MOLECULAR DETECTION OF PORK CONTAMINATION IN BEEF SOLD IN TRADITIONAL MARKETS, YOGYAKARTA, INDONESIA

Beef is one of the animal protein sources needed by the body to meet nutritional requirements. Consequently, fresh beef (whole or milled) is one of the highest-demanded comestibles on the market. Some meat kiosks in Yogyakarta traditional markets are also in service of milling whole meat. However, reports are stating that some kiosks are neglecting aspects concerning halal. One of those aspects is separating tools used to process beef and pork. Reports said that kiosk owners fail to meet that essential requirement causing Muslims to be at risk of consuming contaminated beef without their knowledge. Hence, this study aims to examine whether fresh and ground beef obtained from Pasar Pathuk and Pasar Kranggan, Kota Yogyakarta, is free from pork contamination. The examination was carried out using Polymerase Chain Reaction (PCR) on 14 samples of seven fresh and seven ground beef obtained from both markets. DNA isolation from all samples was done using FavorGen® FavorPrepTM Tissue Genomic DNA Extraction Mini Kit. Isolated DNA was further examined by PCR analysis using P14 and MTCB primers. Results showed that P14 primers could amplify the PRE-1 gene (481 bp) designed as a pork molecular marker only on positive control (fresh pork); meanwhile, MTCB primers could amplify the cytochrome b gene (1141 bp) designed as a mammal molecular marker on all samples involved in this research. Based on the results, we concluded that both fresh and ground beef sold in Pasar Pathuk and Pasar Kranggan, Yogyakarta are not contaminated by pork DNA.


INTRODUCTION
The vast majority of the Indonesian population is Muslim.As a Muslimmajority country, the Indonesian government must ensure that food circulating in the community is safe, of good quality, nutritious and halal-certified (Triasih et al., 2016).One of the halal certification criteria requires that food be free from any pork elements as stated in the Quran  Samples taken from traditional markets were transferred to the laboratory using a cooler box.From each sample, as much as 30 mg were milled and transferred into a 1.5 ml microtube.200 µl FATG1 buffer, proteinase K was added into the microtube.Samples were then vortexed.Samples were incubated at 60C for 1-3 hours.After the first incubation, 200 µl of FATG2 buffer was added and samples were incubated for the second time for 10 minutes.After the second incubation, 200 µl of 96% ethanol was added and samples were vortexed.FATG mini columns were transferred into a collection tube and centrifuged for 1 minute (18,000 rpm).The collection tube was discarded and replaced by a new collection tube.400 µl of W1 buffer was added to the column.Samples were then centrifuged full-speed for another 1 minute.The collection tube was discarded and replaced by a new collection tube.750 µl wash buffer was added into the column and was centrifuged full-speed for 1 minute.The collection tube was discarded and replaced by a new 1,5 ml microtube.100 µl elution buffer was added to the column and the column was incubated at room temperature for 3 minutes.Samples were then centrifuged for 2 minutes.Pellet formed after centrifugation was stored at 4C.

Qualitative and Quantitative Analysis of DNA
Qualitative analysis carried out using 1.5% agarose gel immersed in 0.5x TAE buffer solution.3 µl of DNA sample, 2 µl loading dye, and 5 µl GelRed were used for every well in agarose gel.The electrophoresis tank was set to 100 volts and 15 minutes.Observation of agarose gel was carried out on a UV transilluminator to detect DNA bands.Quantitative analysis was carried out using Nanodrop.

PCR Amplification
PCR was used to amplify the target gene using two pairs of primers as shown in Table 1.P14 primers design was adapted from Fibriana et al. (2012) and the MTCB design was adapted from Naidu et al. (2012).As for PCR condition is shown in Table 2.As much as 1.2 µl DNA sample, 12.5 µl MasterMix, 1 µl primer forward and 1 µl primer reversed were mixed into each of the PCR tubes.9.3 µl ddH2O was added to the tubes.The tubes were then placed into a PCR machine.PCR products were then stored at -20C for further use.

RESULTS AND DISCUSSION
This study aims to identify pork DNA in beef (whole and milled) obtained from Pasar Pathuk and Pasar Kranggan using polymerase chain reaction (PCR).This method has a high sensitivity to detect the smallest amount of DNA present in the samples of interest.In this study, two pairs of primers were used; MTCB (mitochondrial cytochrome b) and P14.
DNA isolation was done using the FavorGen commercial kit FavorGen® FavorPrep TM Tissue Genomic DNA Extraction Mini Kit.DNA isolation consists of 3 main principles: cell destruction (lysis), separation of DNA from solid materials such as proteins and cellulose (extraction) and DNA purification (Nurhayati and Darmawati, 2017).Isolated DNA on fresh pork, fresh beef, whole and ground beef were visualized using gel electrophoresis as a qualitative measure.The results of the visualization are shown in Figure 2.  3. The measurement of DNA concentration and purity is necessary to know the degree of contamination of the isolated DNA.DNA of good quality would have an A260/A280 ratio in the range of 1.8 to 2.0.A260/A280 ratio below 1.8 indicates that DNA contains protein contaminants.Failure to break down (lysis) cell components could be the cause of protein contamination (Pratama, 2015).A260/A280 ratio above 2.0 indicates that DNA contains remnants of the RNA.
Measurement of DNA concentration is also necessary for the next step of the analysis, which is PCR.That is because there is a certain amount of sample concentration that needs to be met to obtain high-quality amplicons (Mustafa et al., 2016 andFatchiyah et al., 2011).The average A260/A280 ratio of 1.807 shown in Table 3 indicates that the purity of DNA isolated in this research was of good quality.Considering the good result of DNA concentration, samples were further analyzed by polymerase chain reaction (PCR).Figure 3 shows PCR products using MTCB primers.Based on the results, all samples, control meat included, were all successfully amplified on ~1140bp.
Figure 3. PCR amplification results using MTCB primers electrified in 1.2% agarose gel 100 volts; M: marker 100 bp; K(+): positive control (fresh beef); K(-): negative control (ddH2O); S1 -S7: fresh meat samples; S8 -S14: ground beef samples Based on the results shown in Figure 3, it is shown that the positive control (fresh beef) was amplified and the negative control (ddH2O) was not amplified.This result indicates that the PCR machine worked as expected eliminating any technical error possibility.Most samples of ground beef (S1-S7) and fresh beef (S8-S14) were successfully amplified.Bands were shown to be sharp and bright indicating high specificity of primers to the target gene.However, below the bands, there were still smears.The appearance of smears can be caused by the quantity of Mg++, dNTP, Taq Polymerase, primer, and excess DNA templates.Another possibility is the inclusion of contaminants on the DNA template which inhibit the activity of Taq polymerase (Fatchiyah et al., 2011).
All amplified samples have a length of ~1140 bp in correspondence to Naidu et.al. (2012) who explained that the target gene, cytochrome b, has a sequence between 1140-1200 bp.It is then proven that 13 samples obtained from Pasar Pathuk and Pasar Kranggan contained meat derived use that according to the provision must be created."Samples were then further analyzed using PCR and P14 primers which target PRE-1 as specific molecular marker for pork.As shown in Figure 3, only positive control (pork meat) was amplified at ~481 bp while all 14 samples were not amplified using P14 primers.This result indicates that P14 primers have high specificity on detecting pork meat.However, it is shown on Figure 3 that the bands were not of good quality even on the positive control.The poor quality of DNA bands can be caused by duration and voltage implemented on electrophoresis (Mustollah, 2016) andTilawah, 2019).

CONCLUSIONS
Based on the results, it is concluded that fresh and ground beef obtained from milling services in Pasar Pathuk and Pasar Kranggan Yogyakarta is not contaminated by pork DNA.Further research is needed especially on the introduction of new samples from various locations.Moreover, investigation of numerous skin care products to uphold their halal status is also required.