Ultra-fast PCR method for the distinguishing between Miichthys miiuy and Sciaenops ocellatus

Abstract

The mi-iuy croaker Miichthys miiuy has immense commercial value in the Republic of Korea. The red drum Sciaenops ocellatus is widely produced by aquaculture, although its price is approximately 25% that of M. miiuy. S. ocellatus has black spots on its tail, enabling it to be distinguished from M. miiuy based on appearance. However, identifying S. ocellatus after simple processing steps, such as skin removal and dicing, is difficult. Certain traders misrepresent and sell S. ocellatus as M. miiuy or cultured M. miiuy for illegal economical gain. Therefore, an accurate and rapid identification method is required to distinguish between M. miiuy and S. ocellatus in the field. Here, a method for rapid field identification was developed based on species-specific primers using a portable ultra-fast PCR instrument. The ultra-fast real-time PCR method can complete the entire analytical procedure, including DNA isolation, amplification, and detection, within 30 min, thus maintaining the accuracy of identifying M. miiuy and S. ocellatus products on site. Forty-nine commercial products were tested, and all samples were successfully identified. Thus, the developed method is rapid, efficient tool for ensuring consumer protection.

Supplementary information: The online version contains supplementary material available at 10.1007/s10068-021-00954-4.

Keywords: Economically motivated adulteration; Miichthys miiuy; Species-specific PCR; Ultra-fast real-time PCR; fishery products.

Fig. 1

Specificity of M. miiuy- and S. ocellatus-specific primer sets. (A) M. miiuy-specific primer set. (B) S. ocellatus-specific primer set. Lane M: 50–800 bp size marker (QIAGEN), lane 1: M. miiuy (124 bp), lane 2: S. ocellatus (124 bp), lane 3: negative control

Fig. 2

Sensitivity of M. miiuy- and S. ocellatus-specific primer sets. (A) M. miiuy-specific primer set. (B) S. ocellatus-specific primer set. Lane M: 50–800 bp size marker (QIAGEN), lane 1–5: 5 to 0.0005 ng of serial-diluted M. miiuy DNA (A) and S. ocellatus DNA (B)

Fig. 3

Species identification for commercial products. (A) M. miiuy-specific primer set. (B) S. ocellatus-specific primer set. Lane M: 50–800 bp size marker (Qiagen), lane R1: M. miiuy DNA, lane R2: S. ocellatus DNA, lane 1–49: template DNA extracted from commercial products listed in Table 2, lane 50: negative control