Identification of Puffer Fish of the Genus Lagocephalus: L. lunaris, L. spadiceus and L. inermis , Using Multiplex PCR

Suh

et al. 2022

Foods

There is a need to identify the species of similar types of fish, especially those that are commercially sold. Particularly, the price of tuna varies depending on its type, which is difficult to determine as they are sold in cut or processed forms. This study developed a multiplex polymerase chain reaction (PCR) assay to identify the five most common tuna species: bigeye, skipjack, Atlantic bluefin, albacore, and yellowfin tunas. Newly designed species-specific primer sets for these five tuna species were created. Subsequently, the amplicon sizes obtained were 270, 238, 200, 178, and 127 base pairs for bigeye, skipjack, Atlantic bluefin, albacore, and yellowfin tunas, respectively. Each primer’s specificity was further tested using 15 other fish species, and no cross-reactivity was observed. To identify multiple targets in a single reaction, multiplex PCR was optimized to increase its resolution and accuracy. The detection levels of the multiplex PCR assay were confirmed to be 1 pg for all the five tunas. Additionally, it was successfully applied to 32 types of commercial tuna products. Therefore, this multiplex PCR assay could be an efficient identification method for various tuna species.

Section: Specificity and Sensitivity Of Multiplex Pcrsupporting

confidence: 74%

Multiplex PCR Assay for Simultaneous Identification of Five Types of Tuna (Katsuwonus pelamis, Thunnus alalonga, T. albacares, T. obesus and T. thynnus)

Suh

et al. 2022

Foods

“…This result indicated that the designed primers did not inhibit each other in a single reaction and had high specificity, resulting in the sensitivity that corresponded to that of the simplex PCR. Compared to the LOD of similar studies of fish products (5 ng for tunas and billfishes, 1 ng for freshwater fish species, and 1 ng for puffer fish), the LOD of our multiplex PCR assay (1 pg) is very sensitive and can detect very small amounts of cod DNA [21][22][23]. These results suggest that this PCR assay can be applied to on-site detection with low DNA extraction efficiency or to fragmented DNA samples such as processed food.…”

Section: Specificity and Sensitivity Of Multiplex Pcrmentioning

confidence: 77%

A Multiplex PCR Assay Combined with Capillary Electrophoresis for the Simultaneous Identification of Atlantic Cod, Pacific Cod, Blue Whiting, Haddock, and Alaska Pollock

Kim

2021

Foods

With an increased consumption of seafood products, food fraud with fish resources has been continuously reported. In particular, codfish has been exploited worldwide as a processed product in fresh, frozen, smoked, canned, or ready-to-eat dish forms. However, it is challenging to identify processed fish products after processing because of their similar morphological characteristics. Substitution and mislabeling of codfish among different species are also happening deliberately or unintentionally. Thus, it is necessary to distinguish cod species to prevent fish adulteration and food fraud. In this study, we developed a multiplex PCR for simultaneously identifying five cod species within Gadidae using capillary electrophoresis. Then, their species-specific primer sets were designed by targeting the mitochondrial cytochrome b gene. Subsequently, the amplicon sizes obtained were 237 bp, 204 bp, 164 bp, 138 bp, and 98 bp for Atlantic cod, Pacific cod, blue whiting, haddock, and Alaska pollock, respectively. The specificity of each primer was further tested using 19 fish species, and no cross-reactivity was observed. The limit of detection of this multiplex PCR assay was 1 pg. The developed multiplex PCR assay can be applied to 40 commercial food products successfully. This detection method will be efficient for managing seafood authentication by simultaneously analyzing multiple cod species.

“…Furthermore, the position of the positive control PCR products on a gel image could be used as a secondary size reference of species-specific bands in addition to the primary size reference provided by the commercial DNA ladders (Figure 2 and 4). Despite the advantages of having the positive control primers in species identification using multiplex primer sets, there have been only a small number of reports that included them in the identification of food products (Catanese et al, 2010;Hellberg et al, 2010;Sangthong et al, 2014;Suwannarat et al, 2017), and in some meat and dairy products from farmed ruminants (Xue et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

Multiplex PCR assay for species identification of meat and dairy products from buffalo (Bubalus bubalis), cattle (Bos indicus and Bos taurus), goat (Capra hircus), and sheep (Ovis aries)

Sangthong¹,

Suwannarat²,

Samipak³

et al. 2021

IFRJ

Self Cite

Cases of fraudulent meat and dairy products have increased worldwide, especially in developing countries. To determine the misrepresented animal species, appropriate tools in routine monitoring should be available for food inspections. In the present work, a multiplex polymerase chain reaction assay for species identification of products from ruminants including buffalo, cattle, goat, and sheep was developed. The primer set KUMUT_cFarmSp1 was composed of five species-specific primers and a pair of positive-control primers. The primer set amplified 106-, 163-, 232-, and 308-bp specific fragments from the cytochrome b (cyt b) gene of buffalo, cattle, goat, and sheep, respectively, and 370-bp positive-control fragment from 16S ribosomal RNA (16S rRNA). The detection limit of this PCR assay is 0.1 ng of DNA template. The developed primer set exhibited strong specificity, sensitivity, robustness, and simplicity for food verification, thus indicating its usefulness for species verification in food quality control and law enforcement.