Construction of a LAMP-CRISPR assay for the detection of Vibrio parahaemolyticus

Loop-mediated isothermal amplification (LAMP) is increasingly used in nucleic acid detection for clinical diagnosis and monitoring of pathogenic microorganisms due to its isothermal properties and high sensitivity. Recently emerging CRISPR/Cas system has also been increasingly applied to the detection of different biological targets. In this study, a detection method was constructed by combining LAMP reaction and CRISPR/Cas12a. The established method has good specificity and sensitivity: qualitative accuracy of 40 tested strains reached 100%, and the detection limit for pure culture and DNA reaches 2.5 CFU/mL and 5 fg/uL, respectively. After 2 h of enrichment culture, the detection of artificially contaminated samples with an initial inoculum of 5 CFU/mL of Vibrio parahaemolyticus can be achieved. After combining LAMP amplification with CRISPR, the entire reaction time is less than 30 min, and the secondary amplification of the detection signal improves the detection limit and provides a variety of endpoint detection methods. The amplification curve can be used to quantify the template more accurately, and samples can also be determined by visual observation utilizing the UV lamp, which is free from the limitations of the instrument and has high application value in places with limited medical conditions.