Multiplex polymerase chain reaction-restriction fragment length polymorphism assay discriminates of rabbit, rat and squirrel meat in frankfurter products

Accepted Manuscript Multiplex polymerase chain reaction-restriction fragment length polymorphism assay discriminates of rabbit, rat and squirrel meat in frankfurter products Md. Eaqub Ali, Mohammad Nasir Uddin Ahamad, Asing, M.A. Motalib Hossain, Sharmin Sultana PII:

S0956-7135(17)30375-4

DOI:

10.1016/j.foodcont.2017.07.030

Reference:

JFCO 5723

To appear in:

Food Control

Received Date: 30 April 2017 Revised Date:

7 July 2017

Accepted Date: 23 July 2017

Please cite this article as: Ali M.E., Uddin Ahamad M.N., Asing , Hossain M.A.M. & Sultana S., Multiplex polymerase chain reaction-restriction fragment length polymorphism assay discriminates of rabbit, rat and squirrel meat in frankfurter products, Food Control (2017), doi: 10.1016/j.foodcont.2017.07.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Multiplex Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Assay Discriminates of Rabbit, Rat and Squirrel Meat in Frankfurter Products

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Md. Eaqub Alia, b, c*, Mohammad Nasir Uddin Ahamada, Asinga, M.A. Motalib Hossaina, Sharmin Sultanaa a Nanotechnology and Catalysis Research Center, Institute of Graduate Studies, University of Malaya, Kuala Lumpur 50603, Malaysia. Institute of Halal Research University of Malaya, University of Malaya, Kuala Lumpur 50603, Malaysia. c

Centre for Research in Biotechnology for Agriculture, University of Malaya, Kuala Lumpur 50603, Malaysia

* Corresponding e-mail: [email protected]; [email protected]

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.

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ABSTRACT

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The demands for rabbit meat are rapidly growing and Rabbitry is becoming a mean of livelihood

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for many youths. Rats and squirrels are very close relatives of rabbits, could be hunted freely or

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raised in domestic farming and so could be substituted in expensive rabbit meat. This study, for

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the first time, developed and validated a tetraplex polymerase chain reaction-restriction fragment

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length polymorphism (PCR-RFLP) assay to identify and discriminate rabbit, rat and squirrel

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meat under raw and processed foods. Four sets of primes amplified 123, 108, 243, and 141 bp

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fragments from rabbit, rat, squirrel and all eukaryotes, respectively. Specificity was confirmed

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through sequencing and RFLP analysis. When PCR products were digested with BtsIMutI and

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BtsCI enzymes, distinctive fingerprints (115 & 8 bp for rabbit; 64 & 44 bp for rat and 176 & 67

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bp for squirrel) were obtained. The detection limit of the assay was 0.1% meat in frankfurter

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formulation.

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Keywords: Rabbitry; Tetraplex PCR-RFLP; Processed foods; Fingerprints; Restriction enzymes.

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1. INTRODUCTION Ensuring food safety and quality from farm to fork requires regulatory laws, public

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awareness and monitoring systems working side by side (Ali et al., 2016). Various food safety

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regulatory agencies have made it illegal to mix undeclared animal materials in any food

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products; the US Federal Meat Inspection Act (FMIA) and the European Parliament Regulation

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(EC) No. 178/2002 strictly prohibit meat and other animal material adulteration in food chain

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(FMIA, 2016; European Parliament, 2002). However, survey reports reveal the practice is

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going on unfettered across all continents; 68% meat products in South Africa, 19.4 % in the

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USA, 33% in the Gulf countries, 22% in Turkey, and 8% in the UK are reported to have

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mislabeled animal materials (Hossain et al., 2016)

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Rabbitry is a growing industry and rabbit (Oryctolagus cuniculus) meat has already got

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popularity in many European and African countries, such as, Malta, Cyprus, Italy, Czech

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Republic, Spain, Belgium, Luxembourg, Portugal, France, Egypt and Algeria (FAOSTAT,

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2010). American restaurants are also experiencing larger and larger demands for rabbit meat

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(Lanz & Margoli, 2013) and it is also permissible in most religions and cultures. Rabbit meat is

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also appreciated as a functional food because of its lower contents of fat, cholesterol and sodium

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but high concentrations of digestible proteins (Dalle Zotte, 2002; Maertens & Coudert, 2006).

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Thus, rabbit meat is sold at a significantly higher price than those of other regular meats such as

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chicken, goat, beef and pork. In the last 50 years, the world’s production of rabbit meat has

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increased by more than 2.5 fold accounting to 1.6 million tons in 2009. China is currently the

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world’s leading producer of rabbit meat (700,000 t/year), followed by Italy (230,000 t/year),

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Spain (74,161 t/year) and France (51,400 t/year) (FAOSTAT, 2010).

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However, like other meat items, rabbit meat is also not free from adulteration risks.

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Recently, one ton of fresh and frozen carcasses of cat were seized by the Chinese Police in an

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enforcement operational raid at Shunjiang in China, where they were being sold as rabbit meat

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(Fang & Zhang, 2016). Definitely, it is a strong piece of evidence that adulteration of such meat

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is really taking place. The rabbit species belongs to the lagomorph family which was previously

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classified under rodents (Fiedler, 1990). Rats and squirrels are prominent rodent species

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widespread in all continents; they could be hunted free of charge or domestically raised in small

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farms without much investment but they do not have any market demands; these have made

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them a lucrative adulterant for substitution in rabbit and other meat items. In 2013, Chinese

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police detained more than 900 people to unmask a ring involved in the chemical transformation

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of rat, mink and fox meat into mutton like appearances and selling them as mutton (The

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Guardian, 2013)

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Commonly black, red, brown and cane rat species are found in the south-east Asian

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regions and in most of the cases they are considered as an agricultural pest (Anonymous, 2016a).

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The black rats (Rattus rattus) are especially ubiquitous because of their high reproductive rates

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and very strong adaptation power in most environments (IUCN, 2016; BPCA, 2016). Despite

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having rejection from most of the societies, some rat species are domesticated and consumed in

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certain communities because of their greater carcass yield (ca. 65%), purported nutritional

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values, soft bones and taste like bird’s meat (Ajayi & Olawoye, 1974; Odebode et al., 2011).

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Wild rats are frequently traded for consumption along road sides shops in many African

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countries (Redhead & Boelen, 1990); Anonymous, 2016b). About, 80 million pieces of cane rats

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are hunted per year only in western Africa, with a yield of 300,000 metric tons of meat (Hoffman

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& Cawthorn, 2012). However, rat meat consumption is greatly risky for public health because

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they are the potential careers of many infectious microbes, such as Leptospira spp, Listeria spp,

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Yersinia enterocolitica, Pasturella spp, Pseudomonas spp, Yersinia pestis and Hantavirus, which

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can cause several life threating listeriosis, yersiniosis, pasturellosis, meilioidosis and plague

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(Anonymous, 2016c). The Indian outbreak of plague in 1994 that took at least 60 lives was

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linked to the rat meat consumption (Deutsch & Murakhver, 2012). Plague outbreak in Vietnam

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in 1967 was also implicated to rat meat consumption (Hauck et al., 1959). Rat meat adulteration

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in common meat items is also a very sensitive and serious issue because it is a taboo in most

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societies and not permissible in most of the religious foods (Doosti et al., 2014).

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On the other hand, squirrel species, especially the plantain squirrels (Callosciurus

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notatus) are widespread across the Penisular Malaysia, Singapore, Thailand, Indonesia and

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Myanmar (Mohd Noor, 1998; Anonymous, 2016d). Certain Southeast Asian and African

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countries consume squirrel meat as a source of animal proteins as well as exotic dishes (Davis et

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al., 1990). Certain attributes such as the distinctive flavor, high proteins, low fat, less cholesterol

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and the absence of health-threatening anabolic steroids in bush meat also encourage the hunting

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of squirrel species (Redhead & Boelen, 1990). Squirrel dishes are also frequently sold in exotic

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restaurants in the UK and USA (Anonymous, 2016e). However, like rat, squirrels are also a

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potential carrier of Salmonella, Borrelia burgdorferi, Francisella tularensis, Leptospir that may

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cause life threating Creutzfeldt-Jakob disease (CJD) syndrome (Neurodegenerative disease)

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(Anonymous, 2016f) and Lyme disease (Anonymous, 2016g).

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As authentication is concerned, squirrels were subjected to phylogeographical (Finnegan

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et al., 2008) and cross-chromosome painting for genome organizations (Li et al., 2004) as well as

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sequencing for structuring, hybridization and population fragmentation analyses (Barratt, et al.,

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1999; Spiridonova et al., 2005). Recently, a real-time PCR has been proposed for the 4|Page

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differentiation of red and gray squirrels (O’Meara et al., 2012). On the other hand, several

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molecular detection schemes have been proposed for the identification of rat (Fang & Zhang,

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2016; Rahmania & Rohman, 2015) and rabbit species (Amaral et al., 2014; Hanapi et al., 2015)

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(Rafayova et al., 2009). However, these methods are mostly based on a single species target and

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long DNA marker that breaks down under food processing treatments, compromising reliability

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and making the analysis costlier (Ali et al., 2015). The recent evolution of multiplex polymerase

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change reaction (mPCR) assays are especially promising because multiple target oligos could be

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identified in a single assay platform, saving both analytical cost and time (Ali, Razzak, et al.,

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2015; Hossain et al., 2016). Recently, mPCR assays have been reported for pig, dog, cat, rat and

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monkey species (Ali et al., 2015); beef, pork, horse and sheep species (Köppel et al., 2011);

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beef, pork, lamb, chicken, ostrich and horse species (Kitpipit, Sittichan, & Thanakiatkrai, 2014)

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and cattle, buffalo and porcine species (Hossain et al., 2016). The species-specific PCR-

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restriction fragment length polymorphism (PCR-RFLP) assays are furthermore interesting

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because they can authenticate the amplified PCR product through restrictive digestion using one

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or more restriction enzymes (REs) (Rashid et al., 2015). Using the existing sequence variation

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within a defined region of DNA, the differentiation of even closely related species has been done

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(Hsieh & Hwang, 2004); cattle, yak, and buffalo (Chen et al., 2010); cattle-buffalo and sheep-

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goat (Girish et al., 2005); swine and wild boar (Mutalib et al., 2012) and various fish species

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(Nebola et al., 2010) have been successfully discriminated by applying PCR-RFLP techniques.

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However, to the best of our knowledge, no multiplex PCR or PCR-RFLP assays have been

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reported for rabbit, rat and squirrel authentication. In this study, these gaps were addressed for

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the first time through the development and validation of a tetraplex PCR-RFLP assay with short-

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lengths of amplicon for the simultaneous identification and discrimination of rabbit, rat and

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squirrel materials under real food matrices, such as frankfurter formulation.

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2. Materials and Methods

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2.1. Sample collection

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Fresh muscle tissues or specimens were obtained from rabbit (Oryctolagus cuniculus), squirrel (Calloscious

notatus), chicken (Gallas gallus), beef (Bos Taurus), buffalo (Bubalus

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bubalis), sheep (Ovis aries), goat (Capra hiscus), pig (Sus scrofa), duck (Anas platyrnychos),

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pigeon (Columba livia), crocodile (Crocodylus niloticus), donkey (Equus asinus), amboina box

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turtle (Cuora amboinensis), chinese edible frog (Hoplobatracus rugulosus), deer (Cervus nippon

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yesoensis), dog (Canis lupus familiaris), cat (Felis catus), tuna (Thunnus orientalis), salmon

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(Salmo salar) and plant species, namely, wheat (Triticum aestivum), cucumber (Cucumis

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sativus), onion (Allium cepa), and chili (Capsicum Capsicum annuum). Where available, meat,

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fish and plant s p i c e s s p e c i m e n s were collected from commercial wet (Pudu Raya) and super

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markets (Aeon, Tesco and Giant) at Kuala Lumpur on three different days to increase the genetic

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diversity of the collected samples. Deer (Cervus nippon) meat was procured in triplicates from

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the Faculty of Veterinary Sciences at the University of Putra Malaysia, located at Serdang in

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Selangor. Stray dog (Canis lupus familiaris), cat (Felis catus) and rat (Rattus rattus) muscle were

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donated by Kuala Lumpur City Hall (KLCH/DBKL) at Air Panas in Kuala Lumpur. Monkey

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(Macaca fascicularis sp) meat was a gift from the Department of Wildlife and National Park

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Malaysia (DWNPM/ PERHILITAN) at Cheras in Kuala Lumpur. It is worthy to note that DBKL

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routinely kills rats, cats and stray dogs for population control and public security purposes in the

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town area; so no animals were killed for this study purposes but sufficient amount of muscle

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tissues were taken from the already killed animals following institutional and country laws.

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Details information of all the collected samples is given in table 1. The identities of all the

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collected samples were confirmed by sequencing. A total of 108 (4 x 9 x 3) commercial

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meatballs of 4 different brand were purchased from Malaysian outlets on three different dates.

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All samples were transported under ice-chilled conditions and w e r e cut into t h e

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possible pieces with surgical blades prior to storage at -20°C until further uses.

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2.2. Preparation of frankfurter

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Model chicken and beef frankfurters were made in the laboratory following Hossain et al.

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(2016) (Table 2). The as prepared frankfurters were deliberately contaminated by spiking with

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1%, 0.5% and 0. 1% meat from each of the squirrel, rabbit and rat species. Frankfurters having

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0.1%

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authenticate the PCR product by RFLP analysis chicken and beef frankfurters were adulterated

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by spiking squirrel, rat and rabbit (SRR) and were heat treated by boiling at 98°C for 90 min and

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autoclaved at 121°C under 15 psi pressure for 2.5 h (Rahman et al., 2014). All samples were

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stored at -20°C until DNA extraction.

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2.3. DNA extraction

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contaminations were autoclaved at 1210C under 15 psi pressures for 2.5 h.

Total DNA was extracted from 30 mg of muscle tissue of each meat and fish species as

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well as their admixed meat products using Yeastern Genomic DNA Mini Kit (Yeastern Biotech

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Co., Ltd. Taipei, Taiwan) as described in our earlier report (Hossain et al., 2016). Plant DNA

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was extracted using DNeasy Plant Mini Kit (QIAGEN GmgH, Hilden, Germany) (Ali et al.,

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2015a). DNA from model and commercial frankfurters was extracted using NucleoSpin Food

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DNA kit (MACHEREY-NAGEL, GmbH & Co., Duren, Germany) (Hossain et al., 2016). The

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purity and concentration of all extracted DNA was determined using a UV-VIS

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spectrophotometer (Biochrom Libra S70, Biochrom Ltd, Cambridge, UK) based on absorbance

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at A260/280 and calculating their ratios (Ali et al., 2015). All extracted DNAs were kept at -20

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°C until further uses.

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2.4. Design of species specific primers

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Species-specific primers of three target species (rabbit, rat and squirrel) were designed by

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targeting mitochondrial Cytb and ATP6 genes that are well protected by multilayer

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mitochondrial membrane (Xin et al., 2006). Usually, mitochondrial genes offer higher degree of

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divergence but sufficient conserved regions within the species due to their maternal inheritance

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and availability in thousands of copies per cell (Xin et al., 2006). Previously, cytb and ATP6

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genes were also used to study inter and intra species discrimination (Kitpipit et al., 2014; Brown

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et al., 1979; Fonseca et al., 2008). The complete genome sequences of each species were

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retrieved form the National Centre of Biotechnology Information (NCBI). The MEGA5 and

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ClustalW sequence alignment tools were used to identify the hyper-variable and conserved

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regions to study the presence of mismatched nucleotides (Hossain et al., 2016). The species

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specific conserved regions were used to design three sets of species-specific primers for rabbit,

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rat and squirrel species, respectively, using Primer3Plus. The theoretical specificity of the

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designed primers was confirmed by Basic Local Alignment Tool (BLAST) in NCBI. Total

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mismatch between the target and non-target species were determined by aligning the primers’

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sequences against 45 species (Table S1) (Tamura et al. 2011). One set of previously documented

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primers that amplified a 141-bp site of eukaryotic 18S rRNA gene were used as internal positive

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control (IAC) (Fajardo et al. 2008). The designed primers were supplied by the 1st Base (1st Base

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Sdn. Bhd., Puchong, Malaysia) and kept at -20 °C until uses. All information about primers could

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be found in table 3.

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2.5. Tetraplex PCR To optimize a tetraplex PCR assay, at first simplex PCR was performed for each target

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species (rbbit, rat and squirrel). The simplex PCR was performed in a 25 µL reaction mixture

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comprising of 5 µL 5X GoTaq Flexi Buffer, 0.2 mM each of dNTPs, 2.5 mM MgCl2, 0.9 U

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GoTaq Flexi DNA Polymerase (Promega, Madison, WI, United States), 0.4 µM of each primer

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and 2 µL (20 ng/µL) DNA template. A 141 bp of endogenous control was used as an internal

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positive control (IAC) for each PCR assay (Fajardo et al., 2008). All PCR assay was performed

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in an ABI 96 well verity Thermal Cycler ( Applied Biosystems, Forest City ,CA, United states)

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with an initial denaturation at 950C for 30s, anneling at 600C for 30-35s ,extension at 720C for 40

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s and the final extention at 720C for 5 min. After completing the PCR assay the PCR products

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were visualized in 2% agarose gel strained with Flourosafe DNA stain (First Base Laboratories

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SdN, Bhd,Selangor, Malaysia) under a gel documentation system (AlphaImager HP,Alpha

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Innotech Crop, California, United States) (data not shown). After optimizing simplex PCR

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assays for individual species, duplex and finally tetraplex PCR assay were sequentially

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developed. The optimized parameters for simplex, duplex and tetraplex PCR assays are shown in

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table 4 and 5.

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2.6. RFLP analysis

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The amplicon sequence of each target species was retrieved from NCBI data base and

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was checked with NEB Curtter Version 2.0 software to find out the restriction sites. Based on the

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software assessment, two restriction endonucleases were chosen for RFLP analysis that cut only

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the target amplicons but did not have any restriction sites in IAC. BtsCI was selected for squirrel

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(243 bp) and rabbit (123 bp) amplicons; but BtsIMutI was chosen for rat (108 bp) amplicons. The 9|Page

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restriction digestion was performed in a reaction mixture composed of 15 µL unpurified PCR

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product from each of the simplex PCRs, 2.5 µL buffer, 1 µL of RE and 0.25 µL BSA and balance

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amount of nuclease free water. The reaction mixture was gently mixed and spun down and

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incubated at 37°C first for 60 min for BtsCI digestion and then 55°C for another 60 min for

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BtsIMutI digestion in a shaking water bath. Finally, the reaction mixture was heated at 80°C for

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20 min to denature the enzymes and stop restriction digestion. However, for tetraplex PCR,

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restriction digestion was executed in 25 µL reaction mixture having 15 µL unpurified PCR

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products, 2.5 µL digestion buffer, 2.5 µL BtsCI enzyme and 1.5 µL BtsIMutI enzyme. The

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digested PCR product was visualized using a QIAxel DNA High Resolution Kit in an automated

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QIAxel Advanced Capillary Elecrophoresis System (QIAGEN GmbH, Hilden, Germany).

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3. Result and Discussion

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3.1. Assessment of DNA quality and concentration

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Total genomic DNA was extracted from pure, admixed and commercial frankfurter

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products under raw and processed condition. The concentration and purity of the extracted DNA

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was determined based on absorbance value at 260 nm and absorbance ratio at 260/280 nm. The

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initial concentration of the extracted DNA was ≥100ng/ul but the lower concentration was

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prepared by adding required amount of nuclease free distilled water since lower concentration

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could not be measured accurately by spectroscopic method. The absorbance ratio of all DNA

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sample at 260/280 nm was 1.7-2.0 that suggested that good quality DNA was extracted from all

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samples (Herrero, Vieites, & Espiñeira, 2012).

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3.2. Confirmation of PCR specificity

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Species Specific PCR assay has been widely used for the determination of species in

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many instances because of its simplicity, low cost and robust result. Determination of specificity

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is first step towards the development of any diagnostic PCR assays. It has been documented that

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critical mismatches in the primer binding regions interferes with PCR efficiency or sometimes

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lead to false negative detection. Therefore, the primer regions were critically evaluated against

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45 species firstly through available bioinformatic softwares analyses and then cross-tested

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against 22 species through practical PCR run using simplex, duplex and tertraplex PCR (Hossain

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et al, 2016). The three primer sets were aligned against 45 potential species that included 5, 10

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and 8 closely related species of the Oryctolagus, Rattus and Calloscious genus, respectively,

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using ClustalW and MEGA5 multiple alignment softwares (Table S1(a-c)). The results reflected

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100% sequence matching only with the respective targets but multiple mismatches ((6-37 nt)

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(14.63-84.77%) against the non-target species; these made it highly unlikely that any non-target

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species would be detected. However, the close species of Oryctolagus, Rattus and Calloscious

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genus contained 5-9, 2-3 and 6-13 bp mismatches, respectively. These mismatched nucleotides

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among the closely related species could be originated from trait modifications and gene

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translocations under challenging environment that results in increased rates of anthropogenic and

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inter species hybridization and introgression among the native and introduced animals within the

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same genus (Spinks et al., 2012). The pairwise distances were also analyzed using neighbor-

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joining method (Table S2 (a-c)). Zero distance was found for the exact species but the distances

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among the 10 rat species were 0-0.12, among the 8 squirrel species were 0-0.20, and among the 5

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rabbit species were 0-0.35. However, the genetic distances among the targets and other non-

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targets were significantly higher (0.2-4.10). These suggested that there is no or very little

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probability of cross-target amplification. When phylogenetic trees were constructed using the

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amplicon sequences, all rabbit, rat and squirrel species clustered in their respective domains

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(Figure S1(a-c)). When 3 D plots were built, huge dissimilarities were observed among the

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targets and non-target species (Figure S2 (a-c)). Thus the theoretical analyses collectively

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reflected that there is no likelihood or probability that any non-target species would be amplified

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by the developed primers. Furthermore, only 2/3 bp mismatches and very close pairwise distance

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among the rat species indicated that rat primers were universal and they would detect all rat

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species.

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Following theoretical confirmation, simplex, duplex and tetraplex PCR assays were

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performed by cross-challenging the primers against the three target and 22 non-target species

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(Figure 1). The amplified PCR products were then sequenced and the obtained sequences were

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subjected to BLAST analyses in NCBI against non-redundant nucleotides. The amplified targets

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showed 99.18, 98.14 and 98.35 % sequence matching with European rabbits, black rats and

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plantain squirrels, respectively. This value was within the acceptable limit because at least 98%

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sequence similarity is required for the accurate identification of species (Cawthorn et al., 2013).

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Primers sequences were further evaluated for melting temperatures (Tm) that play vital roles in

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primer annealing, especially in multiplex PCR that requires quite identical Tm for all primers so

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that they can anneal with respective templates under the same set of PCR conditions. In this

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study, the Tm was about 58 °C for all primers (Table 3).

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Previously, studies were made with the squirrel species for the determination of

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phytogeography (Finnegan et al., 2008), genome organizations through cross-chromosome

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painting (Li et al., 2004), and genetic structure of fragmented populations by sequencing (Barratt

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et al., 1999) and hybridization (Spiridonova et al., 2005). However, these researches are not

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enough for species determination under food matrices. Real-time PCR assay was also 12 | P a g e

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documented for the differentiation of red and gray squirrels but amplicon length was not

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mentioned. However, it is not sure that such amplicon would be stable under food processing

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treatments that usually breakdown long targets. Moreover, their detection limits still remain

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obscure (O’Meara et al., 2012). Several molecular detection schemes were also proposed for the

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authentication of rat (Fang & Zhang, 2016; Rahmania & Rohman, 2015) and rabbit (Amaral et

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al., 2014; Hanapi et al., 2015). However, these methods are also mostly based on a single and

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long DNA marker which often breaks down under food processing treatments as well as under

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the states of natural and environmental decomposition, making them less trustworthy (Ali,

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Asing, et al., 2015). Furthermore, no assays have been reported for the simultaneous

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differentiation of all the three species under food matrices. To the best of our knowledge this

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tetraplex PCR assay is the first systematic document for the identification and discrimination of

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rabbit, rat and squirrel species under food matrices.

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3.3. Specificity and Sensitivity under complex food matrices

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Recent trends demonstrate that meat adulteration practices are more likely done in

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processed food items such as meatballs, burgers, and frankfurters; where the morphological

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aspects of identifications are greatly disrupted through processing treatments (Ali, Razzak, et al.,

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2015; Rahman et al., 2014). To simulate this form of adulteration, the rabbit, rat and squirrel

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specific tetrplex PCR assay was evaluated under chicken and bee frankfurter matrices; a very

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popular food item consumed all over the world (Ali, Razzak, et al., 2015). The chicken and beef

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frankfurters were made in the laboratory having 1%, 0.5% and 0.1% adulteration from minced

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and deboned meat from each of the squirrel, rat and rabbit species. Moreover, frankfurters of

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0.1% adulteration were further autoclaved at 121°C and 15 psi for 2.5 h that is known to

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breakdown target DNA (Ali et al., 2016). However, rabbit (123 bp), rat (108 bp), squirrel (243

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bp) specific PCR products were obtained from both treated and untreated samples having 1%,

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0.5%, and 0.1% adulterations but only IAC (141 bp) that was the signature of eukaryotic DNA

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was amplified from both pure and contaminated chicken and beef frankfurters (Figure 2),

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reflecting the sensitivity and discriminatory attributes of the developed PCR assay even at 0.1%

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contamination.

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3.4. RFLP analysis

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Verifying the authenticity of the amplified PCR products definitely increases assay

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reliability (Yang, Kim, Byun, & Park, 2005) and it could be done through PCR-PFLP, PCR

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product sequencing and probe hybridization techniques (Maede, 2006). Probe hybridization is a

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laborious method and it needs high quality DNA (Mafra, Ferreira, & Oliveira, 2008) which is

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highly unlikely in case of processed foods treated under extensive heat, pressure or chemicals.

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On the other hand, DNA sequencing procedure require expensive laboratory set up and highly

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skilled human capital (Girish et al., 2004; Mafra, Ferreira, & Oliveira, 2008) and so it is not

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suitable for routine food screening program (Albers, Jensen, Bælum, & Jacobsen, 2013).

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However, PCR-RFLP technique is quite simple and could be done in ordinary laboratories (Park,

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Shin, Shin, Chung, & Chung, 2007), offering a great analytical support when a fraudulent

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substitution or any unintentional contaminations are done (Sharma, Thind, Girish, & Sharma,

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2008). In this method, a profile of signature nucleotide fragments is created by restriction

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digestion with one or two endonucleases followed by separation in gel or capillary

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electrophoresis (Ballin, Vogensen, & Karlsson, 2009). In this study, at first each target was

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digested separately with an appropriate restriction enzyme to define its individual restriction

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patterns to eliminate any ambiguities that may arise from the multiplex PCR products. Both

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squirrel (243 bp) and rabbit (123 bp) products ware digested by BtsCI, which generated two

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fragments for each of the targets (176 & 67 bp for squirrel and 115 & 8 bp for rabbit); but rat

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PCR product was cleaved by BtsIMutI that also yielded two fragments (64 & 44 bp) (Figure 3

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and Table 6).

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Finally, the tetraplex PCR products were subjected to restriction enzyme digestion with

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the two enzymes in a single tube, and this collectively generated molecular fingerprints of total

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six fragments (8, 44, 64, 67, 115, 176 bp) plus IAC (141 bp) ( Figure 3). Subsequently the

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optimized tetraplex PCR-RFLP assay tuned for the chicken and beef frankfurter analyses under

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raw, boiled and autoclaved condition. PCR products were obtained from lab made frankfurters

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(81 beef and 81 chicken) (Table 7) having deliberate adulterations (1, 0.5 and 0.1%) of target

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meat items (rabbit, rat and squirrel) were digested and their restriction patterns were observed.

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Expected PCR and RFLP fragments were clearly visualized upon electrophoresis (Figure 4),

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reflecting that variations in food processing treatments could not affect the stability of the

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optimized assay.

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3.5. Analysis of commercial frankfurters

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The replacement of costly meat by cheaper one is frequently done in commercial

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frankfurters (Bargen, Dojahn, Waidelich, Humpf, & Brockmeyer, 2013) in which physical attributes

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of meat are significantly modified and identification cannot be made without analytical supports.

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Frankfurters are also widely consumed both in pure form and as ingredients of other food items

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such as bihun and mee. So the developed tetraplex PCR assay was tested for the screening of

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commercially available 36 beef and 36 chicken frankfurters of four different brands (9x4=36)

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procured from Malaysian outlets. Dummy frankfurters were spiked with 0.1% meat from each

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target and were used as positive control. The rabbit (123 bp), rat (108 bp), squirrel (243bp) and

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IAC (141 bp) specific PCR products were obtained from all positive controls but only IAC was

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amplified from all commercial frankfurts (Table 7), reflecting beef and chicken frankfurters in

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Malaysia did not have any rabbit, rat and squirrel adulteration. Rationally rabbit adulteration in

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chicken and beef products is unlikely because rabbit meat is sold at higher prices than chicken

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and beef. On the other hand, rat and squirrel are not permissible in Malaysian markets, especially

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in halal food items. However, squirrels are sold as exotic dishes in certain places in Malaysia

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such as Jalan Pudu in Kuala Lumpur; but definitely the price is higher than those of the regular

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dishes.

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4. Conclusion

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Wild species, especially those that could be easily hunted or raised, inevitably have

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remained a cheap source of proteins for many population groups, particularly in the developing

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world and substantially contribute to local food supply chain. When traded, these resources can

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further provide cash revenues where few alternative sources of income are available. In these

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contexts, Rabbitry is a growing industry and certain places, such as California, are already

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having the shortage in the supply of rabbit meat. Consequently, the trades of rabbit meat have

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already crossed the geographical boundary and several countries such as China, Italy and France

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have become the global leaders in rabbit meat exports. Therefore, checking of rabbit meat

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against potential adulterant is a timely need. Rat and squirrel species are available in most places

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and so could be easily adulterated in common items including rabbit meat which is genetically

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very close to rats and squirrels. However, the adulteration of rabbit and squirrel has negative and

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huge concerns to public health, religions and cultures.

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The PCR-RFLP method that is developed in this study is a rapid, straightforward and

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cheap way of tracing rabbit, rat and squirrel materials in food chain in a single assay platform.

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The specificity of this assay was confirmed by cross checking against 45 species theoretically

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and 22 non-target species practically. The stability of the target analytes was confirmed through

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different food treatment conditions such as boiling (98 °C for 90 min) and extensive autoclaving

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at 121°C and 15 psi for 2.5 h that is known to breakdown DNA. The assay’s detection limit was

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tested until 0.1 % adulteration that is beyond the profitable margin for commercial adulteration.

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Frankfurter matrices are very complex and so the assay was suitable for the identification of

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target species from any frankfurter products. RFLP option itself is a validation for the

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authenticity of original PCR products. Therefore, the developed assay showed sufficient merits

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to be used by regulatory bodies for the discriminatory identification rabbit, rat and squirrel

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species in food chain or archaeological studies.

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Acknowledgement

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This study was supported by the University of Malaya Grant No. PG257-2016A to M.E.

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Ali. The authors would like to thank to Dewan Bandaraya, Kuala Lumpur and Wildlife and

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National Parks, Malaysia for providing cat, dog, rat and monkey meat samples.

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Conflict of Interest

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All authors declare that they have contributed to this article and they do not have any

conflict of interest to publish it in journal.

Compliance with Ethics requirements

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Ethical clearance of ref. No: NANOCAT/23/07/2013/A(R) was obtained from the

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Institutional Animal Care and Use Committee, University of Malaya (UM IACUC) and all 17 | P a g e

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experiments were conducted following the national and institutional guideline while handling

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animal meats used in this study.

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Figure 1. Specificity analysis of the developed tetraplex PCR assay. In gel images (a) and (b), Lane M, Ladder; and Lane N, negative template control; in (a) Lane 1, tetraplex PCR products of squirrel, rabbit and rat; Lane 2, squirrel (243 bp); ; Lane 3, rabbit (123 bp); Lane 4, rat (108 bp). In the gel images: Lanes 5-14 in (a) and Lanes 2-13 in (b) are endogenous control (141 bp) of the PCR products from chicken, beef ,buffalo, sheep, goat, pork, duck, pigeon, crocodile, donkey, turtle, chinese edible frog, deer, dog, cat, tuna, salmon and four different types of commonly used plant species such as wheat, cucumber, onion and chili, respectively. Eletropherograms of lanes 1-4 of gel image (a) is shown in (c-f), respectively.

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Figure 2. Gel image and the electropherograms of tetraplex PCR for the detection of squirrel, rat and rabbit in deliberately adulterated model beef and chicken frankfurters under raw and processed states. In the gel image, M, Ladder; Lane 1−3, tetraplex PCR of beef frankfurter and Lanes 5−7 tetraplex PCR of chicken frankfurter spiked with 1%, 0.5%, and 0.1% meat from each 22 | P a g e

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of squirrel, rabbit and rat species under raw state. Lanes 4 and 8 tetraplex PCR of heat-treated (autoclaved at 121°C und 15 psi for 2.5 h) 0.1% squirrel, rabbit and rat meat adulterated beef, and chicken frankfurters, respectively; Lane N, negative control. The corresponding electroferograms of Lane 4 and 8 are as shown.

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Figure 3. RFLP analysis of duplex (lanes 1, 3, 5) and tetraplex PCR (lane 7, 8) products before (Lanes 1, 3, 5, and 7) and after (Lanes 2, 4, 6 and 8) restriction digestion. In the gel images: lane M, Ladder; lanes 1-8: endogenous control (141 bp) ; lanes N, negative template control; lanes 1 and 2, squirrel; lanes 3 and 4, rat; lanes 5 and 6, rabbit; lanes 7 and 8, tetraplex PCR of squirrel, rabbit and , rat. The corresponding electropherograms are shown with labels.

629 630 631 632 633 634 635 636 637 638 639 640 641 642

Figure 4. PCR-RFLP analysis of tetraplex PCR products from deliberately adulterated raw (lanes 1, 2, 7, 8), boiled (lanes 3, 4, 9, 10) and autoclaved (lanes 5, 6, 11, 12) beef (Lanes 1−6) and chicken (Lanes 7−12) frankfurters. In gel image, Lanes 1 and 2, squirrel, rabbit and rat meat adulterated raw beef frankfurter before and after digestion, respectively; Lanes 3 and 4, squirrel, rat and rabbit meat-adulterated boiled (98 °C for 90 min) beef frankfurter before and after digestion, respectively; Lanes 5 and 6, squirrel, rat and rabbit meat-adulterated autoclaved (121° C and 15 psi pressure for 2.5 h) beef frankfurter before and after digestion, respectively; Lanes 7 and 8, squirrel, rat and rabbit adulterated raw chicken frankfurter before and after digestion, respectively; Lanes 9 and 10, squirrel, rat and rabbit meat -adulterated boiled (98 °C for 90 min) chicken frankfurter before and after digestion, respectively; Lanes 11 and 12, squirrel, rat and rabbit meat adulterated autoclaved (121°C and 15 psi pressure for 2.5 h) chicken frankfurter before and after digestion, respectively. In the gel images: Lanes 1-12: endogenous control (141 bp); Lane N, negative template control.

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617 618 619 620 621

643 Tables

645

Table 1. Information of collected food samples.

646

Table 2. Composition of model frankfurters used in this study.

647

Table 3. Information about primers used in the study.

648

Table 4. Concentration of various reagents in simplex, duplex and tetraplex PCR.

649

Table 5 .Cycling parameters of the PCR reaction.

650

Table 6. Restriction digests of the PCR product.

651 652

Table 7. Analysis of admixed of commercial meat products with rat, rabbit and squirrel meat specific PCR assay.

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23 | P a g e

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Table 1 Information of collected food samples.

Sources

Geographic coordinates of the sources

Animal Sources

Sample

1 2

Squirrel Rat

Paser Borong, Pudu Raya and Selangor, Malaysia Kuala Lumpur, Malaysia

Dead Dead

Meat Meat

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Rabbit Chicken Cow Goat Pig Pigeon Sheep Duck Buffalo Crocodile Turtle Donkey Deer Monkey Dog

Paser Borong, Pudu Raya and Selangor, Malaysia Midvally,Kuala Lumpur, Malaysia Midvally,Kuala Lumpur, Malaysia Midvally,Kuala Lumpur, Malaysia Midvally,Kuala Lumpur, Malaysia PJ old town, Pataling jaya, Kuala Lumpur, Malaysia PJ old town, Pataling jaya, Kuala Lumpur, Malaysia PJ old town, Pataling jaya, Kuala Lumpur, Malaysia Paser Borong, Pudu Raya and Selangor, Malaysia Old klang road, Selangor, Malaysia Paser Borong, Pudu Raya and Selangor, Malaysia Paser Borong, Pudu Raya and Selangor, Malaysia Kuala Lumpur, Malaysia Cheras, Kuala Lumpur, Peninsular Malaysia, Kuala Lumpur, Malaysia

Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead

Meat Meat Meat Meat Meat Meat Meat Meat Meat Meat Meat Meat Powder Meat Meat

30 30 30 25 20 20 25 20 30 25 25 25 30 20 15

18

Cat

Kuala Lumpur, Malaysia

Dead

Meat

15

19

Chines frog

Wet market Dewan Bandaraya Kuala Lumpur (DBKL Wet market AEON BIG supermarket AEON BIG supermarket Tesco supermarket Wet market AEON supermarket Wet market Wet market Wet market Purl point shopping centre AEON supermarket Wet market Veterinary Department Wildlife and National Parks (DWNP) Dewan Bandaraya Kuala Lumpur (DBKL) Dewan Bandaraya Kuala Lumpur (DBKL) Wet market

Number of samples 30 30

Paser Borong, Pudu Raya and Selangor, Malaysia

Dead

Meat

15

20

Tuna

AEON BIG supermarket

Midvally,Kuala Lumpur, Malaysia

Dead

Meat

15

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No Species

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Table 1 Continued Species

Sources

Geographic coordinates of the sources

Animal Sources

Sample

21 22

Salmon Wheat

AEON BIG supermarket AEON BIG supermarket

Midvally,Kuala Lumpur, Malaysia Midvally,Kuala Lumpur, Malaysia

Dead Powder

23

Cucumber

AEON BIG supermarket

Midvally,Kuala Lumpur, Malaysia

-

24

Onion

AEON BIG supermarket

Midvally,Kuala Lumpur, Malaysia

-

25

Chili

AEON BIG supermarket

Midvally,Kuala Lumpur, Malaysia

-

26

Chicken Frankfurter (Ramly) Chicken Frankfurter (Tesco) Chicken Frankfurter (Ayamas) Chicken Frankfurter (Prima) Beef Frankfurter (Ramly) Beef Frankfurter (Figo Foods) Beef Frankfurter (Saudi Gold) Beef Frankfurter (Farm’s Best)

AEON BIG supermarket

Midvally,Kuala Lumpur, Malaysia

-

Meat Fresh vegetable Fresh vegetable Fresh vegetable Fresh vegetable Meat products

Tesco supermarket

Kuala Lumpur, Malaysia

-

Meat products

3 Kg

AEON BIG supermarket

Midvally,Kuala Lumpur, Malaysia

-

Meat products

2 Kg

AEON BIG supermarket

Midvally,Kuala Lumpur, Malaysia

-

Meat products

2 Kg

-

Meat products

3Kg

30

31

32

33

AEON BIG supermarket

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29

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28

Midvally,Kuala Lumpur, Malaysia

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SC

No

Number of samples 15 1-2 kg 1-2 kg 1-2 kg 1-2 kg 2-3 Kg

AEON BIG supermarket

Midvally,Kuala Lumpur, Malaysia

-

Meat products

2 Kg

Tesco Supermarket

Kuala Lumpur, Malaysia

-

Meat products

3.5 Kg

Kuala Lumpur, Malaysia

-

Meat products

3 Kg

Tesco Supermarket

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Table 2 Composition of model frankfurters used in this study.

chicken 45 45aa 7.5

beef 45a 45a 7.5

squirrel 45a 45a 7.5

rat 45a 45a 7.5

rabbit 45a 45a 7.5

Starch/breadcrumb Chopped onion Chopped ginger Cumin powder Garlic powder Black pepper Tomato pest butter salt othersb

6.5 2.5 0.15 0.75 0.5 0.23 2.0 2.5 SA SA

6.5 2.5 0.15 0.75 0.5 0.23 2.0 2.5 SA SA

6.5 2.5 0.15 0.75 0.5 0.23 2.0 2.5 SA SA

6.5 2.5 0.15 0.75 0.5 0.23 2.0 2.5 SA SA

6.5 2.5 0.15 0.75 0.5 0.23 2.0 2.5 SA SA

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Ingredient Minced meat Soy protein

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To prepare ≥70 g frankfurter specimens,10%, 1%, 0.5%, and 0.1% of chicken, beef, squirrel, rat, and rabbit were mixed with a balanced amount of respective minced meat. bFlavoring agents and enhancers. cSA, suitable amounts

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Table 3 Information about primers used in the study.

Rabbit

Cytb

Rat

ATP6

Endogenous Control

18SrRNA

Sequence (5’-3’)

Tm value

Forward-ATCTCCCCACTCCTTCCAAT Reverse- CGCGGCCTACATGTAAGAAT Forward- TCCGATACCTCCACGCTAAC Reverse- GGAGGATGATGCCAATGTTTC

59.8 °C 60.1 °C

Forward- CATCATCAGAACGCCTTATTAGC Reverse- AGGTTCGTCCTTTTGGTGTATG ForwardGTAGTGACGAAAAATAACAATACAGGAC Reverse-ATACGCTATTGGAGCTGGAATTACC

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Amplicon size (bp) 243

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Squirrel

Target gene Cytb

60.1 °C 61.6 °C

123

60.1 °C 60.3 °C

108

SC

Species

141

Reference Newly developed Newly developed Newly developed Fajardo et al. 2008

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Table 4 Concentration of various reagents in simplex, duplex and tetraplex PCR. dNTP(mM) 0.2 0.25 0.25

MgCl2(mM) 2.5 3.5 4.0

Taq pol(Unit) 0.9 1.0 1.25

Primer(µM) 0.2-0.4 0.12-0.4 0.12-0.7

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PCR Simplex Duplex Tetraplex

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Table 5 Cycling parameters of the PCR reaction. PCR

Initial denaturation

Denaturation

Simplex

94 0C for 3 minutes

95

Duplex

0

C

for

3

95

0

C

30

58 0C for 30 seconds

720 C for 45 seconds

72 0C for 5 minutes

for

45

58 0C for 60 seconds

720 C for 45seconds

72 0C for 5 minutes

for

45

58

720 C for 60 seconds

72 0C for 5 minutes

seconds

0

95 C for 3 minutes

95

0

C

Final extension

for

seconds

minutes

seconds

0

C for 60

seconds

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Multiplex

C

Extension

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94

0

Annealing

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Table 6 Restriction digests of the PCR product.

Squirrel

243 bp

Restriction enzyme BtsCI

Rabbit Rat

123 bp 108 bp

BtsCI BtsIMutI

Restriction site

Fragment size

GGATG NN GGATG NN

176 bp, 67 bp 115 bp, 8 bp 64 bp, 44 bp

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Amplicon size

CAGTG NN

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Target species

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Squirrel-spiked CFF

Pure BFF Rabbit-spiked BFF

Rat-spiked BFF

Squirrel-spiked BFF

.

0 1 0.5 0.1 1 0.5 0.1 1 0.5 0.1 0 1 0.5 0.1 1 0.5 0.1 1 0.5 0.1 -

9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 -

0/9 9/9 9/9 9/9 0/9 0/9 0/9 0/9 0/9 0/9 0/9 9/9 9/9 9/9 0/9 0/9 0/9 0/9 0/9 0/9 -

0/9 0/9 0/9 0/9 9/9 9/9 9/9 0/9 0/9 0/9 0/9 0/9 0/9 0/9 9/9 9/9 9/9 0/9 0/9 0/9 -

-

9 9 9 9 -

0/9 0/9 0/9 0/9 -

0/9 0/9 0/9 0/9 -

9 9 9 9

0/9 0/9 0/9 0/9

0/9 0/9 0/9 0/9

-

Squirrel DNA detection

IAC

Detection accuracy (%)

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Rat DNA detection

AC C

Commercial CFF Ramly Tesco Ayamas Prima Commercial BFF Ramly Figo Foods SAUDI Gold Farm’s best

Rabbit DNA detection

0/9 0/9 0/9 0/9 0/9 0/9 0/9 9/9 9/9 9/9 0/9 0/9 0/9 0/9 0/9 0/9 0/9 9/9 9/9 9/9 -

9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9

SC

Rat-spiked CFF

Number of samples

M AN U

Meat products Pure CFF Rabbit-spiked ACFF

Contamination label (%)

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Table 7 Analysis of admixed of commercial meat products with rat, rabbit and squirrel meat specific PCR assay.

-

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

-

0/9 0/9 0/9 0/9

9/9 9/9 9/9 9/9 -

NA NA NA NA -

0/9 0/9 0/9 0/9

9/9 9/9 9/9 9/9

NA NA NA NA

-

Notes: NA, Not Applicable; CFF, Chicken Frankfurter; BFF, Beef Frankfurter; IAC, Internal Amplification Control.

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(a)

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(d)

(f)

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(e)

Fig. 1. Specificity analysis of the developed tetraplex PCR assay. In gel images (a) and (b), Lane M, Ladder; and Lane N, negative template control; in (a) Lane 1, tetraplex PCR products of squirrel, rabbit and rat; Lane 2, squirrel (243 bp); ; Lane 3, rabbit (123 bp); Lane 4, rat (108 bp). In the gel images: Lanes 5-14 in (a) and Lanes 2-13 in (b) are endogenous control (141 bp) of the PCR products from chicken, beef ,buffalo, sheep, goat, pork, duck, pigeon, crocodile, donkey, turtle, chinese edible frog, deer, dog, cat, tuna, salmon and four different types of commonly used plant species such as wheat, cucumber, onion and chili, respectively. Eletropherograms of lanes 1-4 of gel image (a) is shown in (c-f), respectively.

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Lane 8

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Lane 4

Fig. 2. Gel image and the electropherograms of tetraplex PCR for the detection of squirrel, rat and rabbit in deliberately adulterated model beef and chicken frankfurters under raw and processed states. In the gel image, M, Ladder; Lane 1−3, tetraplex PCR of beef frankfurter and Lanes 5−7 tetraplex PCR of chicken frankfurter spiked with 1%, 0.5%, and 0.1% meat from each of squirrel, rabbit and rat species under raw state. Lanes 4 and 8 tetraplex PCR of heat-treated (autoclaved at 121°C und 15 psi for 2.5 h) 0.1% squirrel, rabbit and rat meat adulterated beef, and chicken frankfurters, respectively; Lane N, negative control. The corresponding electroferograms of Lane 4 and 8 are as shown.

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Lane 2

Lane 4

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Lane 6

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Lane 8

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Fig. 3. RFLP analysis of duplex (lanes 1, 3, 5) and tetraplex PCR (lane 7, 8) products before (Lanes 1, 3, 5, and 7) and after (Lanes 2, 4, 6 and 8) restriction digestion. In the gel images: lane M, Ladder; lanes 1-8: endogenous control (141 bp) ; lanes N, negative template control; lanes 1 and 2, squirrel; lanes 3 and 4, rat; lanes 5 and 6, rabbit; lanes 7 and 8, tetraplex PCR of squirrel, rabbit and , rat. The corresponding electropherograms are shown with labels.

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Fig. 4. PCR-RFLP analysis of tetraplex PCR products from deliberately adulterated raw (lanes 1, 2, 7, 8), boiled (lanes 3, 4, 9, 10) and autoclaved (lanes 5, 6, 11, 12) beef (Lanes 1−6) and chicken (Lanes 7−12) frankfurters. In gel image, Lanes 1 and 2, squirrel, rabbit and rat meat adulterated raw beef frankfurter before and after digestion, respectively; Lanes 3 and 4, squirrel, rat and rabbit meat-adulterated boiled (98 °C for 90 min) beef frankfurter before and after digestion, respectively; Lanes 5 and 6, squirrel, rat and rabbit meat-adulterated autoclaved (121° C and 15 psi pressure for 2.5 h) beef frankfurter before and after digestion, respectively; Lanes 7 and 8, squirrel, rat and rabbit adulterated raw chicken frankfurter before and after digestion, respectively; Lanes 9 and 10, squirrel, rat and rabbit meat -adulterated boiled (98 °C for 90 min) chicken frankfurter before and after digestion, respectively; Lanes 11 and 12, squirrel, rat and rabbit meat adulterated autoclaved (121°C and 15 psi pressure for 2.5 h) chicken frankfurter before and after digestion, respectively. In the gel images: Lanes 1-12: endogenous control (141 bp); Lane N, negative template control.

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Highlights

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First tetraplex PCR-RFLP to discriminate rabbit, rat and squirrel meat in food chain. Cross-specificity tested against 22 species practically and 45 species theoretically Lower limit of detection tested until 0.1% meat under mixed matrices Stability tested under extreme boiling and autoclaving treatments Validated and cross-checked in commercial beef and chicken frankfurter matrices

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