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.

In this study, a donkey-specific primer pair and probe were designed from mitochondrial cytochrome b gene for the detection of raw donkey meat and different processed meat mixtures. The PCR product size for donkey DNA was 99 bp, and primer specificity was verified using 20 animal species. The limit of detection (LOD) was examined by serially diluting donkey DNA. Using real-time PCR, 0.001 ng of donkey DNA could be detected. In addition, binary meat mixtures with various percentages of donkey meat (0.001%, 0.01%, 0.1%, 1%, 10%, and 100%) in beef were analyzed to determine the sensitivity of this real-time PCR assay. At least 0.001% of donkey meat was detected in raw, boiled, roasted, dried, grinded, fried, and autoclaved meat mixtures. The developed real-time PCR method showed sufficient specificity and sensitivity in identification of donkey meat and could be a useful tool for the identification of donkey meat in processed products.

Droplet digital polymerase chain reaction (ddPCR) is one of the newest and most promising tools providing absolute quantification of target DNA molecules. Despite its emerging applications in microorganisms, few studies reported its use for detecting lactic acid bacteria. This study evaluated the applicability of a ddPCR assay targeting molecular genes obtained from in silico analysis for detecting Lactiplantibacillus plantarum subsp. plantarum, a bacterium mainly used as a starter or responsible for fermentation in food. The performance characteristics of a ddPCR were compared to those of a quantitative real-time PCR (qPCR). To compare the linearity and sensitivity of a qPCR and ddPCR, the calibration curve for a qPCR and the regression curve for a ddPCR were obtained using genomic DNA [10²–10⁸ colony-forming units (CFU)/mL] extracted from a pure culture and spiked food sample. Both the qPCR and ddPCR assays exhibited good linearity with a high coefficient of determination in the pure culture and spiked food sample (R² ≥ 0.996). The ddPCR showed a 10-fold lower limit of detection, suggesting that a ddPCR is more sensitive than a qPCR. However, a ddPCR has limitations in the absolute quantitation of high bacterial concentrations (>10⁶ CFU/mL). In conclusion, a ddPCR can be a reliable method for detecting and quantifying lactic acid bacteria in food.

To provide consumers correct information on meat species, specific and sensitive detection methods are needed. Thus, we developed a capillary electrophoresis-based multiplex PCR assay to simultaneously detect red deer (Cervus elaphus), roe deer (Capreolus capreolus), and water deer (Hydropotes inermis). Specific primer sets for these three species were newly designed. Each primer set only amplified target species without any reactivity against non-target species. To identify multiple targets in a single reaction, multiplex PCR was optimized and combined with capillary electrophoresis to increase resolution and accuracy for the detection of multiple targets. The detection levels of this assay were 0.1 pg for red deer and roe deer and 1 pg for water deer. In addition, its applicability was demonstrated using various concentrations of meat DNA mixtures. Consequently, as low as 0.1% of the target species was detectable using the developed method. This capillary electrophoresis-based multiplex PCR assay for simultaneous detection of three types of deer meat could authenticate deer species labeled on products, thus protecting consumers from meat adulteration.