Real-time PCR assays for detection and quantitation of porcine and bovine DNA in gelatin mixtures and gelatin capsules

Cai, Hui; Gu, Xuelin; Scanlan, Mary S.; Ramatlapeng, Dintletse H.; Lively, Chris R.

doi:10.1016/j.jfca.2011.06.008

Cited by 82 publications

(72 citation statements)

References 15 publications

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“…Such methods are infrared spectroscopy coupled with chemometrics of Principal Component Analysis (PCA) for differentiation of porcine and bovine gelatins (Hashim et al, 2010) and those with fish gelatine (Cebi et al, 2016), high performance liquid chromatography coupled with fluorescence detector and chemometrics of PCA (Nemati et al, 2004;Raraswati et al, 2013) and with some types of mass-spectrometer detectors (Zhang et al, 2009;Yilmaz et al, 2013), electrophoretic analysis (Hermanto et al, 2013), Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) combined with PCA (Azira et al, 2014), Enzyme-Linked Immuno-Sorbent Assay (ELISA) (Doi et al, 2009;Venien and Levieux, 2005), conventional method using calcium phosphate precipitation test (Hidaka and Liu, 2003) and Polymerase Chain Reaction (PCR) (Demirhan et al, 2012;Cai et al, 2012). The PCR is an ideal technique to be used for fat and sensitive detection of porcine DNA in gelatin due to the higher stability of DNA compared to protein (Aida et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Real-Time Polymerase Chain Reaction for Halal Authentication of Gelatin in Soft Candy

Sepminarti¹,

Sudjadi²,

Wardani³

et al. 2015

Asian J. of Biochemistry

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Currently, along with the development of science and technology, the diversification of food products occurs in the market. Food products can contain non-halal components like porcine gelatine. One of food suspected to use gelatine is soft candy. Gelatin can be made from pork or beef or other animal. The presence of porcine gelatine in any food products is not allowed for Moslem community, therefore an analytical method offering reliable results must be developed. This study is intended to use Real-Time Polymerase Chain Reaction (RT-PCR) for analysis of porcine gelatine in soft candy. Isolation of DNA was performed with mitochondrial DNA Isolation Kit K280-50 (Bio-Vision). The DNA was analyzed by RT-PCR using primer D-Loop 318. Analysis for the primer specificity was performed on fresh tissue (pig, cows, chickens, goats and rats) and gelatin sources (beef, pigs and catfish). Primer D-loop318 can amplify porcine DNA at the optimum temperature 61.4°C. Repeatability test demonstrated amplification of all positive response samples containing porcine DNA in serial dilution of 10000-1 pg). The Coefficient of Variation (CV) is 6.32%. The repeatability test was also performed on soft candy 100% having CV of 1.06%. The commercial soft candy samples evaluated do not contain porcine DNA.

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Section: Introductionmentioning

confidence: 99%

Real-Time Polymerase Chain Reaction for Halal Authentication of Gelatin in Soft Candy

Sepminarti¹,

Sudjadi²,

Wardani³

et al. 2015

Asian J. of Biochemistry

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“…Oleh karena itu, perlu diupayakan metode yang akurat untuk identifikasi gelatin sapi dan gelatin babi. Metode identifikasi gelatin sapi dan gelatin babi telah banyak dilakukan dan dikembangkan di antaranya dengan teknik presipitasi kimia [8], FTIR [9], LCMS [10], ELISA [11], dan analisis berbasis DNA dengan Real Time PCR [12,13].…”

Section: Pendahuluanunclassified

“…Selain itu, metode ini dapat mendeteksi campuran gelatin babi dan gelatin sapi dengan level kontaminasi 1% [12,13]. Dengan metode ini, kita dapat mengamati secara langsung hasil amplifikasi atau perbanyakan DNA fragmen.…”

Section: Pendahuluanunclassified

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Perbandingan Metode SYBR Green dan Hydrolysis Probe dalam Analisis DNA Gelatin Sapi dan Gelatin Babi Menggunakan Real Time Polymerase Chain Reaction

Zilhadia¹,

Izzah²,

Betha³

2017

J.Sains.Far.Klin

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Gelatin has a large application in the food, pharmaceutical and cosmetics industries. Gelatin is mostly derived from the skin or bone of porcine and bovine. Porcine gelatin is forbidden for Muslim and Jews. For this reason, analytical methods to detect gelatin are needed to make sure the source of gelatin. One of the analytical techniques that can differentiate bovine and porcine gelatin is Real Time Polymerase Chain Reaction (PCR). There are two popular methods of fluorescence dye, namely SYBR green and hydrolysis probe. This study was conducted to compare SYBR green and hydrolysis probe method in analyzing porcine and bovine gelatin DNA using Real Time PCR. The DNA was isolated by commercial kit. The obtained porcine and bovine gelatin DNA were 19.38 ng/μl and 13.63 ng/μl with purity were 1,566 and 1,573, respectively. Then, isolated DNA was analyzed by SYBR green and hydrolysis methods. SYBR green methods was done by annealing temperature of 65 o C for bovine primer and 60 o C for porcine primer. Therefore, hydrolysis probe methods were analyzed by annealing temperature of 60 o C for both porcine primer and bovine primer. The result showed that the hydrolysis probe was higher specificity to identify of porcine and bovine gelatin DNA than SYBR green method. ABSTRAK:Pemanfaatan gelatin secara luas menimbulkan kontroversi dan kekhawatiran bagi masyarakat muslim karena pada umumnya gelatin terbuat dari kulit babi dan sapi. Salah satu teknik analisis yang dapat membedakan gelatin sapi dan gelatin babi adalah Real Time Polymerase Chain Reaction (PCR). Real Time PCR merupakan metode analisis berbasis DNA yang handal, efektif, dan terpecaya. Dalam analisis kualitatif dan kuantitatif, Real Time PCR membutuhkan pewarna fluoresens. Pewarna fluoresens yang umum digunakan adalah SYBR green dan hydrolysis probe. Telah dilakukan perbandingan antara metode SYBR green dan hydrolysis probe dalam analisis DNA gelatin menggunakan Real Time PCR. DNA pada gelatin diisolasi menggunakan kit komersial. Isolat DNA gelatin sapi dan DNA gelatin babi didapatkan sebanyak 19,38 ng/μl dan 13,63 ng/μl dengan kemurnian 1,566 dan 1,573. Isolat DNA yang dianalisis dengan metode SYBR green menggunakan suhu annealing 65 o C untuk primer sapi dan suhu annealing 60 o C untuk primer babi. Isolat DNA yang dianalisis dengan metode hydrolysis probe menggunakan suhu annealing 60 o C untuk primer babi dan primer sapi. Hasil analisis dari kedua metode menunjukkan bahwa metode hydrolysis probe lebih spesifik dalam mengidentifikasi DNA pada gelatin dibandingkan menggunakan metode SYBR green. Keywords PENDAHULUANGelatin adalah polipeptida larut air hasil hidrolisis parsial kolagen dari kulit, tulang dan tulang rawan hewan [1]. Gelatin memiliki sifat yang unik dan berfungsi sebagai zat pembentuk gel, zat pengental, zat pembentuk film, zat pengemulsi, dan zat pensuspensi [2]. Oleh karena itu gelatin digunakan secara luas dalam berbagai sektor industri yaitu industri farmasi, makanan, kosmetik, produk kedokteran dan fotografi. Dalam industri makanan,...

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“…It is indicated that the applied method was not very sufficient and robust. Finally, the results showed that using real-time PCR with commercial DNA extraction kit provides detection of porcine DNA in food products as low as 1%, like in Cai et al (2012), with a 2% false negative rate (Cai et al, 2012;Demirhan et al, 2012). Abd Mutalib et al (2015) reported a technique which is a combination of PCR and southern hybridization in order to be used in analysis of halal authentication of gelatin capsules.…”

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confidence: 99%

Origin Determination and Differentiation of Gelatin Species of Bovine, Porcine, and Piscine through Analytical Methods

Eryılmaz

Işık

Demircan

et al. 2017

Turkish JAF Sci.Tech.

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Gelatin origin determination has been a crucial issue with respect to religion and health concerns. It is necessary to analyze the origin of gelatin with reliable methods to ensure not only consumer choices but also safety and legal requirements such as labeling. There are many analytical methods developed for detection and/or quantification of gelatin from different sources including bovine, porcine and piscine. These analytical methods can be divided into physicochemical, chromatographic, immunochemical, spectroscopic and molecular methods. Moreover, computational methods have been used in some cases consecutively to ensure sensitivity of the analytical methods. Every method has different advantages and limitations due to their own principles, applied food matrix and process conditions of material. The present review intends to give insight into novel analytical methods and perspectives that have been developed to differentiate porcine, bovine and piscine gelatins and to establish their authenticity. Almost every method can be succeeded in origin determination; however, it is a matter of sensitivity in that some researches fail to ensure sufficient differentiation. IntroductionGelatin is a water soluble protein obtained by hydrolysis of collagen to several high molecular weight polypeptides. While the main component of the gelatin is protein (85-92%), it also contains mineral salts and moisture (Baziwane and He, 2003;Schrieber and Gareis, 2007). Gelatin has a great importance in pharmaceutical, cosmetic and especially food industry with its well-known functional properties such as gel formation in jellies, foam formation and stabilization in ice cream, emulsion stabilization in meat products, syneresis stabilization in yogurt (Nhari et al., 2012). Gelatin was produced approximately 326.000 metric tons in 2008 worldwide, and it is estimated to be consumed 395.840 metric tons by the year of 2017 according to Global Industry Analysts, Inc. which is one of the largest and reputed market research companies (Anonymous, 2016).The raw materials used in gelatin production are mainly from bovine and porcine sources: 80% from pig skin, 15% from cattle hide split, the remaining 5 % from bones, fish and other sources (GME, 2016). Gelatin origin determination has been a matter of paramount importance for Muslims, Hindus, Jews, vegetarians and vegans, in that, in Islam and Judaism, porcine derivatives are prohibited to consume, whereas in Hinduism, cow can neither be slaughtered nor consumed. Vegetarians and vegans prefer to eat animal-free foods (Boran et al., 2010;Nhari et al., 2012). Moreover, bovine gelatin has been restricted for human consumption due to Bovine Spongiform Encephalopathy (BSE) outbreak in 1986 in England and other countries (Venien and Levieux, 2005). Therefore, it is necessary to analyze the origin of gelatin with a reliable method. As far as it is concerned; physiochemical, chromatographic, immunochemical, spectroscopic and molecular methods have been developed to determine and differentiate gelatin o...

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Real-time PCR assays for detection and quantitation of porcine and bovine DNA in gelatin mixtures and gelatin capsules

Cited by 82 publications

References 15 publications

Real-Time Polymerase Chain Reaction for Halal Authentication of Gelatin in Soft Candy

Real-Time Polymerase Chain Reaction for Halal Authentication of Gelatin in Soft Candy

Perbandingan Metode SYBR Green dan Hydrolysis Probe dalam Analisis DNA Gelatin Sapi dan Gelatin Babi Menggunakan Real Time Polymerase Chain Reaction

Origin Determination and Differentiation of Gelatin Species of Bovine, Porcine, and Piscine through Analytical Methods

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