In food testing, speciation analysis is an important tool for ensuring food safety and authenticity. It involves identifying the presence of specific species or groups of species in a sample. There are two primary methods for speciation testing: targeted and non-targeted. This month, I look at the pros and cons of each method.
Targeted speciation testing
Targeted speciation testing is a method that focuses on detecting a specific species using quantitative PCR (qPCR). This method is ideal when you know exactly what species you are looking for, such as when testing for the presence of a specific fish species in a fillet or verifying the absence of pig in halal products.
With qPCR, data is captured as an amplification chart and compared to a positive control and often a 1% control sample. Limit of detection (LOD) is typically 0.01% - so able to pick up low levels of DNA.
Limitations of qPCR
The primary limitation of qPCR is that it is qualitative, not quantitative. Additionally, it is limited to a target number of species for which a test is available. Multispecies chips can also be costly to use.
Where best to use qPCR?
Where risks are well managed it is often cheaper to use targeted qPCR to look for the known risks only (rather than the less specific 7 or 9 species chips). Examples may include where you are looking for an expected species – for example, a specific species of fish in a fillet, absence of pig in halal products, or where there are a limited number of known potential contaminants (e.g., chicken handled in a factory also processing turkey).
Non-targeted speciation testing
Non-targeted speciation testing uses Next Generation Sequencing (NGS), which is an untailored method that trawls all available DNA from a sample in a single test. This approach is much more versatile and can be used to detect multiple species in a sample, even those that are unexpected. NGS is suitable for vegan validation, insect identification and detecting the unexpected.
With NGS, the selected primers target sections of DNA that are conserved within species but different between species. These target sections of DNA are then amplified and sequenced. The recovered sequences are compared to reference sequences to identify the species present in a sample. In most cases, it is possible to identify the DNA to a species. In closely related species, discrimination is not always possible, and the results are reported to a genus or family level. This method reports multiple species detected in the sample and the limit of detection (LOD) is typically 0.5% of identified reads.
NGS has the added advantage that you do not need to know what you are looking for, only the kingdom of interest. It is possible to search multiple kingdoms, providing there is a reference sequence. It can be used for detecting surface contamination on the outside of packaging, which is especially useful in tinned products where DNA recovery is expected to be low.
Limitations of NGS
Limitations of NGS include the requirement for DNA and the sensitivity to bias. If DNA is not present or is damaged, NGS will not work. The method also does not detect the quality of raw materials. Occasionally, the sequence can only be identified down to the genus or family level.
Be prepared for the unexpected!
NGS analysis may detect pest species and symbiotic species, as well as the expected species. For example, insect species in vegan products or symbiotic moulds in mushroom samples. It may also detect surface contamination on the outside of packaging, especially in tinned products, where DNA recovery is expected to be low. It is worth noting that rat DNA has been found in tinned products before!
Where best to use NGS?
NGS is suitable for powders and dried products where visual identification is difficult, as well as for comminuted products, ready meals or composite products, vegan products, high-value specialty products, speciation where qPCR is unavailable, complaint investigations, and detection of unknown unknowns.
NGS has been successfully used in samples testing positive on allergen tests to determine the presence of cross-reactive materials known to blends. In this case, NGS is a more cost-effective solution.
Both qPCR and NGS rely on being able to extract DNA from the sample. For highly processed material or where the DNA has been damaged or fragmented, it may not be possible to extract DNA of adequate quality. In addition, presence of DNA inhibitors may prevent amplification of the DNA so there will be times these methods do not work. It’s worth speaking to your laboratory provider to understand why.
Interesting in upping your game when it comes to food fraud? Read the Food Forensics column on how to detect and avoid.