Annals of Food Processing and Preservation

Rapid and Simple Identification of Pork in Meat Products by Using the Isothermal Target and Probe Amplification Assay

Research Article | Open Access

  • 1. Food Research and Development Center, Samsung Welstory, South Korea
+ Show More - Show Less
Corresponding Authors
Jeongsoon Kim, Food Research and Development Center, Samsung Welstory, Inc., 2442-1 Yonggu-daero, Giheunggu, YongIn-si, Gyeonggi-do, South Korea

Species identification of animal tissues in meat products is an important issue to protect the consumer from illegal and/or undesirable adulteration, for economic, religious and health reasons. For example, pork DNA was found in halal chicken sausages served in a primary school in Westminster, central London, the local authority had stated on 14 March 2015. PCR is one of the most widely used techniques in diagnostic applications because it allows a sensitive and rapid diagnosis. However, this technique is not suitable for routine identification of pork in meat products because of the need for expensive thermal cycler equipment and complex operations. The isothermal target and probe amplification (iTPA) analysis has been developed for identifying pork species in meat samples. In this study, the isothermal target and probe amplification assay was developed for rapid, simple and highly specific identification of pork DNA. For the assay, the mitochondrial 16S ribosomal RNA gene and the FRET-based signal probe were amplified at 61o C, followed by measurement of the fluorescent signal. As little as 1 pg of template DNA could be detected without any cross-reactivity with non-target species. Meat mixtures prepared by mixing pork meat with other meats at different ratios (0.01% - 10%) were tested, and the iTPA assay allowed detection of as little as 0.01% pork in meat mixtures. Thus, this work showed that the iTPA assay could be used for specific identification of pork species. This assay only requires a heat block and a fluorescence reader for amplification and detection, respectively, and it has great potential for use as a hand-held device or point-of-care-testing system. The iTPA assay is sensitive and specific for rapid screening of fraudulent adulteration/substitution of meat products


•    Meat products
•    Isothermal Target and Probe Amplification (iTPA)
•    Mitochondrial 16S ribosomal RNA gene
•    Food fraud


Kim J, Shin H (2017) Rapid and Simple Identification of Pork in Meat Products by Using the Isothermal Target and Probe Amplification Assay. Ann Food Process Preserv 2(2): 1016.


Species identification of animal tissues in meat products is an important issue to protect the consumer from illegal and/ or undesirable adulteration, for economic, religious and health reasons [1]. For this purpose, numerous analytical methods have been developed based on protein and DNA analysis. Methods based on protein fractions, including physico-chemical, electrophoretic, and immunological techniques, have been introduced [2]. Unfortunately, these methods are often not suitable for complex food products because they are not sensitive in processed materials to differentiate closely-related species, are time-consuming, inadequate, and/or expensive. DNAbased methods such as PCR are the most specific and sensitive techniques for food component authentication and they are relatively quick. DNA-based techniques have many potential advantages over protein-based techniques such as ELISA, which depends primarily on protein epitopes that are detectable by the antibodies to detect analytes [3]. In DNA-based techniques for meat speciation, genetic markers are used. They may be nuclear gene or mitochondrial gene markers. Among nuclear markers, leptin gene [4], actin gene [5] and melanocortin receptor 1 (MC1R) gene [6] are common, while mitochondrial genes used for this purpose include cytochrome-b [7-10], 12S and 16S ribosomal RNA subunits [11-13], displacement loop region (D- loop) [14,15], and cytochrome oxidase I (COI) [16,17]. On comparison of both these genes, it can be said that mitochondrial genes are more convenient and applicable because mt-DNA isolation is easier due to the presence of multiple copies in a cell; mt-DNA copies range from 100-10,000 per cell, and hence, a very small number of samples can be tested. Other reasons for its preference include more stability of mt-DNA and higher strength in comparison to nuclear DNA. mt-DNA is protected from degradation, even when it is exposed to prolonged environmental conditions.

PCR is a rapid and specific nucleic acid amplification method for the identification of pork species, and a number of PCR assays have been described for food identification. In spite of this, the PCR-based method continues to have some disadvantages, such as complicated operation and requirement of special heating cycle equipment. The iTPA technology was developed [18], and it proved to be a good DNA detection method that amplifies the target gene and the signal probe simultaneously under isothermal conditions, with a high specificity and sensitivity for foods [19- 21] and clinical specimens [22,23]. The iTPA technology relies on the strand displacement activity of DNA polymerase and the RNA degrading activity of RNase H, which specifically hydrolyzes the targeted DNA-RNA hybrid only.

In this study, a species-specific iTPA of mt-DNA method was developed for porcine species identification in meat products. The objective of this study was to develop a technique that could successfully be used in routine control assays to detect fraudulent adulteration/substitution of meat.


The 16S ribosomal RNA gene (GenBank AY920913) was used as the target for iTPA primers and probe design. Four primers, two outer and two inner, and one fluorescence resonance energy transfer (FRET) signal probe that recognized five regions of the target sequence were designed using DNASTAR software (Madison, WI) and synthesized by IDT (San Diego, CA). Oligonucleotide sequences and locations of the primers and the probe are presented in (Table 1). The basic local alignment search tool indicated a high level of specificity (no similarity to any other species). The 20 ul iTPA reaction mix consisted of 15 mM TrisHCl (pH 8.5), 22 mM MgSO4 , 15 mM KCl, 15 mM (NH4 )2 SO4 , 0.1 mg/ml acetylated bovine serum albumin, 6 mM dithiothreitol, 1.4 mM concentration of the deoxynucleoside triphosphates (dNTPs), 0.11 mM concentration of each outer primer, 1.1 mM concentration of each inner primer, 75 nM FRET signal probe, 4 units of Bst polymerase (NEB, Ipswich, MA), 5 units of RNase H (Epicentre, Madison, WI), 6 units of RNase inhibitor (Solgent, Daejeon, South Korea), and 10 ul of DNA template (replaced by 10 of sterilized water for the negative control). The iTPA reaction mix was incubated at 61 for 60 min in a heat block and then cooled to room temperature. After a quick spin-down, the reaction tube (0.2 ml PCR tube) was inserted into a Fluo-100 fluorometer (Allsheng, Hangzhou, China) to read the fluorescence. The F-score was calculated by the following equation:

F-score = [(fluorescence of the sample – fluorescence of the negative control) / fluorescence of the negative control] x 100



In this study, we designed a set of DNA–RNA–DNA chimeric primers and a FRET probe to specifically target the mitochondrial 16S ribosomal RNA gene for porcine species. Thus, a novel and rapid identification system has been developed. By simultaneously utilizing the dual amplification powers of the target DNA and the FRET probe, we have demonstrated that iTPA can be used to rapidly detect 0.01% of pork in meat samples. Four chimeric primers and one FRET probe were designed from five regions of the 16S ribosomal RNA gene for porcine species that are highly specific for pork meat (Table 1). The number of meat samples was tested on a rapid fluorescent detection platform and as a result, 100% inclusivity and 100% exclusivity for porcine species were detected (Figure 1). The iTPA assay is suitable for the identification of pork meat both in laboratories and in field situations because iTPA produces a fluorescence signal in the amplification microtube which is detected directly by any conventional portable fluorescent reader. As no postamplification handling is required for detection, it significantly reduces any cross-contamination risk caused by ampliconcarryover. Thus, the iTPA assay is more cost-effective and practical than both conventional PCR and real-time PCR assays.

In conclusion, we have developed a DNA detection system that can be conveniently used as it only requires a heat block and a fluorescence reader and has great potential in applications for hand-held or point of testing diagnostics. The 16S ribosomal RNA gene-based iTPA assay developed in this study is a specific, sensitive, and rapid method for the detection of porcine species in meat products. This simple method is expected to enable rapid screening for fraudulent adulteration and substitution of foods at a low cost.

Name Sequence (5’-3’) Position#
Outer forward
Outer reverse
Inner forward
Inner reverse
FRET probe
GenBank : AY920913
## Black hole quencher 1



1. Singh VP, Pathak V, Nayak NK, Akhilesh, Verma K, Umaraw P. Recent Developments in Meat Species Speciation-a review. J Livestock Sci. 2014; 5: 49-64.

2. Bhat, Heena Jalal, Parveez A. Para, Syed AB, Subha Ganguly, Asif A. Bhat, et al. Fraudulent Adulteration/Substitution of Meat: A Review. IJRRAS. 2015; 2: 22-33.

3. Liu L, Chen FC, Dorsey JL, Hsieh YHP. Sensitive monoclonal antibodybased sandwich ELISA for the detection of porcine skeletal muscle in meat and feed products. J Food Sci. 2006; 71: M1-M6.

4. Farouk AE, Batcha MF, Greiner R, Salleh HM, Salleh MR, Sirajudin AR. The use of a molecular technique for the detection of porcine ingredients in the Malaysian food market. Saudi Med J. 2006; 27: 1397-1400. 

5. Hopwood AJ, Fairbrother KS, Lockley AK, Bardsley RG. An actin generelated polymerase chain reaction (PCR) test for identification of chicken in meat mixtures. Meat Sci. 1999; 53: 227-231.

6. Fajardo V, Gonza´lez I, Mart?´n I, Rojas M, Herna´ndez PE, Garc?´a T, et al. Real- time PCR for detection and quantification of red deer (Cervus elaphus), fallow deer (Damadama), and roe deer (Capreolus capreolus) in meat mixtures. Meat Sci. 2008; 79: 289-298.

7. Erwanto Y, Abidin MZ, Sugiyono EY, Rohman A. Identification of pork contamination in meatballs of Indonesia local market using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Asian Australas. J Anim Sci. 2014; 27: 1487- 1492.

8. Kumar D, Singh SP, Karabasanavar NS, Singh R, Umapathi V. Authentication of beef, carabeef, chevon, mutton and pork by a PCRRFLP assay of mitochondrial cytb gene. J Food Sci Technol. 2014; 51: 3458-3463

9. Cho AR, Dong HJ, Cho S. Meat Species Identification using Loopmediated Isothermal Amplification Assay Targeting Species-specific Mitochondrial DNA. Korean J Food Sci Anim Resour. 2014; 34: 799- 807.

10. 10 Yang L, Fu S, Peng X, Li L, Song T, Li L. Identification of pork in meat products using real-time loop-mediated isothermal amplification. Biotechnol Biotechnol Equip. 2014; 28: 882-888.

11. Girish PS, Anjaneyulu ASR, Viswas KN, Santhosh FH, Bhilegaonkar KN, Agarwal R. K, et al. Polymerase chain reaction-restriction fragment length polymorphism of mitochondrial 12S rRNA gene: a simple method for identification of poultry meat species. Vet Res Commun. 2007; 31: 447-455.

12. Karlsson AO, Holmlund G, 2007. Identification of mammal species using species -specific DNA pyrosequencing. Forensic Science International. 2007; 173: 16-20.

13. Ergün Sakalar, Seyma Özçirak Ergün, Emine Akar A Simultaneous Analytical Method for Duplex Identification of Porcine and Horse in the Meat Products by EvaGreen based Real-time PCR. Korean J Food Sci Anim Resour. 2015; 35: 3382-388.

14. Karabasanavar NS, Singh SP, Kumar D, Shebannavar SN. Detection of pork adulteration by highly-specific PCR assay of mitochondrial D-loop. Food Chem. 2014; 45: 530-534.

15. Shin-Young Lee, Mi-Ju Kim, Yeun Hong, Hae-Y. Kim. Development of a rapid on-site detection method for pork in processed meat products using real-time loop-mediated isothermal amplification. J foodcont. 2016; 66: 53-61.

16. Kitpipit T, Sittichan K, Thanakiatkrai P. Direct-multiplex PCR assay for meat species identification in food products. Food Chem. 2014; 163: 77-82.

17. Dai Z, Qiao J, Yang S, Hu S, Zuo J, Zhu W, et al. Species Authentication of Common Meat Based on PCR Analysis of the Mitochondrial COI Gene. Appl Biochem Biotechnol. 2015; 176: 1770-1780.

18. Jung C, Chung JW, Kim UO, Kim MH, Park HG. An isothermal target and probe amplification (iTPA) method, based on a combination of an isothermal chain amplification (ICA) technique and a FRET cycling probe technology (CPT). Anal Chem. 2010; 82: 5937-5943.

19. Kim JS, Jahng MS, Lee GG, Lee KJ, Chae HK, Lee JH, et al. Rapid and simple detection of the invA gene in Salmonella spp. by isothermal target and probe amplification (iTPA). Lett Appl Microbiol. 2011; 52: 399-405.

20. Shin H, Kim M, Yoon E, Kang G, Kim S, Song A, et al. Isothermal target and probe amplification assay for the real-time rapid detection of Staphylococcus aureus. J Food Prot. 2015; 78: 723-727.

21. Menghan Zhang, Chengang Teng, Hyewon Shin, Qiang Shen, Joonhyuk Choi and Jeongsoon Kim. Real-Time Detection of Campylobacter jejuni in Food by Isothermal Target and Probe Amplification, J Hum Nutr Food Sci. 2016; 4: 1083.

22. Moon HW, Hur M, Kim H, Kim JY, Park CM, Yun YM. Yun Isothermal target and probe amplification for Mycobacterium tuberculosis identification from broth cultures. Int J Tuberc Lung Dis. 2012; 16: 516-520.

23. Kim H, Lee J, Kwon HJ, Kim KH, Yeo CD, Kim JW, et al. Evaluation of Isothermal Target and Probe Amplification (iTPA) for the Detection of Mycobacterium Tuberculosis. Ann Clin Lab Sci. 2016; 46: 529-536

Received : 19 Jul 2017
Accepted : 30 Aug 2017
Published : 31 Aug 2017
Annals of Otolaryngology and Rhinology
ISSN : 2379-948X
Launched : 2014
JSM Schizophrenia
Launched : 2016
Journal of Nausea
Launched : 2020
JSM Internal Medicine
Launched : 2016
JSM Hepatitis
Launched : 2016
JSM Oro Facial Surgeries
ISSN : 2578-3211
Launched : 2016
Journal of Human Nutrition and Food Science
ISSN : 2333-6706
Launched : 2013
JSM Regenerative Medicine and Bioengineering
ISSN : 2379-0490
Launched : 2013
JSM Spine
ISSN : 2578-3181
Launched : 2016
Archives of Palliative Care
ISSN : 2573-1165
Launched : 2016
JSM Nutritional Disorders
ISSN : 2578-3203
Launched : 2017
Annals of Neurodegenerative Disorders
ISSN : 2476-2032
Launched : 2016
Journal of Fever
ISSN : 2641-7782
Launched : 2017
JSM Bone Marrow Research
ISSN : 2578-3351
Launched : 2016
JSM Mathematics and Statistics
ISSN : 2578-3173
Launched : 2014
Journal of Autoimmunity and Research
ISSN : 2573-1173
Launched : 2014
JSM Arthritis
ISSN : 2475-9155
Launched : 2016
JSM Head and Neck Cancer-Cases and Reviews
ISSN : 2573-1610
Launched : 2016
JSM General Surgery Cases and Images
ISSN : 2573-1564
Launched : 2016
JSM Anatomy and Physiology
ISSN : 2573-1262
Launched : 2016
JSM Dental Surgery
ISSN : 2573-1548
Launched : 2016
Annals of Emergency Surgery
ISSN : 2573-1017
Launched : 2016
Annals of Mens Health and Wellness
ISSN : 2641-7707
Launched : 2017
Journal of Preventive Medicine and Health Care
ISSN : 2576-0084
Launched : 2018
Journal of Chronic Diseases and Management
ISSN : 2573-1300
Launched : 2016
Annals of Vaccines and Immunization
ISSN : 2378-9379
Launched : 2014
JSM Heart Surgery Cases and Images
ISSN : 2578-3157
Launched : 2016
Annals of Reproductive Medicine and Treatment
ISSN : 2573-1092
Launched : 2016
JSM Brain Science
ISSN : 2573-1289
Launched : 2016
JSM Biomarkers
ISSN : 2578-3815
Launched : 2014
JSM Biology
ISSN : 2475-9392
Launched : 2016
Archives of Stem Cell and Research
ISSN : 2578-3580
Launched : 2014
Annals of Clinical and Medical Microbiology
ISSN : 2578-3629
Launched : 2014
JSM Pediatric Surgery
ISSN : 2578-3149
Launched : 2017
Journal of Memory Disorder and Rehabilitation
ISSN : 2578-319X
Launched : 2016
JSM Tropical Medicine and Research
ISSN : 2578-3165
Launched : 2016
JSM Head and Face Medicine
ISSN : 2578-3793
Launched : 2016
JSM Cardiothoracic Surgery
ISSN : 2573-1297
Launched : 2016
JSM Bone and Joint Diseases
ISSN : 2578-3351
Launched : 2017
JSM Bioavailability and Bioequivalence
ISSN : 2641-7812
Launched : 2017
JSM Atherosclerosis
ISSN : 2573-1270
Launched : 2016
Journal of Genitourinary Disorders
ISSN : 2641-7790
Launched : 2017
Journal of Fractures and Sprains
ISSN : 2578-3831
Launched : 2016
Journal of Autism and Epilepsy
ISSN : 2641-7774
Launched : 2016
Annals of Marine Biology and Research
ISSN : 2573-105X
Launched : 2014
JSM Health Education & Primary Health Care
ISSN : 2578-3777
Launched : 2016
JSM Communication Disorders
ISSN : 2578-3807
Launched : 2016
Annals of Musculoskeletal Disorders
ISSN : 2578-3599
Launched : 2016
Annals of Virology and Research
ISSN : 2573-1122
Launched : 2014
JSM Renal Medicine
ISSN : 2573-1637
Launched : 2016
Journal of Muscle Health
ISSN : 2578-3823
Launched : 2016
JSM Genetics and Genomics
ISSN : 2334-1823
Launched : 2013
JSM Anxiety and Depression
ISSN : 2475-9139
Launched : 2016
Clinical Journal of Heart Diseases
ISSN : 2641-7766
Launched : 2016
Annals of Medicinal Chemistry and Research
ISSN : 2378-9336
Launched : 2014
JSM Pain and Management
ISSN : 2578-3378
Launched : 2016
JSM Women's Health
ISSN : 2578-3696
Launched : 2016
Clinical Research in HIV or AIDS
ISSN : 2374-0094
Launched : 2013
Journal of Endocrinology, Diabetes and Obesity
ISSN : 2333-6692
Launched : 2013
Journal of Substance Abuse and Alcoholism
ISSN : 2373-9363
Launched : 2013
JSM Neurosurgery and Spine
ISSN : 2373-9479
Launched : 2013
Journal of Liver and Clinical Research
ISSN : 2379-0830
Launched : 2014
Journal of Drug Design and Research
ISSN : 2379-089X
Launched : 2014
JSM Clinical Oncology and Research
ISSN : 2373-938X
Launched : 2013
JSM Bioinformatics, Genomics and Proteomics
ISSN : 2576-1102
Launched : 2014
JSM Chemistry
ISSN : 2334-1831
Launched : 2013
Journal of Trauma and Care
ISSN : 2573-1246
Launched : 2014
JSM Surgical Oncology and Research
ISSN : 2578-3688
Launched : 2016
Journal of Radiology and Radiation Therapy
ISSN : 2333-7095
Launched : 2013
JSM Physical Medicine and Rehabilitation
ISSN : 2578-3572
Launched : 2016
Annals of Clinical Pathology
ISSN : 2373-9282
Launched : 2013
Annals of Cardiovascular Diseases
ISSN : 2641-7731
Launched : 2016
Journal of Behavior
ISSN : 2576-0076
Launched : 2016
Annals of Clinical and Experimental Metabolism
ISSN : 2572-2492
Launched : 2016
Clinical Research in Infectious Diseases
ISSN : 2379-0636
Launched : 2013
JSM Microbiology
ISSN : 2333-6455
Launched : 2013
Journal of Urology and Research
ISSN : 2379-951X
Launched : 2014
Journal of Family Medicine and Community Health
ISSN : 2379-0547
Launched : 2013
Annals of Pregnancy and Care
ISSN : 2578-336X
Launched : 2017
JSM Cell and Developmental Biology
ISSN : 2379-061X
Launched : 2013
Annals of Aquaculture and Research
ISSN : 2379-0881
Launched : 2014
Clinical Research in Pulmonology
ISSN : 2333-6625
Launched : 2013
Journal of Immunology and Clinical Research
ISSN : 2333-6714
Launched : 2013
Annals of Forensic Research and Analysis
ISSN : 2378-9476
Launched : 2014
JSM Biochemistry and Molecular Biology
ISSN : 2333-7109
Launched : 2013
Annals of Breast Cancer Research
ISSN : 2641-7685
Launched : 2016
Annals of Gerontology and Geriatric Research
ISSN : 2378-9409
Launched : 2014
Journal of Sleep Medicine and Disorders
ISSN : 2379-0822
Launched : 2014
JSM Burns and Trauma
ISSN : 2475-9406
Launched : 2016
Chemical Engineering and Process Techniques
ISSN : 2333-6633
Launched : 2013
Annals of Clinical Cytology and Pathology
ISSN : 2475-9430
Launched : 2014
JSM Allergy and Asthma
ISSN : 2573-1254
Launched : 2016
Journal of Neurological Disorders and Stroke
ISSN : 2334-2307
Launched : 2013
Annals of Sports Medicine and Research
ISSN : 2379-0571
Launched : 2014
JSM Sexual Medicine
ISSN : 2578-3718
Launched : 2016
Annals of Vascular Medicine and Research
ISSN : 2378-9344
Launched : 2014
JSM Biotechnology and Biomedical Engineering
ISSN : 2333-7117
Launched : 2013
Journal of Hematology and Transfusion
ISSN : 2333-6684
Launched : 2013
JSM Environmental Science and Ecology
ISSN : 2333-7141
Launched : 2013
Journal of Cardiology and Clinical Research
ISSN : 2333-6676
Launched : 2013
JSM Nanotechnology and Nanomedicine
ISSN : 2334-1815
Launched : 2013
Journal of Ear, Nose and Throat Disorders
ISSN : 2475-9473
Launched : 2016
JSM Ophthalmology
ISSN : 2333-6447
Launched : 2013
Journal of Pharmacology and Clinical Toxicology
ISSN : 2333-7079
Launched : 2013
Annals of Psychiatry and Mental Health
ISSN : 2374-0124
Launched : 2013
Medical Journal of Obstetrics and Gynecology
ISSN : 2333-6439
Launched : 2013
Annals of Pediatrics and Child Health
ISSN : 2373-9312
Launched : 2013
JSM Clinical Pharmaceutics
ISSN : 2379-9498
Launched : 2014
JSM Foot and Ankle
ISSN : 2475-9112
Launched : 2016
JSM Alzheimer's Disease and Related Dementia
ISSN : 2378-9565
Launched : 2014
Journal of Addiction Medicine and Therapy
ISSN : 2333-665X
Launched : 2013
Journal of Veterinary Medicine and Research
ISSN : 2378-931X
Launched : 2013
Annals of Public Health and Research
ISSN : 2378-9328
Launched : 2014
Annals of Orthopedics and Rheumatology
ISSN : 2373-9290
Launched : 2013
Journal of Clinical Nephrology and Research
ISSN : 2379-0652
Launched : 2014
Annals of Community Medicine and Practice
ISSN : 2475-9465
Launched : 2014
Annals of Biometrics and Biostatistics
ISSN : 2374-0116
Launched : 2013
JSM Clinical Case Reports
ISSN : 2373-9819
Launched : 2013
Journal of Cancer Biology and Research
ISSN : 2373-9436
Launched : 2013
Journal of Surgery and Transplantation Science
ISSN : 2379-0911
Launched : 2013
Journal of Dermatology and Clinical Research
ISSN : 2373-9371
Launched : 2013
JSM Gastroenterology and Hepatology
ISSN : 2373-9487
Launched : 2013
Annals of Nursing and Practice
ISSN : 2379-9501
Launched : 2014
JSM Dentistry
ISSN : 2333-7133
Launched : 2013
Author Information X