A highly sensitive detection of salmonids by real-time PCR targeting salmonid-specific retrotransposon Hpa I element

Abstract

Salmonid fish are known to have the potential to induce allergies. Therefore, from the perspective of food safety, it is crucial to assess the contamination of salmonid components in food. In this study, we aimed to develop a highly sensitive detection method using real-time polymerase chain reaction (PCR) for the accurate identification of minute quantities of salmonid DNA in food. To enhance detection sensitivity, three types of repetitive DNA elements with high copy numbers per genome, that is, the Hpa I element, ribosomal DNA (rDNA), and mitochondrial DNA (mtDNA), were initially selected as candidate PCR targets. The copy numbers of these elements across 11 salmonid species were quantified using real-time PCR. The salmonid-specific retrotransposon with the highest copy number, Hpa I, was chosen as the target gene for the highly sensitive salmonid detection method. By conducting real-time PCR using DNA templates from 11 salmonid species and 71 non-salmonid species, the method demonstrated exceptional specificity to salmonids. By optimizing the conditions of the real-time PCR, the detection limit of salmonid DNA reached 20 fg, and the limit remained unaffected even in the presence of mixed DNA from other species. Compared with existing detection methods for salmonid fish, our approach signifies a substantial 250-fold advancement in detection limits. Application of our method to 16 processed foods containing components of salmonid fish and 5 processed foods devoid of salmonid fish components resulted in successful determination of the presence or absence of salmonid fish in all tested food samples.