Comparative analysis of LC-MS/MS and real-time PCR assays for efficient detection of potential allergenic silkworm.

The silkworm (Bombyx mori) has long been valued food and feed in East Asia for its abundant nutritional and medicinal attributes, conversely, it can elicit allergic responses in susceptible individuals. Therefore, the development of silkworm detection method is required to avert allergenic incidents. In this study, two methodologies, tandem mass spectrometry (LC-MS/MS) and real-time PCR, were developed to achieve effective silkworm detection. These methods exhibited exceptional sensitivity in identifying silkworm presence in processed foods. Furthermore, model cookies spiked with silkworm were used to validate the sensitivities of LC-MS/MS (0.0005%) and real-time PCR (0.001%). Overall, these techniques were useful for trace silkworm detection in food products; therefore, they may help prevent allergic reactions. To the best of our knowledge, this study represents the first comparison of LC-MS/MS and real-time PCR methods for silkworm detection, marking an important contribution to the field. Data are available from ProteomeXchange under identifier PXD042494.

Multiplex PCR detection method of genetically modified canola event (MON94100, LBFLFK, and NS-B50027-4) combined with capillary electrophoresis.

Genetically modified organisms (GMOs) have been continuously developed for their convenience and productivity. In the past three years, three new GM canola events (MON94100, LBFLFK, and NS-B50027-4) have been developed. To efficiently control these GM canola events, the detection methods were needed. Therefore, the multiplex PCR method combined with capillary electrophoresis was developed for three GM canola events. Ten GM canola, eighteen GM soybean, thirty-two GM maize, and ten non-GM crops were used to evaluate the specificity of the method. The detection limit of the multiplex PCR assay was determined to be 0.005 ng in the DNA mixture and 0.1% in the spiked sample. The aim of this study was to establish multiplex PCR coupled with capillary electrophoresis for the newly produced three GM canola events. The developed method is expected to contribute to monitor the commercially available GM canola events. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01377-z.

Development of real-time PCR method for rapid and accurate detection of Centipedes (Scolopendra mutilans) in food.

Centipedes contain pharmacologically active compounds used as important medicinal material. However, the poisons produced by centipedes can cause human diseases; therefore, its use as a food ingredient is prohibited. This is the first report to develop a real-time PCR method for detection of centipedes. The primer and probe targeting the mitochondrial cytochrome c oxidase subunit 1 (COI) gene were newly designed. The specificity was verified using ten species and was confirmed to amplify only the centipede species. The real-time PCR method exhibited good linearity with a high-determination coefficient (R 2 = 0.999) and a detection limit was 0.001 ng. The performance of our method was also verified using five real-time PCR platforms under Universal and Fast PCR conditions. Finally, its applicability to processed food was evaluated using binary insect mixtures, and at least 0.1% of centipedes was detected. Therefore, our method can specifically and sensitively detect centipedes in food, contributing to food safety.

Comparison of Real-Time PCR and Droplet Digital PCR for the Quantitative Detection of Lactiplantibacillus plantarum subsp. plantarum

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.

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

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.

Regulatory policy on genetically modified breeding stack in key countries and the current status in Korea.

With an increasing interest and demand for biotechnology crops in agriculture worldwide, genetically modified (GM) breeding stacks produced by conventional breeding of previously approved GM single events remain popular for farmers in GM crop cultivation countries. However, regulations on stacks vary in each country. Currently, Korea requires approval for all breeding stacks intended for cultivation. To determine whether the stack is subject to a full safety assessment as a new GM crop, molecular characterization, protein expression, composition analysis, and agronomic characterization data are required. Korea's regulatory policy on stacks has not adopted the high-covers-low concept; therefore, subcombinations of already approved higher combination events are subject to breeding stack review if any subcombination was purposefully bred for cultivation use. This review will help promote the efficient management of GM breeding stacks in Korea in the future.

Species Identification of Red Deer (Cervus elaphus), Roe Deer (Capreolus capreolus), and Water Deer (Hydropotes inermis) Using Capillary Electrophoresis-Based Multiplex PCR.

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.

A multiplex PCR assay combined with capillary electrophoresis for the simultaneous detection of tropomyosin allergens from oyster, mussel, abalone, and clam mollusk species.

Tropomyosin present in mollusk species is the most common allergen in humans and causes excessive immune responses. To simultaneously detect tropomyosin allergens in mollusk species, a multiplex PCR assay combined with capillary electrophoresis was developed for the detection of tropomyosin genes of oyster, mussel, abalone, and clam, and the 18S rRNA gene of eukaryotes. The developed multiplex PCR revealed specific amplicons of four mollusk species [oyster (Crassostrea gigas), 150 bp; mussel (Mytilus edulis), 119 bp; abalone (Haliotis discus hannai), 98 bp; clam (Ruditapes philippinarum), 76 bp] and an amplicon of universal eukaryotic primer (eukaryotes, 190 bp); the detection limit of DNA was confirmed to be 16 pg. This multiplex PCR assay was applied for monitoring commercially processed seafood products, achieving successful detection of tropomyosin genes in 19 processed seafood products in Korea. The developed assay is an efficient and useful method for detecting tropomyosin allergens from mollusk species in seafoods.

Development of a Real-Time PCR Assay for the Detection of Donkey (Equus asinus) Meat in Meat Mixtures Treated under Different Processing Conditions.

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.

Simultaneous detection of fruit allergen-coding genes in tomato, apple, peach and kiwi through multiplex PCR.

Fruit allergies have become more common in recent years, and are now a serious health problem. In this study, a multiplex PCR assay was used to detect potential fruit allergens causing food allergy labeling in Korea. For the detection of these allergens, specific primer pairs were designed to amplify the allergen-coding genes Cyclophilin (tomato), Mdtl 1 (apple), Pru p 2.01A (peach) and Pectin methylesterase inhibitor (kiwi), and primer pair targeting the 18S ribosomal RNA gene was additionally used as an endogenous control. Primer specificity was assessed with 23 plant species. A mixture of DNA from the four fruits was serially diluted and used to determine the sensitivity of the multiplex PCR assay, which was approximately 0.08 ng. Eleven commercial fruit products were evaluated to verify the applicability of the multiplex PCR assay. This assay is expected to be a specific and efficient method for detecting fruit allergens in foods.

Rapid on-site detection of shrimp allergen tropomyosin using a novel ultrafast PCR system.

Shrimp is seafood that can commonly trigger allergic reactions. In this study, the ultrafast real-time PCR assay with portable device was developed to detect a shrimp-derived major allergen, tropomyosin, without complicated DNA extraction. For shrimp allergen detection, a specific primer pair was designed based on the shrimp tropomyosin gene and 18S ribosomal RNA gene as internal control. Primer specificity was assessed using 8 common seafood species. Serially diluted shrimp DNA was used to determine the limit of detection of the ultrafast PCR system, which was approximately 3.2 pg. Twenty-three food samples containing shrimp were evaluated to verify the applicability of a direct ultrafast PCR method for detecting shrimp allergens without DNA isolation. It took less than 30 min from sample preparation-to-result analysis to detect shrimp DNA in raw and processed samples. Therefore, this PCR system can be effectively and conveniently utilized in the field to detect shrimp in various food products.

Draft Genome Sequence of Tetragenococcus halophilus Strain FBL3, a Probiotic Bacterium Isolated from Galchijeot, a Salted Fermented Food, in the Republic of Korea.

Tetragenococcus halophilus strain FBL3 is a lactic acid bacterium isolated from galchijeot, a fermented food made from the salted guts of the hairtail fish, in the Republic of Korea. The draft genome of T. halophilus strain FBL3 comprised 87 contigs (≥1 kb) with a total size of 2,420,904 bp and a G+C content of 38.5%.