Eurofins uses NGS testing to identify non-targeted microorganisms
Eurofins Genomics and Eurofins Food Microbiology laboratory in Nantes, France said the non-targeted bacterial screening is for food and feed testing.
It can be used to test raw or processed food matrices and provides a view of the flora in the product, thanks to bioinformatics and database tools.
Using NGS techniques it helps identify microorganisms within e.g. fermented food products and to better understand interactions.
The aim of 16S rDNA screening is identification of microorganism communities in a sample.
The “16S Microbiome Profiling” package created by Eurofins Genomics, is dedicated to the needs of academia, pharma and environmental researchers.
Dr Ilka Haase, analytical service manager, DNA food testing, applied genomics at Eurofins, said 16S rDNA screening can detect pathogens but it is not as much of a focus as authentication.
“As pathogens are normally present only in very small amounts and the sample preparation does not include an enrichment step by overnight culture, the detection of pathogens strongly depends on the sample matrix and the overall microbiota composition of the sample,” she told FoodQualityNews.
“We work on steps for the improvement of the sensitivity of the test, for example like enrichment of bacterial DNA during DNA extraction. Such specials will finally enable us to offer also a test that is more suited for pathogen detection like the screening test.”
NCBI database
All bacterial species of which a sequence is published in the National Center for Biotechnology Information (NCBI) nucleotide database can be identified.
Closely related species with almost identical DNA sequences will not be discriminated and only the genus (or the family) will be reported.
Also degradation of bacterial DNA may occur in highly processed samples or samples with low pH values so no species identification possible.
To avoid false-positive results, a negative control (water) is always co-analysed with the samples.
The classical barcoding by Sanger sequencing is limited to pure samples or mixtures of two species but the NGS approach can be used to analyse unknown mixtures with unlimited species numbers.
Dr Haase said by using NGS it can detect all bacteria (also non-culturable ones) without any enrichment step.
“LC-MS only works with pure cultures and is not possible with mixed bacteria. Hence, a culturing step and growth on agar plates is necessary to separate the bacterial communities and to analyse them separately. With this procedure and also with conventional microbiological techniques, only viable and culturable bacteria will be detected.”
Depending on sample source and subsequent actions, different expansions of 16S microbiome profiling is required.
One example of this is the number of sequences targets used for the analysis. Eurofins can offer up to four different sequence regions of the 16S gene for the analysis.
If an academic customer knows they only need one for clear identification, or if identification on genus level is sufficient, one target is fine.
If the microbial community is very complex, up to four targets might be necessary. For the food screening product the firm uses two targets which was a mixed decision between sensitivity and cost.
For food and feed samples processing steps and additional ingredients in different formulas/recipes may have an influence on the quality (integrity) of bacterial DNA. Especially acidic ingredients (like vinegar) will degrade DNA, said Eurofins.
Turnaround time and sensitivity
Dr Haase said it plans to offer a turnaround times (TAT) of five working days due to the running time of the Illumina MiSeq instrument.
“At the moment we have a variable TAT of up to 10 working days as we collect samples for one run in order to offer the best price for the customers.
“The sensitivity strongly depends on the matrix and the bacterial composition but lies in the lower % range (bacterial species present in relation to the overall bacterial amount).
“The reports give the bacterial species or, if the sequence in formation does not enable a n identification on species level, the bacterial genus.
“Additionally the relative sequence read amounts are given for each result. But it should be noted that the relative read amount is only an indication for the relative amount of the bacterial species DNA present in the microbial community of the sample and is influenced by species composition, different PCR amplification efficiencies and the sample matrix.”
