Skip to main content
SpringerLink
Log in

Detection of Raw Pork Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by Molecular Beacon Probe Real-Time Polymerase Chain Reaction

  • Published:
Food Analytical Methods Aims and scope Submit manuscript

Abstract

Pork identification in raw meat using real-time polymerase chain reaction (PCR) was developed. Total DNA from meat samples were successfully extracted and found to be of high quality and produced clear PCR products. Porcine-specific molecular beacon probe and primers that amplifies 119 bp of the cytochrome b gene fragment of swine (Sus scrofa domestica) was used. Analysis of data showed that the C q (quantification cycle) from 10 ng/μl porcine DNA is (18.70 ± 0.12 to 19.08 ± 0.06). Meanwhile, the other samples exhibited negative result, which confirmed the specificity of the primers. The method also showed that the limit of detection of pork was 0.0001 ng. Based on the regression analysis of the standard curve, the 96% efficiency of real-time PCR was achieved with high correlation coefficient (r 2 = 0.9989). Sensitivity of the assay in discriminating pork as low as 0.1% (w/w) pork in pork–beef mixtures was also obtained. Reproducibility of the assay was successfully validated by applying sample and experimental replicates in every assay being conducted. Thus, this methodology could serve as a fast and sensitive method for detection of pork for meat species verification.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Aida AA, YB Che man, Raha AR, Son R (2007) Detection of pig derivatives in food products for halal authentication by polymerase chain reation-restriction length polymorphism. J Sci of Food and Agri 87:569–572

    Article  CAS  Google Scholar 

  • Aida AA, Che Man YB, Wong CMVL, Raha AR, Son R (2005) Analysis of raw meats and fats of pigs using polymerase chain reaction for halal authentication. Meat Sci 69:47–52

    Article  CAS  Google Scholar 

  • Ballin NZ, Vogensen FK, Karlsson AH (2009) Species determination—can we detect and quantify meat adulteration? Meat Sci 83:165–174

    Article  CAS  Google Scholar 

  • Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  CAS  Google Scholar 

  • Castresana J (2001) Cytochrome b phylogeny and the taxonomy of great apes and mammals. Mol Biol Evol 18:465–471

    Article  CAS  Google Scholar 

  • Che Man YB, Aida AA, Raha AR, Son R (2007) Identification of pork derivatives in food products by species-specific polymerase chain reaction (PCR) for halal verification. Food Control 18:885–889

    Article  Google Scholar 

  • Chen FC, Hsieh YHP, Bridgman RC (2004) Monoclonal antibody-based sandwich enzyme-linked immunoabsorbant assay for sensitive detection of prohibited ruminant proteins in feedstuff. J Food Prot 67:544–549

    CAS  Google Scholar 

  • Chisholm J, Conyers C, Booth C, Lawley W, Hird H (2005) The detection of horse and donkey using real-time PCR. Meat Sci 70:727–732

    Article  CAS  Google Scholar 

  • Chisholm J, Sanchez A, Brown J, Hird H (2008) The development of species-specific real-time PCR assays for the detection of pheasant and quail in food. Food Anal Methods 1:190–194

    Article  Google Scholar 

  • Chou CC, Lin SP, Lee KM, Hsu CT, Vickroy TW, Zen JM (2007) Fast differentiation of meats from fifteen animal species by liquid chromatography with electrochemical detection using copper nanoparticle plated electrodes. J Chromatogr B 846:230–239

    Article  CAS  Google Scholar 

  • Dooley JJ, Paine KE, Garrett SD, Brown HM (2004) Detection of meat species using TaqMan real-time PCR assays. Meat Sci 68:431–438

    Article  CAS  Google Scholar 

  • European Commission (2001) Directive 2001/101/EC of 26 November 2001 amending Directive 2000/13/EC of the European Parliament and of the Council on the approximation of 20 March 2000 on the approximation of the laws of the Member States relating to the labelling, presentation and advertising of foodstuffs. Official Journal of the European Communities L 310:19–21

    Google Scholar 

  • Fajardo V, González I, López-Calleja I, Martín I, Rojas M, Hernández PE, García T, Martín R (2007) Identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) using polymerase chain reaction targeting specific sequences from the mitochondrial 12SrRNA gene. Meat Sci 76:234–240

    Article  CAS  Google Scholar 

  • Fajardo V, González I, Martín I, Rojas M, Hernández PE, García T, Martín R (2008) Real-time PCR for detection and quantification of red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) in meat mixtures. Meat Sci 79:289–298

    Article  CAS  Google Scholar 

  • Feligini M, Alim N, Bonizzi I, Enne G, Aleandri R (2007) Detection of cow milk in water buffalo cheese by SYBR Green real-time PCR: sensitivity test on governing liquid samples. Pakistan J Nutr 6:94–98

    Article  Google Scholar 

  • Ghovvati S, Nassiri MR, Mirhoseini SZ, Heravi MA, Javadmanesh A (2009) Fraud identification in industrial meat products by multiplex PCR assay. Food Control 20:696–699

    Article  CAS  Google Scholar 

  • Hadjinicolaou AV, Demetriou VL, Emmanuel MA, Kakoyiannis CK, Kostrikis LG (2009) Molecular beacon-based real-time PCR detection of primary isolates of Salmonella typhimurium and Salmonella enteritidis in environmental and clinical samples. BMC Microbiol 9:97

    Article  Google Scholar 

  • Hajmeer M, Cliver DO, Provost R (2003) Spinal cord tissue detection in comminuted beef: comparison of two immunological methods. Meat Sci 65:757–763

    Article  Google Scholar 

  • Hsieh H-M, Chiang H-L, Tsai L-C, Lai S-Y, Huang N-E, Linacre A, Lee JC-I (2001) Cytochrome b gene species identification of the conservation animals. Forensic Sci Int 122:7–18

    Article  CAS  Google Scholar 

  • Ihrig J, Lill R, Mühlenhoff U (2006) Application of the DNA-specific dye EvaGreen for the routine quantification of DNA in microplates. Anal Biochem 359:265–267

    Article  CAS  Google Scholar 

  • Lanzilao I, Burgalassi F, Fancelli S, Settimelli M, Fani R (2005) Polymerase chain reaction-restriction fragment length polymorphism of mitochondrial cyt b gene from species of dairy interest. J AOAC Int 88:128–135

    CAS  Google Scholar 

  • Lázaro DR, Lloyd J, Herrewegh A, Ikonomopoulos J, D’Agostino M, Pla M, Cook N (2004) A molecular beacon-based real-time NASBA assay for detection of Mycobacterium avium subsp. paratuberculosis in water and milk. FEMS Microbiol Lett 237:119–126

    Google Scholar 

  • López-Calleja I, González I, Fajardo V, Rodriguez MA, Hernández PE, García T, Martín R (2004) Rapid detection of cows’ milk in sheeps’ and goats’ milk by a species-specific polymerase chain reaction technique. J Dairy Sci 87:2839–2845

    Article  Google Scholar 

  • Martín I, García T, Fajardo V, Rojas M, Pegels N, Hernández PE, González I, Martín R (2009) SYBR-Green real-time PCR approach for the detection and quantification of pig DNA in feedstuffs. Meat Sci 82:252–259

    Article  Google Scholar 

  • Matsunaga T, Chikuni K, Tanabe R, Muroya S, Shibata K, Yamada J, Shinmura Y (1999) A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Sci 51:143–148

    Article  CAS  Google Scholar 

  • Meijerink J, Mandigers C, Locht LVD, Tönnisen E, Goodsaid F, Raemaekers J (2001) A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR. J Mol Diag 3:55–61

    Article  CAS  Google Scholar 

  • Mhlanga MM, Malmberg L (2001) Using molecular beacons to detect single-nucleotide polymorphisms with real-time PCR. Methods 25:463–471

    Article  CAS  Google Scholar 

  • Murugaiah C, Noor ZM, Mastakim M, Bilung LM, Selamat J, Radu S (2009) Meat species identification and Halal authentication analysis using mitochondrial DNA. Meat Sci 83:57–61

    Article  CAS  Google Scholar 

  • Pfaffl MW (2004) In: Bustin SA (ed) Quantification strategies in real-time PCR. A–Z of quantitative PCR. International University Line (IUL), La Jolla, 3:99

    Google Scholar 

  • Rodríguez MA, García T, González I, Hernández PE, Martín R (2005) TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures. Meat Sci 70:113–120

    Article  Google Scholar 

  • Sandhya S, Chen W, Mulchandani A (2008) Molecular beacons: a real time polymerase chain reaction assay for detecting Escherichia coli from fresh produce and water. Anal Chim Acta 614:208–212

    Article  CAS  Google Scholar 

  • Sawyer J, Wood C, Shanahan D, Gout S, McDowell D (2003) Real-time PCR for quantitative meat species testing. Food Control 14:579–583

    Article  CAS  Google Scholar 

  • Vallejo-Cordoba B, González-Córdova AF, Mazorra-Manzano MA, Rodríguez-Ramírez R (2005) Capillary electrophoresis for the analysis of meat authenticity. J Sep Sci 28:826–836

    Article  CAS  Google Scholar 

  • Vet JAM, Marras SAE (2005) Design and optimization of molecular beacon real-time polymerase chain reaction assays. Methods Mol Biol 288:273–290

    CAS  Google Scholar 

  • Wang L, Blasic JR Jr, Holden MJ, Pires R (2005) Sensitivity comparison of real-time PCR probe designs on a model DNA plasmid. Anal Biochem 344:257–265

    Article  CAS  Google Scholar 

  • Wang W, Chen K, Xu C (2006a) DNA quantification using EvaGreen and a real-time PCR instrument. Anal Biochem 356:303–305

    Article  CAS  Google Scholar 

  • Wang Y, Zhu W, Levy DE (2006b) Nuclear and cytoplasmic mRNA quantification by SYBR green based real-time RT-PCR. Methods 39:356–362

    Article  CAS  Google Scholar 

  • Wolf C, Lüthy J (2001) Quantitative competitive (QC) PCR for quantification of porcine DNA. Meat Sci 57:161–168

    Article  CAS  Google Scholar 

  • Ye QY, Zhuang H-S, Zhou C (2009) Detection of trace anthracene in soil samples with real-time fluorescence quantitative immune-PCR using a molecular beacon probe. Environ Toxicol Phar 28:386–391

    Article  CAS  Google Scholar 

  • Zhang C-L, Fowler MR, Scott NW, Lawson G, Slater A (2007) A TaqMan real-time PCR system for the identification and quantification of bovine DNA in meats, milks and cheeses. Food Control 18:1149–1158

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Mohd Hazim Mohd Yusop is a beneficiary of Special Graduate Research Allowance (SGRA) from Universiti Putra Malaysia (UPM). This project was paid by the Research University Grant Scheme (grant vote no. 91763) awarded to Assoc. Prof. Dr. Shuhaimi B. Mustafa.

Author information

Authors and Affiliations

  1. Halal Products Research Institute, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia

    Mohd Hazim Mohd Yusop, Shuhaimi Mustafa, Yaakob B. Che Man & Nur Fadhilah Khairil Mokhtar

  2. Institute of Bioscience, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia

    Abdul Rahman Omar

Authors
  1. Mohd Hazim Mohd Yusop
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shuhaimi Mustafa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yaakob B. Che Man
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Abdul Rahman Omar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nur Fadhilah Khairil Mokhtar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shuhaimi Mustafa.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yusop, M.H.M., Mustafa, S., Che Man, Y.B. et al. Detection of Raw Pork Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by Molecular Beacon Probe Real-Time Polymerase Chain Reaction. Food Anal. Methods 5, 422–429 (2012). https://doi.org/10.1007/s12161-011-9260-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12161-011-9260-y

Keywords

Search

Navigation