Identification of Pork Adulteration in Processed Meat Products Using the Developed Mitochondrial DNA-Based Primers

Jimyeong Ha1,2, Sejeong Kim1,2, Jeeyeon Lee1,2, Soomin Lee1,2, Heeyoung Lee1,2, Yukyung Choi1,2, Hyemin Oh1,2, Yohan Yoon1,2,*
Author Information & Copyright
1Department of Food and Nutrition, Sookmyung Women’s University, Seoul 04310, Korea
2Risk Analysis Research Center, Sookmyung Women’s University, Seoul 04310, Korea
*Corresponding author Yohan Yoon Department of Food and Nutrition, Sookmyung Women's University, Seoul 04310, Korea Tel: +82-2-2077-7585 Fax: +82-2-710-9479 E-mail:

Copyright © 2017, Korean Society for Food Science of Animal Resources. This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: May 17, 2017 ; Revised: Jun 11, 2017 ; Accepted: Jun 12, 2017

Published Online: Jun 30, 2017


The identification of pork in commercially processed meats is one of the most crucial issues in the food industry because of religious food ethics, medical purposes, and intentional adulteration to decrease production cost. This study therefore aimed to develop a method for the detection of pork adulteration in meat products using primers specific for pig mitochondrial DNA. Mitochondrial DNA sequences for pig, cattle, chicken, and sheep were obtained from GenBank and aligned. The 294-bp mitochondrial DNA D-loop region was selected as the pig target DNA sequence and appropriate primers were designed using the MUSCLE program. To evaluate primer sensitivity, pork-beef-chicken mixtures were prepared as follows: i) 0% pork-50% beef-50% chicken, ii) 1% pork-49.5% beef-49.5% chicken, iii) 2% pork-49% beef-49% chicken, iv) 5% pork-47.5% beef-47.5% chicken, v) 10% pork-45% beef-45% chicken, and vi) 100% pork-0% beef-0% chicken. In addition, a total of 35 commercially packaged products, including patties, nuggets, meatballs, and sausages containing processed chicken, beef, or a mixture of various meats, were purchased from commercial markets. The primers developed in our study were able to detect as little as 1% pork in the heat treated pork-beef-chicken mixtures. Of the 35 processed products, three samples were pork positive despite being labeled as beef or chicken only or as a beef-chicken mix. These results indicate that the developed primers could be used to detect pork adulteration in various processed meat products for application in safeguarding religious food ethics, detecting allergens, and preventing food adulteration.

Keywords: food authentication; PCR; processed meat products


Detecting the adulteration of processed meat with unwanted food ingredients is one of the most important food quality-related issues. Food ingredient authentication is related to human health since ingredients may include allergenic or toxic substances. Furthermore, certain groups of people will not eat specific meats because of their religious food ethics and preferences (Ortea et al., 2012). Hsieh et al. (1997) reported that multispecies adulteration were found in commercial meat products. In addition, products labeled beef only are often intentionally adulterated with pork owing to the economic advantage that pork provides. This is found to be especially in the countries having expensive beef such as Korea, Japan, China, and so on (Singh and Neelam, 2011; Soares et al., 2013).

Food labeling regulations in many countries require that the meat species used in processed meat products must be declared for consumers because of religious food ethics, medical purposes, and personal food preferences (Doosti et al., 2014). However, processed meat products are still mislabeled for meat species, especially pork, which is either intentional or accidental (Tanabe et al., 2007). An accurate detection method for pork therefore needs to be developed for the prevention of food adulteration.

Existing detection methods for the identification of pork in processed meat products rely on protein or DNA analysis. Protein-based analytical methods include immunological assays (Anguita et al., 1996; Chen and Hsieh, 2000), chromatography (Chou et al., 2007), and peptide examination (Aristoy and Toldrá, 2004). However, protein-based analytical methods may not be appropriate for processed meat products since proteins can be denatured during processing. On the other hand, DNA-based analyses such as the polymerase chain reaction (PCR), real time PCR, PCR-restricted fragment length polymorphism (PCR-RFLP), and species-specific PCR are used more frequently in identifying fraudulent meat products (Man et al., 2007; Murugaiah et al., 2009; Soares et al., 2010), since these methods can detect small amounts of DNA and amplify specific target regions (Saiki et al., 1988). DNA-based analyses also have numerous advantages including simplicity, rapidity, sensibility, and specificity (Lockley and Bardsley, 2000).

The objective of this study was to design primers for the PCR amplification of a specific region of pig mitochondrial DNA and subsequently use these primers to identify mislabeled processed meat products.

Materials and Methods

Identification of pork-specific mitochondrial DNA sequences for primer design

Pig, cattle, chicken, and sheep mitochondrial DNA sequences were obtained from GenBank (database accession no. AF034253, V00654, AY235570, and AF010406, respectively) and aligned using the MUSCLE multiple sequence alignment program ( The regions most specific and unique to pork were identified among the aligned sequences using BLAST, and primers were subsequently designed. Sequences of the final selected primers were as follows: Pork-Forward (F) (5’- GGT TCT TAC TTC AGG ACC ATC-3’), and Pork-Reverse (R) (5’- GTG TAC GCA CGT GTA TGT AC-3’) (Table 1).

Table 1. Primer sequences specific for the pig mitochondrial DNA D-loop
Primer Sequence (5’ → 3’) Length of base pairs (bp) Length of PCR product (bp)
Download Excel Table

Processed meat sample preparation

To evaluate primer sensitivity, ground meat samples were purchased from butcher’s shop, and processed meat samples were prepared by mixing ground pork (boston butt), ground beef (shank), and ground chicken (breast) as follows: i) 0% pork-50% beef-50% chicken, ii) 1% pork-49.5% beef-49.5% chicken, iii) 2% pork-49% beef-49% chicken, iv) 5% pork-47.5% beef-47.5% chicken, v) 10% pork-45% beef-45% chicken, and vi) 100% pork-0% beef-0% chicken. All mixtures were kneaded by hands, subsequently placed into Eppendorf tube, and cooked in a water bath at 70°C for 3 min. The cooked meat was minced with a knife, and 200 mg was used for DNA extraction.

Monitoring pork adulteration

A total of 35 packaged meat products, such as 14 patties, 8 nuggets, 8 meatballs, and 5 sausages were purchased from a commercial vendor in Itaewon, Korea. Most products were frozen and some products were refrigerated. The product labels indicated that purchase products were manufactured with only chicken, beef, or mixture of these two meats. All products were stored at −20°C until used in the experiments.

DNA extraction

Genomic DNA was extracted from 200 mg of each of the processed meat products using the PowerPrepTM DNA Extraction from Food and Feed Kit (Kogenbiotech Co., Ltd., Korea). Six hundred microliters of lysis buffer A and 40 μL of lysis buffer B were added to 200 mg of each of the processed meat products, followed by incubation at 65°C for 1 h. Thereafter, 400 μL chloroform was added and centrifuged at 12,000 rpm for 10 min. Two hundred microliters of the supernatant was mixed with 200 μL binding buffer and 200 μL isopropanol, and the mixture was transferred into the DNA binding column. The DNA was washed twice with 75% ethanol and finally the DNA was eluted by 100 μL distilled water.

PCR assay

The PCR amplification was performed in a 50 μL reaction volume using 25 μL Taq PCR Master Mix Kit (Qiagen, Germany), 2 μL of each primer, 2 μL extracted DNA, and 19 μL of distilled water. The amplification profile was an initial denaturation step at 95°C for 5 min, followed by 40 cycles at 95°C for 20 s, 55°C for 20 s, and 72°C for 30 s. In order to confirm amplification of the target sequence, the PCR product was electrophoresed on a 2% agarose gel in 1×TAE buffer (Biosesang, Korea) at 100 V for 20 min. A 100-bp DNA Ladder (Dynebio Inc., Korea) was used as the size marker and images were captured in a UV-transilluminator (TF-20M, Vilber Lourmat, France).

Results and Discussion

Food adulteration has been an issue for many years in processed meat products. In particular, pork is often mixed in other meat products such as beef, because it is cheaper. Thus, various pork detection methods have been developed. Among these methods, species-specific oligo Vilber Lourmat nucleotide primers have been used to detect pork in processed meat products. This study targeted the 294-bp long pig mitochondrial DNA D-loop region since mitochondrial DNA is highly conserved in many animal species, is stable from heating, and can be used to detect pork fat (Montiel-Sosa et al., 2000). This suggests that the primers designed in our study could be used to detect pork in heat-treated products.

There are two methods to detect pork adulteration in processed meat products, namely protein and DNA analysis. DNA-based methods are used more frequently than protein-based methods to detect adulterated food, because of the limitations of protein-based methods such as protein denaturation following heating (Fajardo et al., 2010). Meat mixtures were prepared with the inclusion of pork at a concentration of 0%, 1%, 2%, 5%, 10%, and 100%, and subsequently heated to determine primer compatibility and detection limits. Study results indicate the developed primers could detect as little as 1% pork in the heat treated meat products (Fig. 1).

Fig. 1. Agarose gel electrophoresis of PCR products amplified from heated meat; Lane 1: Marker, Lane 2: 0% pork, Lane 3: 1% pork, Lane 4: 2% pork, Lane 5: 5% pork, Lane 6: 10% pork, Lane 7: 100% pork.
Download Original Figure

To monitor pork adulteration in commercial processed meats with the primers developed in our study, 35 processed meat products were purchased including patties, nuggets, meatballs, and sausages labeled as 0% pork. Of the 35 products, three (8.6%) were pork-positive (Table 2). The positive samples were one meatball and two sausage products. The meatball was labeled as beef only, and the sausages were labeled as beef and chicken, and chicken only. Murugaiah et al. (2009) reported that adulteration occurs predominantly in comminute or ground meat products. In our study, all pork-positive samples were from ground meat products.

Most ground meat is accidentally adulterated with unwanted meat species during the process of grinding. It is, thus, essential that grinders are thoroughly cleaned when changing meat species, alternatively different meat grinders should be used for different meat species. However, pork is also intentionally added to beef products because of production costs (Doosti et al., 2014). In many countries, labels declaring the specific meat species used are mandatory because of food allergens, religious food ethics, and prevention of food fraud (Ayaz et al., 2006; Gendel, 2012; Soares et al., 2013); however, as shown in Table 2, adulteration still occurs in commercial products. It is therefore important that an accurate and simple method to detect pork adulteration in processed meat products is developed.

Table 2. Detection of pork adulteration in commercial processed meat products by PCR
Sample no. Product category Labeling Company Presence of pork
1 Patties Beef A -
2 Patties Chicken B -
3 Patties Chicken C -
4 Patties Vegetables D -
5 Patties Chicken E -
6 Patties Beef E -
7 Patties Chicken F -
8 Patties Chicken G -
9 Patties Chicken H -
10 Patties Beef H -
11 Patties Vegetables I -
12 Patties Beef J -
13 Patties Beef K -
14 Patties Beef L -
15 Nuggets Chicken M -
16 Nuggets Chicken N -
17 Nuggets Chicken N -
18 Nuggets Chicken O -
19 Nuggets Chicken B -
20 Nuggets Chicken P -
21 Nuggets Chicken Q -
22 Nuggets Chicken R -
23 Meatball Beef S -
24 Meatball Beef T -
25 Meatball Chicken F -
26 Meatball Chicken F -
27 Meatball Chicken F -
28 Meatball Beef J -
29 Meatball Beef U -
30 Meatball Beef V -
31 Sausage Beef+ Chicken W -
32 Sausage Fish X -
33 Sausage Fish X -
34 Sausage Beef Y -
35 Sausages Beef Z -
Download Excel Table

In conclusion, the primers developed in our study could be used to detect the presence of pork adulteration in various processed meat products, even when heat treated. In addition, pork adulterated commercial meat products were identified, and thus, government agencies should consider monitoring mislabeling of meat products that include pork.


This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through its Export Promotion Technology Development Program, which is funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (315050-2).



Anguita G., Martín R., Garcia T., Morales P., Haza A. I., González I., Hernández P. E. Immunostick ELISA for detection of cow's milk in ewe's milk and cheese using a monoclonal antibody against beef β-casein. J. Food Protect. 1996; 59:436-437.


Aristoy M. C., Toldrá F. Histidine dipeptides HPLC-based test for the detection of mammalian origin proteins in feeds for ruminants. Meat Sci. 2004; 67:211-217.


Ayaz Y., Ayaz N. D., Erol I. Detection of species in meat and meat products using enzyme-linked immunosorbent assay. J. Muscle Foods. 2006; 17:214-220.


Chen F. C., Hsieh Y. H. P. Detection of pork in heat-processed meat products by monoclonal antibody-based ELISA. J. AOAC Int. 2000; 83:79-85.


Chou C. C., Lin S. P., Lee K. M., Hsu C. T., Vickroy T. W., Zen J. M. Fast differentiation of meats from fifteen animal species by liquid chromatography with electrochemical detection using copper nanoparticle plated electrodes. J. Chromatogr. B. 2007; 846:230-239.


Doosti A., Dehkordi P. G., Rahimi E. Molecular assay to fraud identification of meat products. J. Food Sci. Technol. 2014; 51:148-152.


Fajardo V., González I., Rojas M., García T., Martín R. A review of current PCR-based methodologies for the authentication of meats from game animal species. Trends Food Sci. Tech. 2010; 21:408-421.


Gendel S. M. Comparison of international food allergen labeling regulations. Regul. Toxicol. Pharmacol. 2012; 63:279-285.


Hsieh Y. H. P., Chen F. C., Sheu S. C. AAES research developing simple, inexpensive tests for meat products. Highlights Agr. Res. 1997; 44:19-20.


Lockley A. K., Bardsley R. G. DNA-based methods for food authentication. Trends Food Sci. Technol. 2000; 11:67-77.


Man Y. C., Aida A. A., Raha A. R., Son R. Identification of pork derivatives in food products by species-specific polymerase chain reaction (PCR) for halal verification. Food Control. 2007; 18:885-889.


Montiel-Sosa J. F., Ruiz-Pesini E., Montoya J., Roncales P., Lopez-Perez M. J., Perez-Martos A. Direct and highly species-specific detection of pork meat and fat in meat products by PCR amplification of mitochondrial DNA. J. Agr. Food Chem. 2000; 48:2829-2832.


Murugaiah C., Noor Z. M., Mastakim M., Bilung L. M., Selamat J., Radu S. Meat species identification and Halal authentication analysis using mitochondrial DNA. Meat Sci. 2009; 83:57-61.


Ortea I., Pascoal A., Cañas B., Gallardo J. M., Barros-Velázquez J., Calo-Mata P. Food authentication of commercially-relevant shrimp and prawn species: From classical methods to foodomics. Electrophoresis. 2012; 33:2201-2211.


Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988; 239:487.


Singh V. P., Neelam S. Meat species specifications to ensure the quality of meat: A review. Int. J. Meat Sci. 2011; 1:15-26.


Soares S., Amaral J. S., Mafra I., Oliveira M. B. P. Quantitative detection of poultry meat adulteration with pork by a duplex PCR assay. Meat Sci. 2010; 85:531-536.


Soares S., Amaral J. S., Oliveira M. B. P., Mafra I. A SYBR green real-time PCR assay to detect and quantify pork meat in processed poultry meat products. Meat Sci. 2013; 94:115-120.


Tanabe S., Miyauchi E., Muneshige A., Kazuhiro M. I. O. PCR method of detecting pork in foods for verifying allergen labeling and for identifying hidden pork ingredients in processed foods. Biosci. Biotechnol. Biochem. 2007; 71:1663-1667.