is differentiated by melt curve analysis (86.70 ± 0.32 °C and 90.24 ± 0.34 °C for toxR and IAC respectively) and did not impact the analytical sensitivity of the target. Alternatively, the IAC was also applied in a parallel reaction and the assays performed by end-point, colorimetric LAMP. This novel IAC allowed to identify reaction inhibition due to matrix effect, and rule out false negative results. This new assay was combined with the ISO 21872 enrichment protocol, and the evaluation of spiked mussel samples indicated that an LOD95 of 14.5 and 10.8 CFU/25g could be reached after the primary 6h and secondary 24h enrichment steps by real-time LAMP, and 27.9 and 10.9 CFU/25g by naked-eye, color LAMP. It was important to note that in addition to these differences in terms of LOD95, a higher number of samples presenting reaction inhibition was observed after the primary enrichment, compared to the secondary, all successfully identified thanks to the new IAC; and also that statistically longer amplification times were required (Cq values of 19.47 ± 7.87 and 7.90 ± 1.20 respectively). The overall evaluation of the method returned values of diagnostic sensitivity, specificity and accuracy of 100 % along with an index of concordance of 1.00. These results were consistent regardless if the real-time, or the colorimetric, LAMP strategy was followed. • Universal LAMP IAC was developed and demonstrated capable of detecting reaction inhibition. • The novel IAC implemented in an assay to detect V. parahaemolyticus. • The multiplex LAMP assay performed similarly in real-time and colorimetric formats. • V. parahaemolyticus was detected in spiked mussels by both multiplex LAMP assays. • The LOD values were <30 CFU/25g for color LAMP and <15 CFU/25g for real-time.
Source: Food Control
Language: English
Subfield: Biomedical Engineering
Field: Engineering
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $4,430
OpenAlex ID: W4403690627