An end-point polymerase chain reaction (PCR) and capillary electrophoresis (CE) system was developed, according to a study.
Five assays were run under the same PCR cycling conditions on a 96-well plate and assigned a lab chip for amplicon detection.
PCR assays were designed based on specific gene targets from cotton, lupin, maize, soya and sugar beet.
Primers were tested using authenticated positive control material and amplicons analysed using the Agilent 2100 Bioanalyzer.
Each of the five assays was successful in amplifying the specific DNA target in the relevant positive control material using a common PCR cycling programme.
Assays for lupin, maize and soya repeatedly amplified DNA from processed food materials where the crop species were present on the ingredient list.
The cotton and sugar beet assays showed no cross-reactivity with other crop species and demonstrated no false-positives when applied to the range of processed food materials.
They only produced a detectable response when in the presence of target DNA sequence from the positive control but gave no false positives when tested against the processed food materials.
Assay development involved testing specificity and sensitivity of the primers, optimising reaction conditions and applying assays to DNA extracted from processed food materials.
Processed food materials included Trufree digestive biscuits and custard creams, Tuc crackers, Zest cheese bites parmesan, Waitrose gluten free pittas and Genius brown bread.
There are many methods for the detection of allergenic proteins in food products, although DNA-based techniques often have advantages in terms of better sensitivity and greater specificity.
Controls and primers used
Authenticated positive controls for soya, maize, cotton, and sugar beet came as dried Certified Reference Materials (CRMs) from IRMM via LGC Standards (UK).
DNA was extracted from the CRM’s for soya, maize, cotton and sugar beet, and the processed food materials, using a modified CTAB (Cethyltrimethylammonium Bromide) method.
Existing primers were sourced from literature, except for the lupin assay where novel ones were designed using NCBI primerBLAST9 based on sequence data deposited in GenBank.
For optimisation, primer concentrations were varied across 0.25, 0.50, 0.75 and 1.00μM. The annealing temperature (Ta) was changed from 56, 58, 60 and 62°C.
Limit of Detection (LOD) was established using three separate six-point five-fold dilution series of the positive control DNA template.
All assays detected the desired target down to at least 80pg of DNA per PCR with the maize, soya and sugar beet assays working to 16pg.
Further experiments determined the LOD across a six-point two-fold dilution series ranging from 80 to 5pg DNA per PCR for cotton and lupin and 16 to 1pg per PCR for soya, sugar beet and maize.
EU Regulation 1169/2011, which came into force in December last year, states that individual allergenic species must be clearly labelled in the ingredients list.
“In light of these changes it is imperative that Public Analyst and trade laboratories are in a position to utilise appropriate methodologies for the detection of ingredients such as allergens in the event of any legal disputes that arise over food production.
“The present study was successful in developing a simple, singleplex lab-on-a-chip approach for the simultaneous detection of common ingredients in foods, the principles of which can be applied to various fields within the area of food authenticity testing.”
Source: Journal of the Association of Public Analysts
Authors: Eloise Busby and Malcolm Burns