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Duplex Real-Time PCR Assay Using SYBR Green to Detect and Quantify

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Duplex Real-Time PCR Assay Using SYBR Green to Detect and Quantify Food Additives & Contaminants: Part A ISSN: 1944-0049 (Print) 1944-0057 (Online) Journal homepage: http://www.tandfonline.com/loi/tfac20 Duplex real-time PCR assay using SYBR Green to detect and quantify Malayan box turtle (Cuora amboinensis) materials in meatballs, burgers, frankfurters and traditional Chinese herbal jelly powder Asing, Eaqub Ali Md., Sharifah Bee Abd Hamid, Motalib Hossain Md., Mohammad Nasir Uddin Ahamad, S. M. Azad Hossain, Nina Naquiah & I. S. M. Zaidul To cite this article: Asing, Eaqub Ali Md., Sharifah Bee Abd Hamid, Motalib Hossain Md., Mohammad Nasir Uddin Ahamad, S. M. Azad Hossain, Nina Naquiah & I. S. M. Zaidul (2016) Duplex real-time PCR assay using SYBR Green to detect and quantify Malayan box turtle (Cuora amboinensis) materials in meatballs, burgers, frankfurters and traditional Chinese herbal jelly powder, Food Additives & Contaminants: Part A, 33:11, 1643-1659, DOI: 10.1080/19440049.2016.1236403 To link to this article: http://dx.doi.org/10.1080/19440049.2016.1236403 Accepted author version posted online: 19 Submit your article to this journal Sep 2016. Published online: 17 Oct 2016. Article views: 31 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac20 Download by: [Jordan Univ. of Science & Tech] Date: 19 November 2016, At: 18:46 FOOD ADDITIVES & CONTAMINANTS: PART A, 2016 VOL. 33, NO. 11, 1643–1659 http://dx.doi.org/10.1080/19440049.2016.1236403 Duplex real-time PCR assay using SYBR Green to detect and quantify Malayan box turtle (Cuora amboinensis) materials in meatballs, burgers, frankfurters and traditional Chinese herbal jelly powder Asinga, Md. Eaqub Alia,b, Sharifah Bee Abd Hamida, Md. Motalib Hossaina, Mohammad Nasir Uddin Ahamada, S. M. Azad Hossaina, Nina Naquiaha and I. S. M. Zaidulc aNanotechnology and Catalysis Research Center (NANOCAT), University of Malaya, Kuala Lumpur, Malaysia; bCentre for Research in Biotechnology for Agriculture (CEBAR), University of Malaya, Kuala Lumpur, Malaysia; cDepartment of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University, Kuantan, Malaysia ABSTRACT ARTICLE HISTORY The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected species widely Received 12 July 2016 used in exotic foods and traditional medicines. Currently available polymerase chain reaction Accepted 2 September 2016 (PCR) assays to identify MBT lack automation and involve long targets which break down in KEYWORDS processed or denatured tissue. This SYBR Green duplex real-time PCR assay has addressed this Malayan box turtle; SYBR research gap for the first time through the combination of 120- and 141-bp targets from MBT and Green real-time PCR; limits eukaryotes for the quantitative detection of MBT DNA in food chain and herbal medicinal of detection and preparations. This authentication ensures better security through automation, internal control quantification; protected and short targets that were stable under the processing treatments of foods and medicines. A species; herbal jelly powder melting curve clearly demonstrated two peaks at 74.63 ± 0.22 and 78.40 ± 0.31°C for the MBT and eukaryotic products, respectively, under pure, admixed and commercial food matrices. Analysis of 125 reference samples reflected a target recovery of 93.25–153.00%, PCR efficiency of 99–100% and limit of detection of 0.001% under various matrices. The quantification limits were 0.00001, 0.00170 ± 0.00012, 0.00228 ± 0.00029, 0.00198 ± 0.00036 and 0.00191 ± 0.00043 ng DNA for the pure meat, binary mixtures, meatball, burger and frankfurter products, respectively. The assay was used to screen 100 commercial samples of traditional Chinese herbal jelly powder from eight different brands; 22% of them were found to be MBT-positive (5.37 ± 0.50–7.00 ± 0.34% w/w), which was reflected through the Ct values (26.37 ± 0.32–28.90 ± 0.42) and melting curves (74.63– 78.65 ± 0.22°C) of the amplified MBT target (120 bp), confirming the speculation that MBT materials are widely used in Chinese herbal desserts, exotic dishes consumed with the hope of prolonging life and youth. Introduction invigorating elements for the long-term restoration of youth and sexual life (Hempen & Fischer 2009; Regulatory laws, analytical tests and public awareness Graham-Rowe 2011). They fall under the reptile work side by side to ensure safety and quality in foods umbrella. There are approximately 460 varieties of and pharmaceutical products from farm to fork freshwater turtles and tortoises around the world (Shackell 2008). Recent market survey and media (Fund 2002); currently, all are enlisted under the reports reflect that adulteration and mislabelling of most vulnerable clades of vertebrates (Spinks et al. animal products, such as sausages, ground meat, meat- 2012), and out of 293 International Union for balls, deli meat, dried meat, game meat, bush meat and Conservation of Nature and Natural Resources burgers, are widespread across the globe (20–70% in (IUCN) Red-Listed freshwater turtles and tortoises, 88 Mexico, Turkey and Africa; 8% in the UK, and 19.4% species are found in Asia. According to Fund (2002), in the USA) (Brodmann et al. 2001; Ayaz et al. 2006; 3% of the world’s turtle species are already extinct, 9% Fajardo et al. 2010; Brown 2013; Cawthorn et al. 2013; are critically threatened, 18% are threatened and 2% D’Amato et al. 2013; Özpınar et al. 2013). The bones, are at high risk in various habitats. About 1% of the shells, skins and eggs of turtle and tortoise species are Asian turtles are already extinct, 20% are critically believed to have active healing attributes and CONTACT Md. Eaqub Ali [email protected] Nanotechnology and Catalysis Research Center (NANOCAT), University of Malaya, Kuala Lumpur, Malaysia © 2016 Informa UK Limited, trading as Taylor & Francis Group 1644 ASING ET AL. endangered, 31% are endangered and 25% are represents a great threat to nesting resources (WWF vulnerable. 2015). The enormous illegal trade cannot be sustained The Malayan box turtle (MBT) is the most common and its negative impact has already been reflected hard-shelled chelonian turtle species in Asia, with through a series of collapses in regional turtle stocks extensive habitats across Malaysia, Indonesia, India, in several countries (Fund 2002). Furthermore, the Bangladesh, Thailand, Myanmar, Vietnam, turtle materials in food chains and medicines involve Philippines, Singapore, Laos and Cambodia (Schoppe both health and social risks because these animals are 2008; Schoppe & Das 2011). It belongs to the Cuora natural scavengers of waste materials and hosts of genus, which encompasses a total of 12 turtle species, several microbes and heavy metals (Aguirre et al. all of which have habitats across the Asian peninsulas 2006; Magnino et al. 2009), and they are also prohib- (Spinks et al. 2012). Unfortunately, all the Cuora spe- ited from consumption for Muslims (Ali et al. 2015a). cies are enlisted in the most vulnerable category by the The Malaysian government is also highly committed to IUCN and Appendix II of the CITES database ensure halal ingredients in foods and medicines. (Schoppe 2008). Each year, more than 10 million live Therefore, this study focused on the development of Asian box turtles (Cuora) (ABT) are imported into an automated PCR method to determine the low levels southern China from Southeast Asian countries (Fund of MBT tissue expected in foods and traditional 2002), and the Taiwan statistical report revealed that medicines. more than 120 metric tons of turtle shells were Animal materials in the food chain may be authen- imported between 1992 and 1998 from mainland ticated using protein- (Ayaz et al. 2006; Karabasanavar China (Lo et al. 2006), indicating overhunting of et al. 2014), lipid- (Ghovvati et al. 2009) and DNA- Cuora species is rampant across Asia. (McKenna et al. 2010) based analytical techniques; but Since 2005, the Malaysian government and DNA-based molecular schemes have evolved as the Department of Wildlife and National Parks method of choice because of the exceptional stability, (PERHILITAN) of Malaysia have jointly banned the uniformity and polymorphic features of the DNA export of MBT and other turtle species to other coun- molecule itself. The short-length mitochondrial DNA tries. However, the MBT species is extensively captured biomarkers are especially important because of DNA’s by local hunters for the meat, shells and bones for stability attributes, maternal origin, abundance in mul- trades in local and international markets. The burgeon- tiple copies per cells and additional protection given by ing demands in local and international markets also the specialised mitochondrial membranes (Ali et al. might encourage illegal trafficking of MBT and other 2014). Several DNA-based approaches such as spe- turtles and could be used in meat products and tradi- cies-specific PCR (Hsieh et al. 2003; Yan et al. 2005; tional medicines (Schoppe 2008; Rashid et al. 2015; Ali Karabasanavar et al. 2014; Davy et al. 2015), multiplex et al. 2015a). Recently, a huge amount of illegal fresh PCR (Ghovvati et al. 2009; Ali et al. 2015b), PCR water turtle meats was seized at Karachi seaport by the product sequencing (Lo et al. 2006; McKenna et al. Pakistani customs department while being trafficked as 2010), PCR-restriction fragment length polymorphism fish meat for Hong Kong (Woodhouse 2015). (PCR-RFLP) (Rashid et al. 2015), randomly amplified Concomitantly, Malaysian police and customs depart- polymorphic DNA (RAPD) (Saez et al. 2004), real-time ments seized 10,000 turtle eggs in Sabah in 2008 and PCR (Rojas et al. 2011; Davy et al. 2015; Safdar & 4.3 metric tons of reptiles including lizards, snakes, Junejo 2015) and DNA barcoding (Ardura et al. 2010; freshwater turtles and tortoises at the Thailand– Liu et al. 2013) have been proposed for both forensic Malaysia border area in 2010 (Anonymous 2008).
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