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. 2020 Jul 18;57(11):4286–4292. doi: 10.1007/s13197-020-04641-w

Detecting mislabelling in meat products using PCR–FINS

Manju Soman 1,✉, Robin J Paul 1, Mini Antony 1, Soumya Padinjarattath Sasidharan 1
PMCID: PMC7520481  PMID: 33071350

Abstract

Economically motivated adulteration (EMA) or misrepresentation of meat products is of concern, especially in developing countries, due to obvious health hazards and religious sensitivities. As Indian cooking involves prolonged heat treatments and addition of spices and condiments, species authentication of food, especially meat products, may be challenging. This study evaluated the efficacy of Polymerase Chain Reaction-Forensically Informative Sequencing (PCR-FINS) in meat speciation of highly processed meat. Further the prevalence of mislabelling in processed and deeply cooked meat products being sold in supermarkets and restaurants in a south Indian city was investigated. FINS targeting the mitochondrial cytochrome b gene and the ATP synthase gene was applied to identify meat species of 106 meat products labelled as chicken, beef, carabeef, mutton and pork. Mislabelling was detected in more than half of mutton (52.3%) and carabeef (55.5%), and in under a third (27.2%) of beef products. PCR-FINS is a reliable method for meat species identification even in highly processed food but there is a need for appropriate universal primers which can target all common species used in meat products. This study is the first of its kind from the South Indian state of Kerala.

Electronic supplementary material

The online version of this article (10.1007/s13197-020-04641-w) contains supplementary material, which is available to authorized users.

Keywords: Meat species authentication, PCR–FINS, NCBI BLAST, Mislabelling, Phylogenetic tree

Introduction

India, ranked fourth in the world in meat production, produced about 9.2 million metric tons of meat (chicken, beef and carabeef) in the year 2019 and is the third largest exporter of (boneless) carabeef and beef (USDA 2020). India has a total meat processing capacity of over 1 million tons per annum and the country has exported 405.64 metric tons of processed meat during the year 2018–19 (APEDA 2020). Indian cuisine is eclectic, highly influenced by region, religion and ethnicity. The addition of a variety of spices and herbs result in distinctive tastes and strong flavours. Traditional products such as Kebab, Korma and Biriyani are popular in national and international markets, as are European meat products such as sausages, ham and smoked meat. As people are becoming increasingly conscious of food safety, traceability and authenticity, accurate labelling and description of food becomes crucial especially in highly processed food. Although EMA is likely to be more prevalent in developing countries due to the large populations, poverty and rising prices, the problem is not unique to the third world. Examples of meat adulteration reported from rich countries include the British and European ‘horse meat scandal’ of 2013. Such adulteration may lead to health hazards associated with exposure to drug residues such as phenyl butazone in horse meat, and allergens, bacteria and parasites in porcine meat (Gamble 1997). In India, it is also a cause for cultural concern in view of religious taboos attached to certain meats.

Meat speciation is technically challenging, especially for extensively processed products. This challenge can be best met by DNA based techniques such as Southern Blotting with species-specific satellite DNA probes, Random Amplified Polymorphic DNA fingerprinting (RAPD), PCR–Restriction Fragment Length Polymorphism (PCR–RFLP), Multiplex PCR and PCR–FINS (Matsunaga et al. 1999; Lin et al. 2005; Aida et al. 2005; Minarovič et al. 2010; Li et al. 2011; Sakalar et al. 2015).

The PCR–FINS technique involves PCR amplification of a highly conserved gene sequence followed by nucleotide sequencing and analysis using NCBI BLAST (Basic Local Alignment Search Tool). FINS has been reported to be a definitive approach to molecular identification of biological materials. The success of FINS depends on accurate selection of the DNA region and reference species (Li et al. 2011). The genes commonly targeted are mitochondrial cytochrome b gene (cyt b) (Lago et al. 2011), cytochrome c oxidase subunit 1 gene (CO1) (Haider et al. 2012), rRNA genes (5S, 12S, 16S, 18S) (Girish et al. 2004; Karabasanavar et al. 2010) and d-loop region (Karabasanavar et al. 2011). Of these, mitochondrial cyt b gene and CO1 gene are the most preferred for speciation and phylogenetic studies. Bartlett and Davidson (1992) and Lago et al. (2011) have reported FINS as a reliable method for species identification of processed meat.

Indian food processing industry holds promise for Indian farmers in aiding the export of surplus agro-products. Animal product export contributes significantly to the Indian economy and as demand for Indian meat products in international markets has increased, authentication and traceability of meat products has become increasingly vital. The Food Safety Standards Authority of India (FSSAI) has imposed new laws on labelling and packaging of food products. The current study investigates mislabelling in meat products in a southern Indian city, using PCR–FINS targeting the mitochondrial cyt b gene and the ATP synthase gene, to identify meat of domestic cattle, buffalo, goat/sheep, swine and chicken.

Methods

Meat sampling—Various branded meat products were purchased from local retailers and restaurants in Ernakulam, India. One hundred and six meat products labelled as cattle (Bos taurus/ Bos indicus) (22), buffalo (Bubalus bubalis) (9), goat (Capra hircus) (11), sheep (Ovis aries) (10), pig (Sus scrofa) (11) and chicken (Gallus gallus) (43) were analysed. The products included uncooked or partially cooked Indian products such as meat balls, cutlets, salami, samosa, kebab, keema, minced meat, smoked meat, curry cuts and deeply cooked products such as curry, biriyani and pickles and western products such as sausages, meat popcorns, nuggets, burger patties, meat ham fingers, spring rolls, meat kieves and Frankfurters. Frozen cattle, buffalo, goat, sheep, chicken and pig meat samples were used as controls. The samples were stored at − 20 °C immediately after collection, until analysis. All the branded meat products were tested as blind samples in the laboratory.

DNA extraction and PCR amplification

Two commercially available DNA extraction kits Gen Elute Mammalian genomic DNA Mini Prep Kit (Sigma) and Pure link Genomic DNA Kit (Invitrogen) were used to extract DNA.

Universal primers, L14724-5′CGAAGCTTGATATGAAAAACCATCGTTG-3 (Irwin et al.1991) and H15149-5′AAACTGCAGCCCCTCAGAATGATATTTGTCCTC-3′ (Kocher et al.1989) were used to amplify a 486 bp nucleotide fraction of the cyt b gene of cattle, buffalo, goat/sheep and pig, as described by Hsieh et al. (2005). The PCR reaction was performed in a final volume of 50 µl containing 2.5units of Taq polymerase, 2.5 mM MgCl2, and 200 µM dNTP cycle and 8 pmol of each oligonucleotide primer. The cycle comprised initial denaturation cycle at 95 °C for 10 min followed by 35 cycles of denaturation (95 °C for 45 s), annealing (50 °C for 45 s) and extension (72 °C for 90 s), followed by a final extension cycle of 72 °C for 10 min.

Chicken DNA was amplified using species specific primers designed for ATP Synthase F0 Subunit 8 gene partial CDS of chicken viz., F 5′-GGGACACCCTCCCCCTTAATGACA-3′ and R 5′-GGAGGGCTGGAAGAAGGAGTG-3′ as per Doosti et al. (2014). The PCR reaction was performed in a final volume of 50 µl containing 2 units of Taq polymerase, 2 mM MgCl2, and 200 µM dNTP cycle and 25 pmol of each oligonucleotide primer. The PCR cycle comprised initial denaturation at 94 °C for 5 min, 30 cycles of denaturation (94 °C for 1 min), annealing (69 °C for 1 min), extension (72 °C for 1 min) and final extension (72 °C for 6 min).

The primers were custom synthesised from IDT, USA and the reactions were carried out in an Eppendorf Gradient S Mastercycler thermocycler.

Species identification and nucleotide sequence analysis

The amplified DNA products were sequenced at the DNA sequencing facility at Agrigenome, Kakkanad, Cochin by the dideoxy-chain-termination method (Sanger et al. 1977), analysed using NCBI BLAST, and submitted to the GenBank database. A 402 bp sequence, numbering from 14747 to 15148 bp of the human mitochondrial DNA, was used for sequence comparison. Sequences with maximum similarity were spotted to ascertain the identity of the meat DNA to the nearest related species. Representative nucleotide sequences of each species, from the test samples as well as from GenBank, were used to investigate the phylogeny using the MEGA 6.06 software. Sequence alignment was done by ClustalW method in the BioEdit software and a Neighbour-Joining phylogenetic tree/ dendrogram was constructed. The degree of confidence assigned to the nodes in the phylogenetic tree was estimated by bootstrapping with 1000 replicates and the evolutionary distance was computed using the Kimura 2-parameter method and P-distance method.

Results

Both DNA extraction kits yielded intact DNA (Figs. 1 and 2) from the meat products. The chosen universal primers successfully amplified the 486 bp fragment of the mitochondrial cyt b gene of cattle, goat, sheep, pig and buffalo (Fig. 3). Even though distinct amplicons were detected at the 486 bp level on amplification of chicken DNA, these could not be accurately sequenced. The species-specific primers, for ATP Synthase F0 Subunit 8 gene partial CDS of chicken, amplified the 266 bp fragment of the chicken DNA (Fig. 4) which could be sequenced.

Fig. 1.

Fig. 1

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Isolated DNA of cattle and buffalo samples on agarose gel. Lane 1—positive control (raw beef DNA), Lane 2 to Lane 8—DNA of BF22, Bf18, Bf21, Bf20, Bf19, B7, B8, Lane 9—negative control, Lane 10—1 kbp ladder

Fig. 2.

Fig. 2

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Isolated DNA of cattle, buffalo, chicken, sheep, goat and pig samples on agarose gel. Lane 1—100 bp ladder, Lane 2 to Lane 7—DNA of BF1, B6, C1, G3, G1 and P1

Fig. 3.

Fig. 3

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Amplified products of cyt b gene partial CDS of different samples using universal primers. Lane 1—positive control (raw beef amplicon), Lane 2 to Lane 9—amplicons of samples Bf 19, Bf 20, Bf 21, B7, B8, P11, B 9 and B 22. Lane 10—100 bp DNA ladder, Lane 11—Negative control

Fig. 4.

Fig. 4

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Amplification of ATP Synthase F0 Subunit 8 gene partial CDS of chicken. Lane 1—100 bp ladder, Lane 2 to Lane 5—Amplicons of samples C1, C2, C3 and C4

The following GenBank accession numbers have been assigned to the 90 nucleotide sequences submitted to GenBank.

The results of the present study described in Tables 1 and Supplementry Table 2 reveal high percentage of species misrepresentation in Carabeef, Beef and Mutton products. Out of the 43 chicken products, 41 products were amplified and sequenced. All the 41 sequences (95.34%) showed 100% BLAST identity to Gallus gallus species. Out of 22 beef products, mislabelling was detected in six (27.27%). The six products showed 97–100% BLAST identity to Bubalus bubalis. The rest of the 16 products showed 99 to100% identity to Bos genus which included Bos indicus (15) and Bos Taurus (1). Of the 9 buffalo products, 5 (55.55%) were identified as beef. These 5 products showed 99 to 100% sequence identity to Bos genus which included Bos indicus (3), Bos taurus (1) and Bos grunniens (1). The rest 4 showed identity to Bubalus bubalis. Of the 21 mutton labelled products, mislabelling was detected in 11 (52.37%) products. Of this, one product (4.76%) viz., raw meat previously suspected of mislabelling showed 100% identity to Bos indicus. Ten products (47.61%) showed 99–100% identity to Capra hircus. The rest of the 10 products showed 99–100% sequence identity to Ovis aries. Of the 11 pork labelled products, all 11 showed 99–100% identity to Sus scrofa.

Table 1.

Species identified and percentage of mislabeling detected in the meat products

Processed meat tested No. of samples Species identified
by BLAST		 Number of species identified by BLAST % Of identified species % Mislabelling
Chicken 43 Gallus gallus 41 95.34 4.65 (Not identified by species specific PCR)
Beef 22 Bos indicus 15 68.18 27.27 Beef mislabelled as carabeef
Bos taurus 1 4.54
Bubalus bubalis 6 27.27
Carabeef 9 Bubalus bubalis 4 44.44 55.55 Carabeef mislabelled as beef\
Bos indicus 3 33.33
Bos taurus 1 11.11
Bos grunniens 1 11.11
Mutton 21 Ovis aries 10 47.61 52.37 mislabelling (47.61% chevon and 4.76% beef)
Capra hircus 10 47.61
Bos indicus 1 4.76
Pork 11 Sus scrofa 11 100 0
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The words shown in bold are names of the different species that was detected in the mislabelled meat products

The dendrogram for mammalian species displayed 2 clusters, one representing the ruminant species viz., Bos indicus, Bubalus bubalis, Capra hircus and Ovis aries and the other representing the porcine species Sus scrofa. Within the first cluster, sequences related to Capra hircus and Ovis aries were clustered separately from Bos indicus and Bubalus bubalis with a common ancestor. The dendrogram for chicken displayed two clusters both genetically related to Gallus gallus with a common ancestor.

Discussion

The food supply chain today is a complex and dynamic global network. Food safety and traceability are particularly important in processed foods where ingredients may be indiscernible. Indian cooking involving high temperatures and addition of several spices and condiments may lead to low DNA concentrations (Sakalar et al. 2012). Hence the major challenge in speciation of meat products by genomic methods is extracting intact DNA. Ahmed et al. (2018) reported the inability to extract and amplify DNA from pickled products. In this study 2 products labelled as chicken samosa and chicken pickle could not be amplified by species specific PCR. But as all other pickled and samosa products had identifiable DNA, it may be safely assumed that these 2 products did not contain chicken DNA. Compared to nuclear DNA, mitochondrial DNA is protected by a double layered mitochondrial membrane and better resists processing injury and thus a superior choice for speciation studies. Mitochondrial DNA undergoes several mutations within its sequences thus facilitating unique identification of different species (Linacre and Lee, 2005). This study targeted the mitochondrial cyt b and ATP synthase genes for speciation. Universal primers, L14724 and H15149 for the 486 bp fragment of mitochondrial cyt b gene were chosen based on their reported efficacy in identifying DNA from highly degraded specimens (Hseih et al. 2001). This primer pair identified DNA of all mammalian species but failed to identify chicken DNA. Chicken products were identified using species specific primers for a 266 bp fragment of ATP Synthase gene partial CDS. The primer pair LCO1490 and HCO2198 for mitochondrial cytochrome C oxidase subunit 1(CO1) gene fragment, commonly referred to as the DNA barcode is regarded as gold standard for meat species identification (Botti and Giuffra 2010). Haider et al. (2012) used these primers to identify meat of cow, chicken, turkey, pig, sheep, buffalo, camel and donkey using PCR RFLP.

More than 95% of chicken products showed 99–100% identity to Gallus gallus. But as species-specific primers were used to amplify chicken DNA, the results only indicate the presence or absence of chicken DNA in these products. Poultry is the most consumed meat in India due to relatively lower prices, religious restrictions on beef and pork, and limited availability of fish in non-coastal areas. But processed poultry products make up only 2–3 percent of the total meat produced in India mainly because Indian consumers prefer freshly slaughtered chicken meat. (Mehta and Nambiar 2007).

All the 11 pork products showed 99–100% identity to Sus scrofa. While 27.2% of beef products were identified as carabeef, 55.5% of buffalo products, were identified as beef. Since many Indian states have imposed a ban on cattle (Bos indicus/Bos taurus) slaughter and beef consumption, the carabeef sector now constitutes the bulk of the red-meat industry—depending primarily on unproductive water buffaloes and water-buffalo bulls from the dairy sector (Intodia 2017). In 2012, the buffalo population in India and Kerala was estimated at 108.7 million and 0.1 million respectively while cattle population (Bos indicus/Bos taurus) was 190.9 million and 1.27 million respectively (NDDB 2016). This relatively small buffalo population in a time of high demand for buffalo meat in the current scenario may be one of the reasons for such adulteration or mislabelling.

More than 50% of mutton products showed mislabelling. Of this, one raw-meat sample (4.8%) showed 100% identity to Bos indicus. This appears to validate known consumer concerns on the authenticity of this product. Such adulteration may be common as veal is cheaper than mutton and chevon. Veal when soaked in goat milk and cooked, is believed to have a texture and smell similar to mutton and chevon. Nearly 50% of products labelled as mutton were found to be chevon (goat meat) with 99–100% identity to Capra hircus. In Kerala, both goat meat and sheep meat are called ‘mutton’ in the vernacular and this may partly explain the inaccurate labelling of goat products as mutton.

The FINS technique facilitated the study of phylogeny of the different species used in the local meat processing industry. BLAST was able to reveal, to an extent, the animal breeds that were used in the meat products.

A major drawback of FINS using Sanger based DNA sequencing is that it can identify only the predominant species of a mixed meat product and fails to describe the species composition of mixed food. Hence the methods used in this study could indicate only the predominant species present in the meat product and could not have detected lower-level adulterations. Newer high throughput Next Generation Sequencing (NGS) techniques such as Illumina, 454 pyrosequencing and Ion Torrent, may detect even low-represented species, in highly processed multispecies products through a metagenomic approach (Bertolini et al. 2015; Giusti et al.2017).

Conclusion

This pilot study on species authentication of meat products in Kerala reports varying degrees of mislabelling in meat products. PCR–FINS proves to be a reliable test for species identification even in highly processed meat. The primer pair L14724 and H15149 effectively identified mammalian DNA but failed to identify avian DNA. Universal primers that identify all food animal and bird DNA may be best suited for speciation studies. Also, a metagenomic approach using NGS technology may be appropriate for meat product authentication. EMA is a challenging problem as most adulterants are indiscernible, and novel adulterants are being introduced to escape newer detection techniques. This demand better regulatory systems and appropriate detection methods.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file 1 (DOCX 29 kb) (29.4KB, docx)
Supplementary file 2 (DOCX 22 kb) (23KB, docx)

Acknowledgements

The authors would like to thank Dr. N.N. Sasi, Director Animal Husbandry department, Kerala for his support in conducting the study. The authors would like to thank Neema Job for her contributions to the study.

Funding

This work was funded by the Department of Animal Husbandry, Govt of Kerala.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal rights

This article does not contain any studies with human or living animal subjects.

Footnotes

Publisher's Note

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Supplementary file 1 (DOCX 29 kb) (29.4KB, docx)
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