Authentication of flying-fish-meal content of processed food using PCR-RFLP

Abstract

Nucleotide sequence and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the 3 0 -portion of the mitochondrial 16S RNA gene (rDNA) coding sequence were used to differentiate between flying fishes and other fishes that are frequently used in ago-noyaki production. In this study, we successfully distinguished between flying fishes and the other fishes by combining amplified DNA fragments with universally designed primers and digesting the PCR products with AfaI and MfeI restriction endonucleases. PCR-RFLP of 15 ago-noyaki, 2 noyaki, and 8 other processed flying-fish products were analyzed to authenticate flyingfish content among processed seafood products. After digestion of the PCR products with both enzymes, we found that all ago-noyaki and processed flying fish products contained either two or three DNA fragments of *200, 300, and 530 bp, and noyaki samples (which do not contain flying fish) contained only one fragment of *530 bp. Here, we present a new procedure to confirm the content of flying-fish meal in ago-noyaki.

Analytical Methods for Quantification of Relative Flying Fish Paste Content in Processed Sea Food (ago-noyaki)

“…With the use of the same primers, 16S-F and 16S-R, single PCR products of an estimated size of 530 bp were successfully amplified from seven flying fish species and five other fishes which are used in ago-noyaki (Nagase et al, 2009). This method uses DNA fragments from flying fish paste produced by AfaI and MunI digestions.…”

“…Although accuracy needs to be improved, this method is simple allowing for easy application. Recently, a quantitative PCR method using real-time PCR has been developed for authentication in forensic science (Maeta et al, 2009). However, due to the high cost of the system, use of this method is limited.…”

“…For the T100 sample containing only flying fish paste, two DNA fragments (200 and 300 bp) were found; for the T20 -T80 samples containing both flying fish and Alaska pollack pastes, three DNA fragments (200, 300 and 500 bp) were found; and for the T0 sample containing only Alaska pollack paste, one DNA fragment (500 bp) was found. As the relative flying fish paste content increased, fluorescence intensity of the 500-bp frag-R (5'-GTCTGAACTCAGATCACGTAGGAC-3') primers (Nagase et al, 2009), and 2.5 U of Ex Taq polymerase (Takara Bio Co., Shiga, Japan). PCR involved an initial denaturation at 94℃ for 2 min, followed by 30 cycles at 94℃ for 30 s, annealing at 60℃ for 30 s and extension at 72℃ for 30 s; with a final extension at 72℃ for 10 min.…”

“…In our previous reports, we established a PCR-RFLP method to confirm the content of flying fish paste in agonoyaki (Nagase et al, 2005(Nagase et al, , 2009). However, these reports provide only qualitative and not quantitative results.…”

Fraud identification in fishmeal using polymerase chain reaction (PCR)

“…Fishmeal is a generic term for a nutrient-rich feed ingredient used primarily in diets for domestic animals, sometimes used as a high-quality organic fertilizer (Shi et al., 2009). The vitamin content of fishmeal is highly variable and influenced by several factors, such as origin and composition of the fish, meal processing method and product freshness (Nagase et al, 2009). The lipids in fishes can be separated into liquid fish oils and solid fats.…”