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Detecting adulterants in food

Dr Monee Shamsher & Dr Angus Knight of Leatherhead Food Research report on the progress within next generation sequencing (NGS) in authenticity screening and its practical and analytical limitations.

Food fraud and adulteration has existed for centuries. Well documented recent cases include Spanish Toxic Oil Syndrome and Sudan I. However, the horsemeat scandal of 2013 provoked a widespread distrust in the traceability of foods and food ingredients and raised the profile of food adulteration. In response to this, the British Retail Consortium (BRC) introduced vulnerability assessment within Global Standard Food Safety Issue 7; this requires companies to assess and annually review the vulnerability of raw materials to adulteration within the supply chain. Vulnerability assessment requires and is supported by a requirement to use the most suitable test methods.

Limitations of traditional detection methods

Authenticity testing seeks to confirm that all ingredients claimed on a product label are present in that product. It is an essential part of vulnerability assessment and can provide due diligence defence and deterrence value in supporting quality assurance. In reality, for fraud to be economically viable, adulteration is anticipated to be present at a significant level (>15% w/w). Quantification of any adulteration in processed foods will always present problems since there are no universal standards for complex food matrices and the relationship between DNA measurement and meat or fish content based upon protein measurement and nitrogen factors is not known. There are many sources of measurement uncertainty in molecular methods for food authentication[1].

To date, testing for meat and fish speciation has generally focused on the use of protein based ELISA assays or DNA based PCR assays. ELISA assays can provide a fast dipstick test particularly suitable for on-site use with raw materials with a limit of detection of 1% w/w protein. PCR based assays are available in different laboratory based formats. Quantitative Real-Time PCR (qPCR) allows the detection and quantification of target DNA fragments. Different quantitative PCR assays have been developed to distinguish between various amounts of species in processed and canned foods and for foods derived from genetically modified crops.

For most analytical formats, the use of ELISA or PCR based testing is a targeted approach requiring a ‘guesstimate’ at which adulterant may be present. Similar to ELISA assays, qPCR is a non-targeted approach also requiring a ‘guesstimate’ of what might be present. This limits the use of qPCR as a screening tool for detecting unknown and unsuspected adulterants. Furthermore, real-time qPCR is carried out under relatively nonspecific amplification conditions and is reliant on fluorescence based detection leading to concerns over false positive results, especially when DNA is extracted from processed food matrices. Both ELISA and qPCR fail to spot unsuspected adulteration or fraud. Hence, to date,

food and beverage manufacturers have been hampered in their attempts to detect food fraud by tests which can only confirm or deny known adulterants."

Searching for unknown contaminants can be like looking for a needle in a haystack.

Detecting any species

For many years, DNA sequencing was predominantly reliant on the sequencing method developed by Frederick Sanger and colleagues in 1977, which provided the majority of sequence data for the next 25 years. Since then, new sequencing methods have been developed providing more rapid determination of DNA sequences and approaches to quantitative sequence analysis – these are collectively known as ‘next generation’ sequencing technologies (NGS).

NGS has two very significant advantages over current ELISA or real-time PCR approaches for the identification of DNA sequences. Firstly the approach is non-targeted, allowing the identification of any species that might be present. Secondly, the approach is self-validating and generates data that can be directly compared with database sequences leading to greater confidence in species identification.

Sequencing of selected PCR amplified regions of DNA is usually directed at mitochondrial or chromosomal genes. When sufficient DNA of good quality can be extracted, NGS can allow sequencing of all DNA sequences present directly from a sample; this is known as deep sequencing. Recent data[2] has demonstrated the feasibility of the application of this approach for the quantitative identification of plant, animal and bacterial DNA in food. At present this analysis is too complex and costly for food laboratories but shows promise for the future. Intriguingly the data showed some of the hidden dangers of sequence analysis finding sequence matches with human, mouse and whale DNA due to redundant ancient fragments of DNA or to nuclear mitochondrial translocations (NUMTs) that can complicate DNA analysis[3].

Mitochondrial sequences are present in multiple copies in every cell which results in increased sensitivity and is therefore preferred for highly processed foods e.g. canned foods or gelatin identification. Typical NGS sequencing approaches use PCR based amplification using conserved flanking primers to amplify species specific variable intervening sequences, which are then sequenced in thousands of individual sequencing reactions. This metagenomics approach allows non-targeted analysis, so that guessing which species might be present is not required. Individual sequence reads are compiled and assembled using software tools and compared with database sequences to identify the species present.

NGS sequencing platforms currently range from those designed for de novo whole genome sequencing (WGS) to those for resequencing and particularly for sequences of interest identified by WGS. WGS is currently very costly to purchase, run and maintain for routine applications in the food industry and is presently not quantitative. Different platforms for WGS have been reviewed[4]. WGS generates large amounts of complex data requiring expert data interpretation. Alternative NGS platforms, such as pyrosequencing for resequencing, allow resequencing of the same DNA sequence from different sources in order to identify specific mutations or instances of adulteration.

DNA based test for adulterants

Recognising the food industry’s need for a test which looks for evidence of any type of adulterant in a food sample in a timely and cost effective way, Leatherhead has developed the Adulteration and SPECiation Test or ASPECT. It combines highly efficient DNA extraction with non-targeted pyrosequencing.

ASPECT characteristics:
  • non-targeted,
  • applicable to meat, poultry, fish,herbs and spices and highly processed and canned foods,
  • detection limit of 1% w/w adulterant DNA from the authentic species,
  • rapid turnaround

Pyrosequencing[5,6], is a rapid NGS technology resulting in quantitative sequence data in the form of a sequence profile. Pyrosequencing is also used as an NGS sequencing chemistry for WGS. Both peak height and peak position correspond to sequence data at different nucleotide positions and can be used to determine the species of origin and the presence of admixtures. Pyrosequencing for food authentication was first described in this laboratory for plant speciation by Ortola and colleagues in 2007[7]. Since then the method has been refined and a number of different assays for meat, poultry and fish speciation have been developed. Examples of different fish sequence profiles are shown in Figure 1. The accuracy of the method is such that the ASPECT assay has been able to identify the source of gelatin in sweets (Figure 2).

Figure 1: Example of Pyrograms showing variation in sequence data for different fish
100% Atlantic cod reference sample

Figure 2. Identification of gelatine sources from sweets using ASPECT
100% Beef Gelatine

ASPECT provides semiquantitative analysis by comparing peak height and positions. Pyrosequencing profiles can also be generated in silico and used to model mixtures allowing the resolution of mixtures of different species. ASPECT assays can therefore resolve unknown and unsuspected DNA mixtures providing an extremely versatile tool for food authentication and a practical tool for manufacturers in their battle against food fraud. Next generation sequencing (NGS) holds the key to nontargeted testing, thus enabling detection of unknown and unsuspected adulterants.


1. Burns, M., Wiseman, G., Knight, A., Bramley, P., Foster, L., Rollinson, S., Damant, A., Primrose, S. (2016) Measurement issues associated with quantitative molecular biology analysis of complex food matrices for the detection of food fraud. Analyst 141: 45-61

2. Ripp, F., Krombholz, F., Liu, Y., Weber, M., Schafer, A., Schmidt, B., Koppel, R., Hanklen, T. (2014) All-Food-Seq (AFS): a quantifiable screen for species in biological samples by deep DNA sequencing. BMC Genomics 15:639

3. Song, H., Buhay, J.E., Whiting, M.F., Crandall, K.A. (2008) Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are co-amplified.

4. Quail, M.A., Smith, M., Coupland, P., Otto, T.D., Harris, S.R., Connor, T.R., Bertoni, A., Swerdlow, H.P., Gu, Y. (2012) A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics 13:341

5. Ahmadian, A., Gharizadeh, B., Gustafsson, A.C. Sterky, F., Nyren, P., Uhlen, M., Lundeberg, J. (2000) Single nucleotide polymorphism analysis by Pyrosequencing™. Analytical Biochemistry 280: 103-110.

6. Ronaghi, M., (2001) Pyrosequencing™ sheds light on DNA sequencing Genome Research 11 3-11.

7. Ortola-Vidal, A., Schnerr, H., Rojmyr, M., Lysholm, F., Knight, A (2007). Quantitative Identification of plant genera in food products using PCR and pyrosequencing™ Technology. Food Control 18, 921-927.

Dr Monee Shamsher and Dr Angus Knight, Leatherhead Food Research, Yew Tree Bottom Road, Great Burgh, Epsom, Surrey, KT18 5XT.
Leatherhead Food Research offers services ranging from consumer insight, sensory testing and ground-breaking ingredient and product innovation to expert advisory work around food safety and global industry regulations. It also operates an internationally recognised membership programme for the food and drinks industry. Leatherhead Food Research is a Science Group (AIM:SAG) company.
Web: Email: Tel: +44 (0)1372 822 274

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