Rapid and simultaneous detection of virulence factors of Vibrio parahaemolyticus by multiplex microfluidic-LAMP (MMLAMP)

Vibrio parahaemolyticus (VP) is an essential foodborne bacterium who can cause gastroenteritis. However, current detection methods generally still have the difficulty for quickly screening VP strains and accurately detecting VP. In this work, a sensitive and specific multiplex microfluidic-LAMP (MMLAMP) method was constructed to rapidly detect VP using a centrifugal microfluidic chip combined with LAMP. The system enables simultaneous detection of two hemolysin genes (tlh and trh) and one VP regulatory factor (toxR) in a single chip improving the accuracy for detecting VP. The MMLAMP system detected 2.42 x 10(4) copies/mu L of the plasmid (tlh-pUC57), 4.44 x 10(3) copies/mu L of the plasmid (trh-pUC57) and 4.86 x 10(4) copies/mu L of the plasmid (toxR-pUC57) in less than 35 min, respectively. Meanwhile, MMLAMP system was successfully applied for VP detection in artificially contaminated samples, the LOD is 220 CFU/g, which is 10 times lower than that of qPCR within the same detection time. The system also demonstrated 100 % specificity and good precision (C.V. < 5 %) in detecting the three targets of the VP, providing a new approach for point-of-care testing (POCT) of VP.