Tae Sun Kang

Specific PCR assays to determine bovine, porcine, fish and plant origin of gelatin capsules of dietary supplements.

Gelatin, a purified protein derived mostly from pig skin and bovine tissue, is used widely in both food and pharmaceutical industries. Here, to determine the species of origin of capsule gelatin, we developed a sensitive and reliable test using the polymerase chain reaction (PCR) method, which included 1) species-specific or universal primer sets, designed to detect short 16S ribosomal RNA (rRNA) gene sequences from cow, pig, and fish (tilapia) as well as genes encoding the large subunit of plant ribulose-1,5-bisphosphate carboxylase oxygenase and 2) species-specific PCR coupled with whole-genome amplification. This method was used to verify manufacturing label claims of 28 gelatin capsule samples sold as dietary supplements. The results from 27 samples were consistent with gelatin-related information on the manufacturer label, while one sample that mentioned tilapia gelatin was found to contain only bovine DNA. This rapid method can therefore be used to verify the authenticity of gelatin capsules.

A rapid real-time PCR method to differentiate between mottled skate (Beringraja pulchra) and other skate and ray species

Skates and rays are commercially important fish in South Korea, and among them, Beringraja pulchra has the highest economic value. However, the similar morphological traits among skates and rays are often exploited for seafood fraud. Here, we designed both Beringraja pulchra-specific and skate-universal primer sets, capable of detecting short sequences in the cytochrome oxidase subunit I gene, and developed highly sensitive and reliable quantitative real-time PCR (qPCR) assays to differentiate between Beringraja pulchra and other skate and ray species. AΔCq method based on differences in the amplification efficiency was developed, validated, and then used to confirm the presence of Beringraja pulchra in twenty-six commercial skate products. The averageΔCq value obtained for other skate species (18.94 ± 3.46) was significantly higher than that of Beringraja pulchra (1.18 ± 0.15). For on-site applications, we developed an ultra-fast qPCR assay, allowing for completion of the entire analytical procedure within 30 min.

Development of Primer Sets for the Detection of Polygonum multiflorum, Cynanchum wilfordii and C. auriculatum

ABSTRACT - The aim of this study was to develop rapid screening method for the identification of Chineseherbal medicine species with similar appearance, Polygonum multiflorum, Cynanchum wilfordii and C. auriculatum,by using genetic markers. As a genetic marker, psbA-trnH gene in chloroplast was selected due to differences insequence among the three species. Species-specific primers were designed based on the sequences of the marker geneof P. multiflorum, C. wilfordii, and C. auriculatum, and the expected size of PCR products was 160, 147, and 119 bp,respectively. Under the developed conditions, cross-reaction was not detected among these three plant species. Toconfirm the efficiency of our species-specific primers, the optimized method was applied to a variety of processedproducts composed of mostly P. multiflorum and C. wilfordii, demonstrating that our method was a rapid and easyscreening assay. Our findings suggest this screening method can be utilized to prevent the distribution of economicallymotivated adulteration food and to improve consumers right.Key words : species-specific primer, PCR (polymerase chain reaction), adulterated food

Comparison of quantitative methods based on SYBR Green real-time qPCR to estimate pork meat adulteration in processed beef products

Quantitative real-time PCR (qPCR) is a modern technique that has been widely used for the detection of species used in meat products. For obtaining accurate and reliable qPCR results, we assessed two common DNA quantification methods for isolated DNA and five quantification approaches for qPCR products. DNA dilution based on spectrofluorometric results showed better qPCR results than those based on spectrophotometry in terms of linear correlation, amplification efficiency, and linear dynamic range. Binary pork-beef mixtures were used to construct standard curves of SYBR Green-based qPCR products using five quantification approaches, and they were validated and compared using in-house pork models. 18S rRNA gene normalization methods showed better trueness (-11.79% to -6.73%) than that of methods using absolute and relative standard curves (-28.52% to -18.64%) in a model burger. These normalized reference methods successfully estimated the quantities of pork meat in the range of 100%-0.01% in commercial beef products.

Development of four PCR-based methods to differentiate tilefish species (Branchiostegus japonicus and B. albus)

The red tilefish (Branchiostegus japonicus) is an important ingredient and fishery resource in South Korea. Branchiostegus japonicus-specific and tilefish primer sets were designed, and four PCR-based methods were developed to differentiate B. japonicus and B. albus species. The specificity of the conventional and quantitative real-time PCR (qPCR) developed was confirmed using twenty species, showing no cross-activity, and the limit of detection was 0.1-0.001 ng/µL. Their accuracy was validated using a forensically informative nucleotide sequencing method on forty-seven red tilefish products. These two methods were further improved to develop direct triplex PCR and ultra-fast qPCR for the on-site food analysis, which could complete the entire analytical procedure within either 90 or 30 min, while maintaining the same accuracy. Therefore, these four PCR methods can be efficiently customized in various analytical areas and conditions, including field analysis, rapid screening, quality control, and labeling compliance, required by food manufacturing industry and regulatory authorities.

The complete chloroplast genome of Codonopsis lanceolata (Campanulaceae)

Abstract The complete chloroplast genome sequence of Codonopsis lanceolata was determined by next generation sequencing. The total length of chloroplast genome of C. lanceolata was 169,447 bp long, including a large single-copy (LSC) region of 85,253 bp, a small single-copy (SSC) region of 8060 bp, and a pair of identical inverted repeat regions (IRs) of 38,067 bp. A total of 110 genes was annotated, resulting in 79 protein-coding genes, 27 tRNA genes, and 4 rRNA genes. The phylogenetic analysis of C. lanceolata with related chloroplast genome sequences in this study provided the taxonomical relationship of C. lanceolata in the genus Campanula.

The complete chloroplast genome of Caltha Palustris (Ranunculaceae)

Abstract The complete chloroplast genome sequence of Caltha palustris, a species of the Ranunculaceae family, was characterized from the de novo assembly of HiSeq (Illumina Co.) paired-end sequencing data. The chloroplast genome of C. palustris was 155,292 bp in length, with a large single-copy (LSC) region of 84,120 bp, a small single-copy (SSC) region of 18,342 bp, and a pair of identical inverted repeat regions (IRs) of 26,415 bp. The genome contained a total of 114 genes, including 80 protein-coding genes, 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. The phylogenetic analysis of C. palustris with 14 related species revealed the closest taxonomical relationship with Hydrastis canadensis in the Ranunculaceae family.

Monitoring of the Source of Gelatin in Dietary Supplement Capsules Sold on the Internet

상업적으로 유통되는 젤라틴 캡슐 제품은 소비자의 건강(예: 광우병) 및 종교적 신념에 대한 우려를 야기시킬 수 있다. 젤라틴은 대부분 소, 돼지 등에서 유래한 원료 물질을 가공한 것으로서, 가공 후 그 원료 물질을 분석하는 것은 대단히 어렵다. 따라서 정부 규제기관의 표시사항 준수여부 모니터링 연구가 주기적으로 필요하다. 본 연구에서는 인터넷에 유통되는 건강기능식품(n = 181)을 대상으로 젤라틴 캡슐의 원료 물질을 종 특이 PCR 방법으로 분석했다. 55개 제품의 경우 표시사항에 젤라틴 캡슐원료 물질에 대한 정보를 명시하였으나(예: bovine-, fish-and plant-derived gelatin), 126개 제품의 경우 사용 원료에 대한 정보 없이 “gelatin”으로 표시하였다. 이 126개 제품의 젤라틴 캡슐 분석 결과 51개 제품은 소 유래의 젤라틴을, 31개 제품은 돼지 유래의 젤라틴을, 그리고 44개 제품은 소와 돼지의 원료를 혼합하여 제작한 블렌딩 젤라틴을 사용한 것으로 밝혀졌다. 따라서 소비자의 알 권리, 종교적 신념 및 건강을 보호하기 위해 젤라틴 캡슐에 사용된 원료 물질을 표시사항에 제공하는 것은 매우 중요하다.