Detection of Porcine Meat in Meat Products by using
Polymerase Chain Reaction Technique
(PELACAKAN DAGING BABI PADA PRODUK DAGING DENGAN TEKNIK POLYMERASE CHAIN REACTION)

RETNO D. SOEJOEDONO
Laboratorium Imunologi,
Departemen Penyakit Hewan dan Kesehatan Masyarakat Veteriner
Fakultas Kedokteran Hewan, Institut Pertanian Bogor

Abstract

Antibody based techniques in analyzing food components and contaminants have also utilized recently in some developed countries. However, antibody based tecniques often suffer from false positive reactions due to cross-reactivity with closely related animal species and false negative reaction due to the degradation of the protein. By those above reasons, using Polymerase Chain Reaction technique (PCR) with Restriction Fragment Length Polymorphism (RFLP), which are quick and accurate, can distinguish between closely related species.
Key words : porcine meat, PCR, and RPFL

ABSTRAK
Di beberapa negara yang sedang berkembang, teknik berbasis antibodi telah dipakai belakangan ini untuk menganalisis komponen dan cemaran makanan. Namun, uji ini sering memunculkan hasil positif semu yang diakibatkan oleh adanya reaksi silang antara hewan yang berkerabat dekat. Selain itu reaksi negatif semu juga sering muncul akibat degradasi protein. Uji polymerase chain rection (PCR) dengan teknik Restriction Fragment Length Polymorphism (RFLP) yang cepat dan akurat dapat membedakan komponen daging dari hewan yang berkerabat dekat.

Kata kunci : Daging babi, PCR, dan RFLP

INTRODUCTION
Many highly specific and sensitive test methods that have been used in serological diagnostic are based on the techniques in analyzing foods components and contaminants have also utilized recently in some moslems countries. However, antibody based tecniques often suffer from false positive reactions due to cross-reactivity with closely related animal species.
For the quality of the product, including of the authenticity, is an important concern for modern consumers and different levels of the food production chain. For example sealing cheaper meat, religious and health reason.. PCR differs the posibillity to detect animal food ingredients and to identify them are being derived from certain animal species. Specific PCR, amplyfing a fragment of the mitochondrially encoded cytochrome-b genes are studied for this purpose. Realizing that majority of Indonesian population are moslems, that used of pork or component in food or food processing is very sensitive issues. Also important for religious or moral reasons to identify certain meat species like pork . Otherwise, people allergic to meat species ( chicken, fish) need ways to identify this species.

MATERIALS AND METHODS
1. Different Indonesia samples were obtained from local shop
The Indonesia sample material investigated are : Meat ball ( frozen meat, bakso sapi); Chicken ball ( frozen meat, bakso ayam); Abon (is cooked meat spices) and Dendeng ( dendeng sapi is fresh dried meat and spices)

2. The Internal Reference Materials (IRM)
The Internal Reference Materials (IRM) were prepared the Institut of Veterinary Food Science, Giessen, Germany; with the concentration of 0,01 %, 0,1%, 1%, 2%, 5% and 10% ( of pork in beef mixtures )
Preparation of the Internal Reference Materials (IRM)
Twenty five g of pork , 200 were g mince beef meat and 25 ml aquabidest steril were added to as a base of the IRM, mixed by using Blender (Moulinette, Moulinex) for 3 – 4 times. Then it was transferred to plastic bag
(double plastic bag) applied in Stomacher 400 ( Laboratory Blender) for 120 sec. at the normal speed. This mixture was used as a base for preparing: 0,01%, 0,1%, 1%, 2%, 5% and 10% of the pork as IRM (Table 1).

Table 1 :The concentration of pork in beef mixtures : 0,01%, 0,1%, 1%, 2%, 5% and 10%
A base mix(g) Distillated water(ml) Pork meat Beef meat %
200 - 25 - 10 (a)
25(a) 5 - 20 5
20(a) 10 - 70 2
10(a) 10 - 80 1(b)
10(b) 10 - 80 0,1(c)
10(c) 10 - 80 0,01

3. The DNA isolation
DNA isolation by using DNeasy tissue kit for the IRM and the Indonesia samples ( Qiagen kit )
Thirty mg of the IRM ( 0,01%, 0,1%, 1%, 2%, 5% and 10%) and abon, meat ball, chicken ball and dendeng used and placed in 2 ml microcentrifuge tube, 180 µl buffer ATL was added. Twenty µl Proteinase K was added, mixed by vortex and placed in the shaking waterbath at 55 °C for 1 – 3 hours. During the incubation period, the samples were vortexed Added 200 µl buffer AL, mixed by vortex and incubated in the shaking waterbath at 70 °C, for 10 min. Added 200 µl ethanol 96%, mixed. Placed the mixture to DNeasy mini column sitting in a 2 ml collection tube, centrifuged at 8000 rpm for 1 min. Discarded flow through and the collection tube. Placed the DNeasy mini column in a new 2 ml collection tube. Added 500 µl buffer AW1, centrifuged for 1 min, at 8000 rpm. Discarded flow through and collection tube. Placed the DNeasy mini column in a new collection tube, added 500 µl buffer AW2, centrifuged for 3 min, at full speed to dry the DNeasy membrane. Placed the DNeasy mini column in a clean 2 ml microcentrifuge tube and added 200 µl AE buffer directly onto the DNeasy membrane. Incubated at room temperature for 1 min. and then centrifugated for 1 min at 8000 rpm to elute.

4. Universal Oligonucleotide Primer.
The samples as well as the IRM and Indonesia samples were investigated by use
universal oligonucleotide primer sequence (Kocher at al. , 1989).
The oligonucleotide primer 4 ctyB-1 –5´CCATCCAACATCTCAGCATGATGAAA 3´ and primer 4 cty-B-II –5´CCCCTCAGAATGATATTTGTCCTCA 3´ were used to amplify the DNA from IRM and Indonesia samples.
The reaction mixture ( 50 µl ) contained 1 µl Primer 1 ( 10 pmol/µl ); 1 µl Primer 2 ( 10 pmol/µl ), 1 µl dNTP (10 mmol, Roche Diagnostic, Mannheim, Germany, 5 µl 10x thermophilic-buffer (PE Applied Biosystem, Weiterstadt,Germany) , 0,2 µl Taq DNA Polymerase ( 5 U/ µl , PE Applied Biosystem, Weiterstadt,Germany) and 39,3 µl double-distilled water . Finally, 2,5 µl DNA preparation (10%, 5%, 2%, 1%, 0,1% and 0,01% ) were added to each tube. And were utilized beef control, pork control , non template control and marker XIV
The PCR was applied in a thermal cycler ( Hybaid) ) with the following program : 1 x 3 min precycle at 93 °C , 35 x for ( 30 sec at 94 °C, 30 sec at 52 °C and 30 sec at 72 °C followed by a final extension incubation at 72 °C for 5 min. Five µl of the PCR product was added loading buffer (10 ml 0f 15 % Ficoll solution and 0,25 ml of 5% Bromophenolblue).
The presence of PCR product was electrophoresed in 1,5% agarose gel (Sigma) , at 100 volt, for 1 hour and stained with ethidium bromide ( 50 µl /1,5 l aquadest ) for 30 min, washed in aquadest , 10 min and visualised under UV light.

5. Restriction Fragment Length Polymorphism (RFLP)
5.1. The PCR product of the IRM and Indonesia samples were digested with three
enzymes Hinf I, Hae III and Rsa I
The PCR were digested using the following protocol :
21,5 µl of the PCR product mixed 2,5 µl of the 10 x restriction enzyme buffer and added 1 µl ( 10 units) of the restriction enzyme. Final volume was 25 µl and incubated at 37°C for overnight. The marker is used marker XIII. 10 µl the DNA fragments were separated by electrophoresis in 3 % agarose gel ( Metaphor gel, Bio Whittaker Molecular Application, Rockland,USA) for 2,5 hours at 80 volt .

5.2. The meat ball was digested with two enzymes Alu I, Taq I
The PCR were digested using the following protocol :
21,5 µl of the PCR product mixed 2,5 µl of the 10 x restriction enzyme buffer and added 1 µl ( 10 units) of the restriction enzyme . Final volume was 25 µl and incubated at 37°C for overnight. For Taq I enzyme was incubated at 65°C for 4 hours. The marker is used marker XIII. 10 µl the DNA fragment were separated by electrophoresis in 3 % agarose gel (Metaphor gel, Bio Whittaker Molecular Application, Rockland,USA) for 2,5 hours at 80 volt

RESULTS AND DISCUSSION.

Table 2 : Predicted fragments sizes of the partial cytochrome b gene of different food animal
species after PCR-RFLP analysis.

Species Size of DNA fragment (bp) after digestion with indicated restriction enzymes Sequence references
Hae III Hinf I Rsa I Alu I Taq I
Ostrich 227132 233126 205149 Abdulmawjood and Bülte 2000
Chicken 15912674 18816110 210149 359 359 Desjardins and Morais 1990
Turkey 1261037455 196161 149109101 359 359 Meyer et al 1995
Pig 15313274 359 359 244115 218141 Meyer et al 1995
Cattle 28574 19811744 359 190169 359 Meyer et al 1995
Sheep 15912674 198181 359 359 359 Meyer et al 1995
Goat 2307455 198161 359 359 218141 Meyer et al 1995
Buffalo 28574 359 190169 191168 Meyer et al 1995
* Abdulmawjood and Bülte, 2000 ; Meyer et al 1995

This study :
Samples Size of DNA fragment (bp) after digestion with indicated restriction enzymes Sequence species
Hae III Hinf I RSA I Alu I Taq I
Meat ball 28574 359 190169 191168 Buffalo
Chicken ball 15912674 18816110 210149 359 359 Chicken
Dendeng 28574 19811744 359 190169 359 Cattle

Fig. 1. PCR amplicons of part of cytochrome b gene by using universal oligonucleotide
primers of IRM : 1. marker XIV, 2. 0,01%, 3. 0,1%, 4. 1%, 5. 2%, 6. 5%, 7. 10%
8. bovine, 9. pork, 10. non template control, 11. marker XIV.

Fig. 2. PCR amplicons of part of cytochrome b gene by using universal oligonucleotide
primers of Investigated samples : 1. marker XIV, 2. abon 3. meat ball, 4. chicken ball.
5. dendeng, 6. chicken, 7. turkey, 8. sheep, 9. pork, 10. bovine, 11. goat, 12. non tem-plate control, 13 marker XIV

Fig. 3. PCR amplicons of part of cytochrome b gene by using universal oligonucleotide
primers of Investigated samples : 1. marker XIV, 2. abon 3. meat ball 4. chicken ball,
5. dendeng, 6. 5% mixed meat , 7. abon, 8. meat ball, 9. chicken ball, 10. dendeng,
11. 5% mixed meat, 12. chicken; 13. turkey , 14. sheep, 15 .pork, 16. bovine, 17. goat,
18. non template control .

Fig. 4 PCR amplicons of part of cytochrome b gene by using universal oligonucleotide
primers of IRM and Investigated samples : 1. marker XIV, 2. 0,01%, 3. 0,1%, 4. 1%,
5. 2%, 6. 5%, 7. 10% , 8. pork, 9. bovine, 10. abon, 11. meat ball , 12. chicken ball,
13. dendeng, , 14. .pork, 15. bovine, 16. sheep ,17. goat,18. chicken; 19. turkey ,
20. marker XIV .

Fig. 5. PCR amplicons of the part of cytochrome b gene by using universal oligonucleotide
primers Investigated samples : 1. marker XIV, 2. meat ball, 3. meat ball, 4. bovine,
5. pork, 6. sheep , 7. goat, 8. chicken, 9. turkey , 10. non template control,
11. marker XIV .

Fig. 6. Restriction profil of the part of cytochrome b gene digested with Hae III
restriction enzyme : 1. marker XIII, 2. 0,01%, 3. 0,1%, 4. 1%, 5. 2%, 6. 5%,
7. 10% , 8. bovine, 9. pork, 10. marker XIV .

Fig. 7. Restriction profil of the part of cytochrome b gene digested with Hinf I
restriction enzyme : 1. marker XIII, 2. 0,01%, 3. 0,1%, 4. 1%, 5. 2%,
6. 5%, 7. 10% , 8. bovine, 9. pork, 10. non template control, 11. marker XIII .

Fig. 8. Restriction profil of the part of cytochrome b gene digested with RSA I
restriction enzyme : 1. marker XIII, 2. meat ball 3. chicken ball 4. dendeng,
5. chicken , 6. turkey, 7. sheep , 8. pork, 9. bovine, 10. goat, 11. marker XIII.

Fig. 9. Restriction profil of the part of cytochrome b gene digested with Hinf I
restriction enzyme : 1. marker XIII, 2. meat ball 3. chicken ball 4. dendeng,
5. chicken , 6. turkey, 7. sheep , 8. pork, 9. bovine, 10. goat, 11. marker XIII.

Fig. 10. Restriction profil of the part of cytochrome b gene digested with Hae III
restriction enzyme : 1. marker XIII, 2. meat ball 3. chicken ball 4. dendeng,
5. chicken , 6. turkey, 7. sheep , 8. pork, 9. bovine, 10. goat, 11. marker XIII.

Fig. 11 Restriction profil of the part of cytochrome b gene digested with RSA I
restriction enzyme : 1. marker XIII, 2. meat ball 3. dendeng, 4. 5% meat
mixed, 5. meat ball, 6. chicken ball, 7. 5% meat mixed. 8. chicken, 9. turkey,
10. sheep,11. pork, 12. bovine, 13. goat, 14. marker XIII.

Fig. 12 Restriction profil of the part of cytochrome b gene digested with Hae III
restriction enzyme : 1. marker XIII, 2. meat ball 3. dendeng, 4. 5% meat
mixed, 5. meat ball, 6. chicken ball, 7. 5% meat mixed, 8. chicken, 9. turkey,
10. sheep, 11. pork, 12. bovine, 13. goat, 14. marker XIII.

Fig. 13. Restriction profil of the part of cytochrome b gene digested with Hinf I
restriction enzyme : 1. marker XIII, 2. meat ball 3. dendeng, 4. 5% meat
mixed, 5. meat ball, 6. chicken ball, 7. 5% meat mixed, 8. chicken, 9. turkey,
10. sheep, 11. pork, 12. bovine, 13. goat, 14. marker XIII.

Fig. 14. Restriction profil of the part of cytochrome b gene digested with RSA I
restriction enzyme : 1. marker XIII, 2. 0,01% , 3. 0,1% 4. 1% 5. 2%, 6. 5% ,
7.10% , 8. pork, 9. bovine, 10 pork, 11. bovine 12.sheep, 13. goat, 14. chicken
15. turkey, 16 .marker XIII.

Fig. 15. Restriction profil of the part of cytochrome b gene digested with Hae III
restriction enzyme : 1. marker XIII, 2. 0,01% , 3. 0,1% 4. 1% 5. 2%, 6. 5% ,
7.10% , 8. pork, 9. bovine, 10 pork, 11. bovine 12.sheep, 13. goat, 14. chicken
15. turkey, 16 .marker XIII.

Fig. 16. Restriction profil of the part of cytochrome b gene digested with Hinf I
restriction enzyme : 1. marker XIII, 2. 0,01% , 3. 0,1% 4. 1% 5. 2%, 6. 5% ,
7.10% , 8. pork, 9. bovine, 10 pork, 11. bovine 12.sheep, 13. goat, 14. chicken
15. turkey, 16 .marker XIII.

Fig. 17. Restriction profil of the part of cytochrome b gene digested with RSA I
restriction enzyme : 1. marker XIII, 2. meat ball 3. chicken ball, 4. dendeng,
5. pork, 6. bovine , 7. sheep. 8. goat, 9. chicken, 10. turkey, 11. marker XIII.

Fig. 18. Restriction profil of the part of cytochrome b gene digested with Hae III
restriction enzyme : 1. marker XIII, 2. meat ball 3. chicken ball, 4. dendeng,
5. pork, 6. bovine , 7. sheep. 8. goat, 9. chicken, 10. turkey, 11. marker XIII.

Fig. 19. Restriction profil of the part of cytochrome b gene digested with Hinf I
restriction enzyme : 1. marker XIII, 2. meat ball 3. chicken ball, 4. dendeng,
5. pork, 6. bovine , 7. sheep. 8. goat, 9. chicken, 10. turkey, 11. marker XIII.

Fig. 20. Restriction profil of the part of cytochrome b gene digested with Alu I
restriction enzyme : 1. marker XIII, 2. meat ball 3. bovine, 4. pork, 5. sheep.
6. goat, 7. chicken, 8. turkey, 9. marker XIII.

Fig. 21. Restriction profil of the part of cytochrome b gene digested with Taq I
restriction enzyme : 1. marker XIII, 2. meat ball 3. bovine ,4. pork, 5. sheep.
6. goat, 7. chicken, 8. turkey, 9. marker XIII.

The amplification of the gene encoding cytochrome b using the universal oligonucleotide primers Cytb-I and Cytb-II yielded a 359 bp amplicon. As an alternative to sequencing, the present study describes a PCR –RFLP analysis of a conserved region of the cytochrome b gene. Meyers et al. 1995, used the same primers to amplify 10 different food animal species, the sequences were analyzed to find conserved and variable regions for the selection the restriction enzyme.
The specifity of the primers cyt b I-II was tested amplfying genomic DNAs and all gave the predicted 359 bp fragment after enzymatic amplification and visualized on an 1,5 % agarose gel ( Fig. 1)
The amplification products were digested with Hae III restriction endonuclease showed the band of 0,1% and 0,01 % 285 bp and 74 bp, 1%, 2%,5%, 10% all gave the predicted 285 bp, 153 bp, 132 bp and 74 bp fragment were produced (Fig 6) . It were digested with Hinf I restriction enzyme were found 198 bp, 117 bp and 44 bp fragments and 5 %, 10% were found and predicted 359 bp, 198 bp and 117 bp ( Fig 7).
Using PCR-RFLP analysis of the cytochrome b gene for the samples ( meat ball, chicken ball and dendeng ( Fig 2) resulted in sufficient amplification and meat ball digested using Rsa I yielding three fragments 359, 210 and 149 bp; dendeng 359 bp was produced ( Fig. 8) . The restriction enzyme Hinf I digested the meat ball with a size of 359 fragment, chicken ball 188 bp and 161 bp fragments and dendeng 359 bp fragment were produced ( Fig. 9). The restriction Hae III digested the meat ball two fragments with a size of 284 bp and 74 bp; chicken ball to three fragments with a size of 159 bp, 126 bp and 74 bp and dendeng to two fragments with a size of 285 bp and 74 bp (Fig. 10).
After enzymatic amplification and visualized on an 1,5 % agarose gel for meat ball, dendeng, 5% mixed meat gave the predicted 359 bp fragment of sizes ( Fig 3). The result of Rsa I restriction endonuclease digested of meat ball, 5% mixed meat 359 bp fragment were produced, the amplification product of chicken ball was digested with Rsa I enzyme and all gave the predicted 210, 149 bp fragments sizes ( Fig 11). Using with Hae III for digested meat ball and 5 % mixed meat the predicted 159, 126 and 74 bp fragments were produced (Fig 12) and with Hinf I restriction enzyme of the meat ball produced one fragment resulting 359 bp (Fig. 13).
Using the primer Pig I and Pig II (Institut of Veterinary Food Science, Giessen, Germany) a species – specific product of a size approximately 250 bp could be amplified for the IRM and pork but no amplification from any of the other species (beef, sheep, goat, chicken and turkey).
The DNA fragment of 359 bp could be amplified of cytochrome b gene, and no amplification was obtained from the abon tested ( Fig. 4).
Figure 14 and 17 shows the results obtained after restriction analysis of the IRM and the Indonesia samples PCR product. Using the restriction enzyme Rsa I, no restriction site was founded for the IRM yielding 359 bp ; two fragments of 210 bp and 149 bp for chicken ball and 359 bp for dendeng were founded. The restriction enzyme Hae III digested the IRM amplicon and detected in the limit concentration is about 0,01% -1 % with RFLP ( Fig. 15). Using the restriction enzyme Rsa I was produced for meat ball to with a size of one fragment with a size of 359 bp, chicken ball two fragments of a size of 210 bp and 149 bp; and dendeng one fragment resulting 359 bp ( Fig. 17). Using the restriction enzyme Hae III, for digest in the meat ball to two fragments with a size of 285 bp and 74 bp ; chicken ball to three fragments with a size of 159 bp, 126 bp and 74 bp ; two fragments with a size of 285 bp and 74 bp for dendeng ( Fig. 18). Using the restriction enzyme Hinf I, the IRM resulting three fragments with the size of 198 bp, 117 bp and 44 bp ( Fig. 16). The restriction enzyme Hinf I for meat ball showed one fragment with a size of 359 bp ; with the chicken ball showed two fragments with a size of 188 bp and 161 bp. The dendeng was digested with enzyme Hinf I to three fragments with a size of 198 bp, 117 bp and 44 bp (Fig 19).
Using PCR-RFLP analysis of the cytochrome b gene for meat ball resulted in sufficient amplification ( Fig. 5). The result is shown in the weak signals of Alu I restriction fragment obtained with a size of 190 bp and 169 bp from meat ball ( Fig. 20). The restriction enzyme Taq I digested the PCR amplicon ( meat ball) to two fragments with a size of 191 bp and 168 bp ( Fig 21). Meat ball sample showed restriction patterns identical to buffalo type, chicken ball to chicken and dendeng to cattle. Sequece analysis revealed identical sequences when compared to species type ( Table 2).

Conclusion.
The aim of this study was to evaluate a PCR based method for detection of pork in meat and meat products. For this propose an universal oligonucleotide primers were used to this amplify a 359 bp fragment of mitochondrial cytochrome b gene of all animal species investigated. RFLP analysis was performed with different restrictions enzymes to differentiate the animal species.
The detection limit of this primer was 0,01% of pork in meat mixtures. No amplification was obtained from any of the other animal species ( bovine, sheep, goat, chicken and turkey ) tested.
According to the results of both species-specific primers and RFLP fragment profil of different restriction enzymes used the IRM and the Indonesia samples were correctly identified. On the other hand, meat ball sample showed restriction patterns identical to buffalo species. The sample : abon, could be not tested neither by PCR –RFPL nor by species-specific primers probably because it might be this product contained no meat.
The advantage of the PCR based method of the the cytochrome b gene is faster and even more sensitive then the other serological diagnostic. The speceis-specific PCR method detected pork in meat mixtures at level limit of 0,01% of pork in meat mixtures.

Acknowledgements
Sincere thanks are due to Prof. M. BŰLTE for facilities in the Molecular Biology Laboratory, Institute of Veterinary Food Science, Justus–Liebig–Universität Giessen and Dr. Amir ABDULMAWJOOD for discussion, valuable suggestion and the preparation of the figures. Thanks are also due to Mrs. WALTER and Mrs. SIMON for hers useful technical assistances. A frank gratitude is also sends to Prof. Mirnawati SUDARWANTO who had recommended me to study in those above mentioned laboratory. This work was supported by funds from German Academic Exchange Services and School of Veterinary Medicine, Bogor Agricultural University, Indonesia and in collaboration with Institute of Veterinary Food Science, Justus–Liebig–Universität Giessen, Germany.

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