Multicolor Melting Curve Analysis-Based Multilocus Melt Typing of Vibrio parahaemolyticus

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis outbreaks. To track the source of these diseases in a timely manner, a high throughput typing method is critical. We hereby describe a novel genotyping method for V. parahaemolyticus, termed multilocus melt typing (MLMT), based on multilocus sequence typing (MLST). MLMT utilizes melting curve analysis to interrogate the allelic types of a set of informative single nucleotide polymorphisms (SNPs) derived from the housekeeping genes used in MLST. For each SNP, one allelic type generates distinct Tm values, which are converted into a binary code. Multiple SNPs thus generate a series of binary codes, forming a melt type (MT) corresponding with a sequence type (ST) of MLST. Using a set of 12 SNPs, the MLMT scheme could resolve 218 V.parahaemolyticus isolates into 50 MTs corresponding with 56 STs. The discriminatory power of MLMT and MLST was similar with Simpson's index of diversity of 0.638 and 0.646, respectively. The global (adjusted Rand index = 0.982) and directional congruence (adjusted Wallace coefficient, MT→ST = 0.965; ST→MT = 1.000) between the two typing approaches was high. The entire procedure of MLMT could be finished within 3 h with negligible hands on time in a real-time PCR machine. We conclude that MLMT provides a reliable and efficient approach for V. parahaemolyticus genotyping and might also find use in other pathogens.