Quantitative PCR assay for the simultaneous identification and enumeration of multiple Karenia species

Quantitative PCR (qPCR) is the method of choice for specific detection and quantification of harmful algal bloom (HAB) species. Development of qPCR assay for simultaneous enumeration of species that frequently co-exist in HABs is required. A high sensitivity TaqMan qPCR assay, using probe and primers, located at ITS1-5.8S-ITS2 rDNA region, detecting, specifically,Karenia selliformis,K. bidigitata, andK. mikimotoi, was designed. ITS1-5.8S-ITS2 rDNA region copy numbers perKareniacell genome were estimated to 217.697 +/- 67.904, allowing cell quantification. An application of the designed methodology in field samples has been conducted, and it showed high sensitivity (detection of around 10(-1)cell/100 mg of bivalve mollusk tissue, equivalent to about 20 copies of the target sequence). We suggest that the optimized method could contribute to early detection of three closely relatedKareniaspecies in seafood cultivating areas to promote control quality, guarantee a fast and effective intervention, and improve public health prevention.