PCR

PCR (Polymerase Chain Reaction)

Biomolecular methods are complementary optimisations to the results obtained by light microscopy. Since publication of EU regulation 51/2013 amending EC/152/2009 PCR has become an official method for the detection of ruminant DNA in aquafeed.

Through genetic amplification, the PCR method enables well-defined taxonomic related DNA sequences to be detected: at species level (bovine, pig, chicken, and sheep) or at supra-species level (ruminants, fishes).

Basically the method involves the following steps.

  • Extraction of the DNA fragments from the feed.
  • Multiplication of the number of copies of the defined DNA targets (amplicons) by successive PCR heating cycles. The number of copies is doubled at each cycle. Real time PCR allows following over the cycles –i.e. overtime– the increase of amplicon numbers by reaction to a fluorescent DNA probe.
  • Analysis of data referring to threshold values and delivery of a qualitative result, it means a result expressed as “present” or “absent”.

Kinetics of real time PCR are monitored while running on the thermocyclers. Several methods exists depending on the extraction mode, the differences among DNA targets for a given species (e.g. more than 10 different targets for porcine) and the type of equipment. Studies have demonstrated that in general these different methods succeeded at detecting bovine PAPs at levels of 0.1% in feed.

Advantages

  • Identification of species and taxonomic groups
  • Low levels of contamination are detectable (0.1%)
  • Almost perfect specificity.
  • Rapid method
  • Moderate human expertise level

Disadvantages

  • Indirect method: the DNA from the animal product is detected, not the proteins.
  • Inability to distinguish between authorized and prohibited products: a positive signal for bovine can originate from PAPs but also from dairy products
  • Restrictions in the choice of suitable DNA target sequence: have to be short enough (<100bp) and to originate from multicopy sequences (e.g. mitochondria)
  • Cut-off value specific of the PCR platform (combination of equipment and reagents) and of the target
  • Needs further development
  • Quantification of the amount of animal proteins is impossible: there is no correlation between the number of DNA amplicons and the effective protein content