16S rRNA Gene Primer Validation for Bacterial Diversity Analysis of Vegetable Products

ABSTRACT High-throughput sequencing of the 16S rRNA gene enhances understanding of microbial diversity from complex environmental samples. The 16S rRNA gene is currently the most important target in bacterial evolution and ecology studies, particularly for determination of phylogenetic relationships among taxa, exploration of bacterial diversity in a given environment, and quantification of the relative abundance of taxa at various levels. However, some parts of the conserved region of the bacterial 16S rRNA gene are similar to the conserved regions of plant chloroplasts and eukaryotic mitochondria. Therefore, if DNA contains a large amount of nontarget DNA, this nontarget DNA can be coamplified and consequently produce useless sequence reads. We experimentally assessed the primer pair 335f/769r and the widely used bacterial primer pair SD (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21). The primer pair 335f/769r was examined for its ability to amplify bacterial DNA in plant and animal feed samples by using the single-strand confirmation polymorphism method. In our present study, these primer pairs were validated for microbial community structure analysis with complex food matrices by using next-generation sequencing. The sequencing results revealed that the primer pair 335f/769r successfully resulted in fewer chloroplast and mitochondrial sequence reads than generated by the universal primer pair SD and therefore is comparatively suitable for metagenomic analyses of complex food matrices, particularly those that are rich in plant DNA. Additionally, some taxonomic groups were missed entirely when only the SD primer pair was used.