Diversity is one of many very desirable characteristics of processed muscle foods. Processed meats consist of sausages, cured and smoked, non-comminuted meats (ham and bacon), restructured products, and canned products. Processed meats can be successfully manufactured from beef, pork, lamb, venison, chicken, turkey, and seafood [1].

The major authenticity concerns for Muslim consumer in meat and meat products include pork substitution, undeclared blood plasma, using prohibited ingredients, pork intestine casings and non-halal method of slaughter [2]. But in most countries, food manufactures choose to use porcine derivatives because they are cheap and readily available [3].

Pork and its derivative are Haram (unlawful or prohibited) to be consumed by muslims. Pork is a typical meat in Malaysian market while, wild boar (Sus scrofa) or babi hutan is found in the Malaysian rain forest [4]. According to Nakyinsige [2] porcine derivatives used in the meat processing industry include; pork fat (lard), mechanically recovered meats (MRM), porcine gelatine and porcine blood plasma.

Problems related to adulteration of meat species in ground and comminute products have been a widespread problem in some retail markets, while meat species identification is a major global concern [5]. In order to protect Muslim consumers from fraud and adulteration several analytical approaches have been made to identify animal species in food products [6].

PCR identification of species using mitochondrial DNA (mt-DNA) gives a series of advantages due to its present in thousands of copies per cell [7]. DNA molecule being the targeted compound for species identification compared to protein due to their high stability and present in most biological tissues.

Protecting Muslim consumer from adulterated food product with non Halal ingredient is a must. The aim of this study was to detect the porcine DNA and species identification using mitochondrial DNA for Halal authentication.

Sample for analysis

A total of 31 (n=31) samples randomly selected from supermarket in Selangor and Kuala Lumpur area which are finished meat product; and two of them made from pork meat; pork sausages and breakfast meatball.

DNA extraction

The DNA extraction for each samples were extracted using Qiagen Blood and Tissue kit. Sample weighing about 100-150 mg of ground finished product were placed into 2.0 ml sterile micro centrifuges tubes. 180 μl ATL buffer and 20 μl Proteinase K was added into the micro centrifuges tubes with sample and vortex. The tube was incubated overnight (16-18 hours) at 56°C for cell lyses. After the incubation process complete, the micro centrifuges tube was vortex for 15 seconds to mix thoroughly. Then 200 μl AL buffer was added and vortex again. After that, the micro centrifuges tube was incubated at 70°C for 10 minutes. After 10 minutes, the sample in tube was vortex and undergo centrifuge at 8000 rpm for 1 minute. 200 μl ethanol (100%) was added and mix thoroughly by vortex. All the mixed was transferred into spin column and centrifuged at 8000rpm for 1 minute. Flow through was discarded. 500 μl AW1 was added to spin column and centrifuges for 1 minute at 8000 rpm. Collection tube was changed and flow through was discarded. Then, 500 μl AW2 was added and centrifuges at maximum speed (13000 rpm) for 5 minutes. The flow through was discarded and centrifuged again at maximum speed for just 1 minute to dry the membrane. After that, the spin column was inserted into micro centrifuges tube and ready for elution step. 100 μl of AE buffer was added on top of membrane and incubated at room temperature for 1 minute. Then, centrifuge at 8000 rpm for 1 minute. The DNA was collected in the micro centrifuges tube. The DNA was stored at -20°C prior to use as DNA template in PCR amplification.

PCR amplification

83-bp primers: PCR amplification was performed in 20 μl reaction volume containing 10 μl ready to use Atlas Taq 2x PCR mix, 1 μl of each forward and reverse primers, 4 μl of 50 ng/μl DNA template and H₂O PCR grade up to 20 μl. The reaction was performed in Eppendorf thermal cycler with the following cycling condition: after an initial heat activation at 95°C for 9 min, 45 cycles were programmed as follows: 92°C for 30 s, 55°C 30 s, 72°C 30 s and final extension at 72°C for 5 minutes. The PCR products were separated by electrophoresis through 2.5% agarose gel with pre-stain red safe.

531-bp primer: Mitochondrial (mt) DNA D-loop region fragments namely Pork1 and Pork2 were amplified by PCR. Amplification of DNA were carried out in a final volume of 50 μL in tubes containing 100ng/μL DNA template, 2X PCR Master Mix (BioAtlas), 1 μl of each 100mM primers and nuclease free water (NFW) mark up to volume. Initial denaturation perform for 3 min at 92°C; followed by 30 cycles of amplification at 92°C for 20 s (denaturation), 58°C for 20 s (hybridization) and 72°C for 30 s (elongation) were carried out using thermal cycler (Eppendorf).

The production and consumption of poultry products have been on the increase globally (Lasekan et al. 2013) [8]. Recently, issues related to adulteration of food products arose in Malaysia. So identifying the species of meat in the finished meat products is the main target to fulfil Halal requirement and Islamic laws.

Mitochondrial DNA sequence is highly conserved in different species of animals [9]. These present studies describe a conventional PCR method for detection of porcine materials in meat product using different sizes of amplicon of mitochondrion (mt) DNA. This method previously described by Montiel-sosa [7], was used for identification of species-specific in pork meat and fat in meat which targeted D- loop region of mt-DNA. Yoshida founded mtATP6 primers with amplicon length 83-bp able to detect the porcine DNA in feedstuff [10].

Result demonstrated on agarose gel shows that the amplification of PCR was done successfully when the band of desired amplicon size was appeared on the gel image. Negative control and positive control always present in every batch of amplification process to determine the ability of the primer used.

Analysis was started with the samples with positive porcine DNA result which are pork sausages and breakfast meatball (pork meat). The result was used to compare with other finished meat product that bought from the markets. Based on the Figure 1 and Figure 2 all pork samples shows the band with 83-bp and 531-bp complimentary with positive control which is the DNA obtain from fresh pork meat.

Figure 1: Gel electrophoresis of porcine DNA detection isolated from pork products using primers mtATP6. Lane M; Marker (100 bp ladder); Lane 1: Negative control; Lane 2-5: Sample positive with porcine DNA (83 bp) and; Lane 6: Positive Control.

Figure 2: Gel electrophoresis of porcine DNA detection isolated from pork products using primers mt-DNA D-loop with 531 bp band size. Lane M; Marker (100 bp ladder); Lane 1: Negative control; Lane 2-5: Sample positive with porcine DNA (531 bp) and; Lane 6: Positive Control.

Based on the figure above, both mt-DNA primers are appears to be highly species-specific that able to detect the presence of porcine DNA in processed food. Studies by Yoshida [10] shown that mtATP6 clearly detected damage or fragmented porcine DNA in feed effectively. Thus, the amplification process was expended to various types of meat products. Table 1 below shows the results for all samples tested.

Table 1: Shows the results of Porcine DNA in food products using mt DNA primers.

Based on Table 1 all processed food, meat based and seafood product are free from contamination, addition or substitution with porcine derivatives except for pork products. The mt-DNA primer gives a series of advantages due to its present in thousands of copies per cell.

In this study, different sizes of primers length, 83-bp and 531-bp was used but results obtain shows both primers are highly specific for pork mt-DNA. Food products which contain fish, seafood, beef and chicken based does not amplified at 83-bp and 531-bp due to highly species-specific primes.

In conclusion, processed food products with damage or fragmented DNA also can be used for DNA detection using specific primers. Both mitochondria oligonucleotide primers from Monteil and Yoshida et al. [7,10] are designed for detection of pork and their derivatives. This method is reliable for porcine detection in meat product and can be used to detect fraud and to ensure that foods comply with religious regulations.