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Objectives  Sequencing of 16S rDNA V3–V4 region is widely applied for food community profiling. However, two different universal forward primers (named here MUYZER-primer1 and KLINDWORTH-primer2) targeting an identical conservative sequence upstream of the V3 region of 16S rRNA gene, and only distinguished by a single mismatch are both used. This study was carried out to compare whether the accuracy of food microbiota analysis would depend on the choice of one of these two primers.    Results  Alignment of both primers with common food-borne bacteria 16S sequences revealed that the mismatch between both primers might specifically affect the amplification of  Leuconostoc, Oenococcus  and  Fructobacillus  species but not  Weissella  species. Food products containing either  Leuconostoc  and/or  Weissella  were selected for a detection test. As expected from our in silico analysis, our study showed that this mismatch induced a strong biased amplification specifically associated to the OTUs belonging to the genus  Leuconostoc  but not to the genus  Weissella . In presence of Muyzer-primer1, none of the sequences expected for  Leuconostoc  genus was detected whereas those sequences were correctly amplified with Klindworth-primer2. Since  Leuconostoc  is an important genus in food, agro-environments and in digestive tract of animals, we recommend that Muyzer-primer1 should thus be abandoned for the bacterial characterization of their associated microbiota.

bmc research notes

Detection of an amplification bias associated to Leuconostocaceae family with a universal primer routinely used for monitoring microbial community structures within food products