Applied Microbiology and Biotechnology

, Volume 98, Issue 23, pp 9805–9816 | Cite as

Detection of pork adulteration in processed meat by species-specific PCR-QIAxcel procedure based on D-loop and cytb genes

  • Hassan BarakatEmail author
  • Hoda A. S. El-Garhy
  • Mahmoud M. A. Moustafa
Methods and protocols


Detection of pork meat adulteration in “halal” meat products is a crucial issue in the fields of modern food inspection according to implementation of very strict procedures for halal food labelling. Present study aims at detecting and quantifying pork adulteration in both raw and cooked manufactured sausages. This is by applying an optimized species-specific PCR procedure followed by QIAxcel capillary electrophoresis system. Manufacturing experiment was designed by incorporating pork with beef meat at 0.01 to 10 % substitution levels beside beef and pork sausages as negative and positive controls, respectively. Subsequently, sausages were divided into raw and cooked sausages then subjected to DNA extraction. Results indicated that PCR amplifications of mitochondrial D-loop and cytochrome b (cytb) genes by porcine-specific primers produced 185 and 117 bp pork-specific DNA fragments in sausages, respectively. No DNA fragments were detected when PCR was applied on beef sausage DNA confirming primers specificity. For internal control, a 141-bp DNA fragment of eukaryotic 18S ribosomal RNA (rRNA) gene was amplified from pork and beef DNA templates. Although PCR followed by either QIAxcel or agarose techniques were efficient for targeted DNA fragments differentiation even as low as 0.01 % (pork/meat: w/w). For proficiency, adequacy, and performance, PCR-QIA procedure is highly sensitive, a time-saver, electronically documented, mutagenic-reagent free, of little manual errors, accurate in measuring PCR fragments length, and quantitative data supplier. In conclusion, it can be suggested that optimized PCR-QAI is considered as a rapid and sensitive method for routine pork detection and quantification in raw or processed meat.


PCR Adulteration Halal processed meat cytb gene D-loop gene QIAxcel procedure 


  1. Ali ME, Hashim U, Mustafa S, Che Man YB (2012) Swine-specific PCR-RFLP assay targeting mitochondrial cytochrome B gene for semiquantitative detection of pork in commercial meat products. Food Anal Methods 5:613–623CrossRefGoogle Scholar
  2. Ali ME, Hashim U, Mustafa S, Che Man YB, Yusop MHM, Kashif M, Dhahi TS, Bari MF, Hakim MA, Latif MA (2011) Nanobiosensor for detection and quantification of DNA sequences in degraded mixed meats. J Nanomater 2011:1–11CrossRefGoogle Scholar
  3. Armand R, Channon JY, Kintner J, White KA, Miselis KA, Perez RP, Lewis LD (2004) The effects of ethidium bromide induced loss of mitochondrial DNA on mitochondrial phenotype and ultrastructure in a human leukemia T-cell line (MOLT-4 cells). Toxicol Appl Pharmacol 196:68–79PubMedCrossRefGoogle Scholar
  4. Barakat H, Spielvogel A, Hassan M, El-Desouky A, El-Mansy H, Rath F, Meyer V, Stahl U (2010) The antifungal protein AFP from Aspergillus giganteus prevents secondary growth of different Fusarium species on barley. Appl Microbiol Biotechnol 87:617–624PubMedCrossRefGoogle Scholar
  5. Bartlett SE, Davidson WS (1992) FINS (forensically informative nucleotide sequencing): a procedure for identifying the animal origin of biological specimens. Biotechniques 12:408–411PubMedGoogle Scholar
  6. Bonne K, Verbeke W (2008) Muslim consumer trust in halal meat status and control in Belgium. Meat Sci 79:113–123PubMedCrossRefGoogle Scholar
  7. Calvo JH, Osta R, Zaragoza P (2002) Quantitative PCR detection of pork in raw and heated ground beef and pâté. J Agric Food Chem 50:5265–5267PubMedCrossRefGoogle Scholar
  8. Calvo JH, Zaragoza P, Osta R (2001) Random amplified polymorphic DNA fingerprints for identification of species in poultry pate. Poult Sci 80:522–524PubMedCrossRefGoogle Scholar
  9. Che Man YB, Mustafa S, Khairil-Mokhtar NF, Nordin R, Sazili AQ (2012) Porcine-specific polymerase chain reaction assay based on mitochondrial D-loop gene for identification of pork in raw meat. Int J Food Prop 15:134–144CrossRefGoogle Scholar
  10. Chikuni K, Ozutsumi K, Koishikawa T, Kato S (1990) Species identification of cooked meats by DNA hybridization assay. Meat Sci 27:119–128PubMedCrossRefGoogle Scholar
  11. Di Pinto A, Forte VT, Conversano MC, Tantillo GM (2005) Duplex polymerase chain reaction for detection of pork meat in horse meat fresh sausages from Italian retail sources. Food Cont 16:391–394CrossRefGoogle Scholar
  12. Girish PS, Anjaneyulu ASR, Viswas KN, Shivakumar BM, Anand M, Patel M, Sharma B (2005) Meat species identification by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of mitochondrial 12S rRNA gene. Meat Sci 70:107–112PubMedCrossRefGoogle Scholar
  13. Graf C, Rüegg-Kuslyte A, Kuhn R (2011) Species determination for meat using PCR-RFLP analysis on the QIAxcel® system. Application NoteGoogle Scholar
  14. Hird H, Chisholm A, Sanchez M, Hernandez R, Goodier K, Schneede C, Boltz B, Popping B (2006) Effect of heat and pressure processing on DNA fragmentation and implications for the detection of meat using a real-time polymerase chain reaction. Food Addit Contam 23:645–650PubMedCrossRefGoogle Scholar
  15. Karabasanavar NS, Singh SP, Kumar D, Shebannavar SN (2014) Detection of pork adulteration by highly-specific PCR assay of mitochondrial D-loop. Food Chem 145:530–534PubMedCrossRefGoogle Scholar
  16. Karabasanavar NS, Singh SP, Umapathi V, Girish PS, Shebannavar SN, Kumar D (2011) Authentication of carabeef (water buffalo, Bubalus bubalis) using highly specific polymerase chain reaction. Eur Food Res Technol 233:985–989CrossRefGoogle Scholar
  17. Kesmen Z, Gulluce A, Sahin F, Yetim H (2009) Identification of meat species by TaqMan-based real-time PCR assay. Meat Sci 82:444–449PubMedCrossRefGoogle Scholar
  18. Kesmen Z, Sahin F, Yetim H (2007) PCR assay for the identification of animal species in cooked sausages. Meat Sci 77:649–653PubMedCrossRefGoogle Scholar
  19. Kumar D, Singh SP, Singh R, Karabasanavar NS (2011) A highly specific PCR assay for identification of goat (Capra hircus) meat. Small Rumin Res 97:76–78CrossRefGoogle Scholar
  20. Mane BG, Mendiratta SK, Tiwari AK (2013) Pork specific polymerase chain reaction assay for authentication of meat and meat products. J Meat Sci Technol 1:21–27Google Scholar
  21. Marois C, Dufour-Gesbert F, Kempf I (2001) Comparison of pulsed-field gel electrophoresis with random amplified polymorphic DNA for typing of Mycoplasma synoviae. Vet Microbiol 79:1–9PubMedCrossRefGoogle Scholar
  22. Matsumoto T, Koshii Y, Sakane K, Murakawa T, Hirayama Y, Yoshida H, Kurokawa M, Tamura Y, Nagai T, Kawase I (2013) A novel approach to automated genotyping of Mycobacterium tuberculosis using a panel of 15 MIRU VNTRs. J Microbiol Methods 93:239–241PubMedCrossRefGoogle Scholar
  23. McMurray CL, Hardy KJ, Hawkey PM (2010) Rapid, automated epidemiological typing of methicillin-resistant Staphylococcus aureus. J Microbiol Methods 80:109–111PubMedCrossRefGoogle Scholar
  24. Melake NA, Shaker GH, Salama MA (2012) Incidence of Helicobacter pylori infection and their clarithromycin-resistant strains in otitis media with effusion regarding phenotypic and genotypic studies. Saudi Pharm J 20:345–353PubMedCentralPubMedCrossRefGoogle Scholar
  25. Mercimek-Mahmutoglu S, Sinclair G, van Dooren SJM, Kanhai W, Ashcraft P, Michel OJ, Nelson J, Betsalel OT, Sweetman L, Jakobs C, Salomons GS (2012) Guanidinoacetate methyltransferase deficiency: first steps to newborn screening for a treatable neurometabolic disease. Mol Genet Metab 107:433–437PubMedCrossRefGoogle Scholar
  26. Moghazy EA, El-Shaarawy MOA (2001) Quality attributes of beef burger as affected by using proplies and frozen storage. Egypt J Agric Res 79:1499–1512Google Scholar
  27. Moghazy EA, Sharaf SM, El-Seesy TA (2004) Effect of substitutines mutton tailfat with vegetable oil on quality attributes of healthy beef sausage. Egypt J Appl Sci 19:191–205Google Scholar
  28. Montiel-Sosa JF, Ruiz-Pesini E, Montoya J, Roncalés P, López-Pérez MJ, Pérez-Martos A (2000) Direct and highly species-specific detection of pork meat and fat in meat products by PCR amplification of mitochondrial DNA. J Agric Food Chem 48:2829–2832PubMedCrossRefGoogle Scholar
  29. Olive PL, Wlodek D, Durand RE, Banáth JP (1992) Factors influencing DNA migration from individual cells subjected to gel electrophoresis. Exp Cell Res 198:259–267PubMedCrossRefGoogle Scholar
  30. Rehbein H, Kress G, Schmidt T (1997) Application of PCR-SSCP to species identification of fishery products. J Sci Food Agric 74:35–41CrossRefGoogle Scholar
  31. Rojas M, González I, Pavón MA, Pegels N, Lago A, Hernández PE, García T, Martin R (2010) Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds. Food Addit Contam A 27:749–763CrossRefGoogle Scholar
  32. Skarpeid H, Moe RE, Indahl UG (2001) Detection of mechanically recovered meat and head meat from cattle in ground beef mixtures by multivariate analysis of isoelectric focusing protein profiles. Meat Sci 57:227–234PubMedCrossRefGoogle Scholar
  33. Tanabe S, Hase M, Yano T, Sato M, Fujimura T, Akiyama H (2007a) A real-time quantitative PCR detection method for pork, chicken, beef, mutton, and horseflesh in foods. Biosci Biotechnol Biochem 71:3131–3135PubMedCrossRefGoogle Scholar
  34. Tanabe S, Miyauchi E, Muneshige A, Mio K, Sato C, Sato M (2007b) PCR method of detecting pork in foods for verifying allergen labeling and for identifying hidden pork ingredients in processed foods. Biosci Biotechnol Biochem 71:1663–1667PubMedCrossRefGoogle Scholar
  35. Toorop R, Murch SJ, Ball RO (1997) Methodology and development of prediction equations for the determination of pork substitution in veal. Food Res Int 30:629–636CrossRefGoogle Scholar
  36. Wissiack R, de la Calle B, Bordin G, Rodriguez AR (2003) Screening test to detect meat adulteration through the determination of hemoglobin by cation exchange chromatography with diode array detection. Meat Sci 64:427–432PubMedCrossRefGoogle Scholar
  37. Xiao M, Kong P, Jin Q, Wang K, Xiao N, Jeoffreys G, James GL, Gilbert GL (2012) Comparison of two capillary gel electrophoresis systems for Clostridium difficile ribotyping, using a panel of ribotype 027 isolates and whole-genome sequences as a reference standard. J Clin Microbiol 50:2755–2760PubMedCentralPubMedCrossRefGoogle Scholar
  38. Yang I, Kim Y, Byun J, Park S (2005) Use of multiplex polymerase chain reactions to indicate the accuracy of the annealing temperature of thermal cycling. Anal Biochem 338:192–200PubMedCrossRefGoogle Scholar
  39. Yusop MHM, Mustafa S, Che Man YB, Omar A, Mokhtar N (2012) Detection of raw pork targeting porcine-specific mitochondrial cytochrome B gene by molecular beacon probe real-time polymerase chain reaction. Food Anal Methods 5:422–429CrossRefGoogle Scholar
  40. Zhang S, Zhao S, Wang Z, Li C (2013) Investigation of parent-of-origin SNPs in 5 imprinted genes for forensic purpose. For Sci Int Genet Suppl Ser 4:e304–e305CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Hassan Barakat
    • 1
    Email author
  • Hoda A. S. El-Garhy
    • 2
    • 3
  • Mahmoud M. A. Moustafa
    • 2
    • 3
  1. 1.Food Science Department, Faculty of AgricultureBenha UniversityMoshtohorEgypt
  2. 2.Genetics Department, Faculty of AgricultureBenha UniversityMoshtohorEgypt
  3. 3.Agricultural Biology Lab., Faculty of AgricultureBenha UniversityMoshtohorEgypt

Personalised recommendations