AUTHENTICATION OF RAW CHICKEN MEAT FROM PORK CONTAMINATION USING GENE CYT-B WITH DUPLEX-POLYMERASE CHAIN

Halal is one of important aspects in consumer protection. Meat and processed meat products are food that should be controlled strictly because those are prone to be adulterated by pork contamination. Therefore, it is necessary to provide detection technique which is accurate, fast and cheap. The objective of this research was to identify the presence of impurities of pork meat on raw chicken meat using gene Cyt-b with duplex-PCR analysis. This research used six samples of raw chicken meat and raw pork. Raw chicken meat was bought from supermarkets in the city of Surakarta and raw pork was obtained from pig slaughterhouse. The percentage of raw pork contamination on raw chicken meat was designed as much as 1, 5, 10, and 25%, respectively. The DNA genome was isolated according to DNA isolation protocol from Genomic DNA Mini Kit. In addition, duplex-PCR was performed based on protocol of KAPA2G Fast Multiplex PCR kit. The data was descriptively analyzed by directly looking the DNA bands on the gel documentation apparatus. The result showed that specific DNA bands for chicken and pig were completely appeared on 1.5% of agarose gels. Duplex-PCR detect contamination of pork on raw meat of chicken at all contamination levels. This research proved that the duplex-PCR detect the contamination of pork until the level of 1%. (


Introduction
The authentication of meat is a sensitive and important issue concerning food products, especially in Moslem countries (Erwanto et al., 2014). It is closely related to food safety, i.e. the issues of pork contamination and its derivatives on food product (Murugaiah et al., 2009;Koppel et al., 2011).
Pork contamination causes controversial reactions in society because pork is clearly prohibited for the Islamic community, many people are highly hypersensitive to pork, and there are vegetarian life styles (Rahmawati et al., 2015). In Indonesia, the pork contamination could be occurred during meat processing. Grinding process is a common activity to produce meat products, and it is susceptible to pork contamination in raw meat. Erwanto et al. (2014) investigated consumers in Yogyakarta obtained ground meat from the traditional market which is grinded using the traditional equipment that also may be used for pork. Therefore, an effective method should be developed to specifically and selectively identify the pork on raw meat from other species.
Recently, authenticity testing and analytical techniques have immeasurably improved and now it can draw on a wide variety of techniques and methods, each method is appropriate and specific to deal with a particular problem (Ballin, 2010). Methods based on DNA identification have been applied to meat specification such as polymerase chain reaction (Aida et al., 2005;Sahilah et al., 2011), Duplex-PCR (Soares et al., 2010), Multiplex-PCR (Koppel et al., 2011), PCR-RFLP (Erwanto et al, 2014) and real time PCR (Dooley et al., 2004;Kesmen et al., 2009;Ali et al., 2012) were methods to detect the contamination in pork which have been developed.
Methods used to analyze contaminants in pork processed food products namely Polymerase chain reaction (PCR) is one of molecular biology approaches used to authenticate and to detect species origin in meat by amplifying spesies specific sequence in the genome such as fragment of mt-DNA Cyt-b gene (Girish et al., 2005). Contaminants analyze which is used was mitochondrial DNA (mt-DNA) is done based on the existence of a sequence-specific DNA fragment of species on meat (Aida et al., 2005;Ling-Sun, 2003).
Cytochrome b (Cyt b) is one of gen in mitochondria DNA and it was often used to identify species from raw material in marine product (Sotelo et al., 2001). The PCR analysis of species-specific mitochondrial DNA sequences is the most common method currently used for identification of meat species in food and seems to be the best as a 'routine' test, because it is easy, rapid and allows the discrimination of several species at the same time (Bottero and Dalmasso, 2011), and it only requires a small sample size (Che Man et al., 2007;Nakyinsige et al., 2012). The use of mitochondrial DNA in PCR analysis can also enhance sensitivity because each cell has about a thousand mitochondria and each mitochondria has ten copies of DNA (Jain, 2003). Therefore, the objective of this research was to identify the presence of impurities of pork meat on raw chicken meat using Cyt-b gene by duplex-PCR analysis.

Sample collection
Pork and chicken meat samples were obtained from commercial sources. Both of samples was labeled to ensure that all samples were perfectly separated, and then samples were stored in the refrigerator until used for further analysis (Ni'mah et al., 2016).
The grinding process of chicken meat and pork was done separately. The meat samples were cut into small size 1x1 cm, and then the meats were ground using meat grinders. Moreover, the percentage of raw pork contamination on raw chicken meat was designed as much as 1, 5, 10, and 25%, respectively. The meat samples were weighed and manually mixed until homogeneous for DNA extraction analysis.

DNA extraction
The isolation of DNA was conducted according to the Protocol of the Genomic DNA Mini Kit for animal tissues (Geneaid Biotech Ltd., Taiwan). Firstly, 30 mg of meat tissue was placed into 1.5 ml tube and mashed by micropestle, and then 200 µl of GT buffer was added into the tube and homogenized by using a grinder. Furthermore, 20 µl of proteinase K was added to the tube, homogenized back and incubated at 60°C for 30 minutes. After that, 200 µl of GBT buffer was added into tube, and it was homogenized and incubated at 60ºC for 20 minutes. Bulletin of Animal Science, DOI: 10.21059/buletinpeternak.v41i2.13376 Supernatant was moved into a new 1.5 ml tube and added 200 µl of ethanol absolute and it was homogenized. Furthermore, the sample was moved to GD column which is placed on top of 2 ml collection tube, and it was centrifuged at 14,000-16,000 rpm for 2 minutes and then the 2 ml collection tube was replaced with the new one. In addition, 400 µl of Buffer W1 was added into the sample, and it was back centrifuged at 14,000-16,000 rpm for 1.5 minutes. The rest of the mixture was dumped, and GD column was inserted back into tube 2 ml collection. Furthermore, 600 µl of wash buffer was added to the GD column and centrifuged for 3 minutes at 14,000-16,000 rpm. GD column was put into a new 1.5 ml tube and added 100 µl of elution buffer heated at 60°C, and it was centrifuged at 14,000-16,000 rpm during 1.5 minutes. Finally, the DNA was ready to use for subsequent analysis. The DNA concentration was determined by observing the intensity of DNA bands on 1% of agarose gels.

Simplex-and Duplex-PCR
The PCR was performed with a total volume of 25 µl consisting of 10.5 µl aquabidest; 0.5 µl of each primer (10 M); 12.5 µl 2X Fast KAPA2G Multiplex Mix (Kapa Biosystems, Inc., United States); and 1 µl of DNA template. The PCR was carried out to amplify fragments of DNA mitochondrial Cyt-b gene, and the primer used in this study was previously published by Matsunaga et al. (1999) which is presented in Table 1. PCR program was performed using the GeneAmp® PCR System 9700 (Singapore) as follows: initial temperature of denaturation at 95 o C for 3 minutes, followed by 30 cycles of denaturation at 95 o C for 15 seconds, annealing temperature at 60 o C for 30 seconds and extension at 72 o C for 30 seconds and also final extension at 72 o C for 1 minute. The results of duplex-PCR were visualized in 1.5% agarose gel using gel documentation (Vilber Lourmat Infinity 1100126M, France).

DNA extraction
As many as 6 raw meat samples were designed and used for the isolation of DNA in this study. The results showed that the DNA of the genome was successfully extracted from raw meat in all levels contamination of pork on raw chicken meat (Figure 1).
The DNA genome extracted in this study was low concentration indicated by smear DNA bands on the 1% agarose gel (Figure 1). The low DNA concentration yielded in this study may due to the meat samples have been cooked before isolation process. Previous study reported that genomic DNA can still be extracted from cooked meat such as sausage, cutlet and leftover meals (Matsunaga et al., 1999;Nakyinsige et al., 2012;Zhang, 2013). Basic principle of DNA extraction is by breaking up the cells from tissues until the DNA genome was completely separated from other cell components like protein, DNA and RNA (Faatih, 2009).

Simplex-and Duplex-PCR
This research conducted both simplexand duplex-PCR methods. Simplex-PCR was applied to optimize PCR conditions while duplex-PCR was performed to detect contamination of pork in chicken meat raw. The existence of pork contamination in the meat mixtures was indicated by a 398 bp of DNA band. On the other hand, a species of chicken was shown by the existence of 227 bp of DNA band.    This research was conducted to detect contamination of pork in chicken raw meat up to the level of 1%. Previous study reported that both raw and cooked meats could be detected by using multiplex-PCR from mt-DNA Cyt-b gene up to level of 8% (Matsunaga et al., 1999;Ni'mah et al., 2016). Multiplex-PCR was also used successfully as one way to authenticate a good product of meat raw or processed products, and it could be used by a company as the reference of quality control to produce food (Nakyinsige et al., 2012;Zhang, 2013).

Conclusion
Pork contamination in raw chicken meat could be detected by Duplex-PCR of mt-DNA Cyt b gene. The sensitivity of authentication method was able to detect pork contamination in raw chicken meat until 1%.