title: Specific amplification of bacterial DNA by optimized so-called universal bacterial primers in samples rich of plant DNA.
abstract: Universal primers targeting the bacterial 16S-rRNA-gene allow quantification of the total bacterial load in variable sample types by qPCR. However, many universal primer pairs also amplify DNA of plants or even of archaea and other eukaryotic cells. By using these primers, the total bacterial load might be misevaluated, whenever samples contain high amounts of non-target DNA. Thus, this study aimed to provide primer pairs which are suitable for quantification and identification of bacterial DNA in samples such as feed, spices and sample material from digesters. For 42 primers, mismatches to the sequence of chloroplasts and mitochondria of plants were evaluated. Six primer pairs were further analyzed with regard to the question whether they anneal to DNA of archaea, animal tissue and fungi. Subsequently they were tested with sample matrix such as plants, feed, feces, soil and environmental samples. To this purpose, the target DNA in the samples was quantified by qPCR. The PCR products of plant and feed samples were further processed for the Single Strand Conformation Polymorphism method followed by sequence analysis. The sequencing results revealed that primer pair 335F/769R amplified only bacterial DNA in samples such as plants and animal feed, in which the DNA of plants prevailed.
authors: Chair of Animal Hygiene, WZW, TUM, Weihenstephaner Berg 3, 85354 Freising, Germany; Chair of Food Safety, Faculty of Veterinary Medicine, LMU, Schönleutnerstr. 8, 85764 Oberschleißheim, Germany. Electronic address: D.Samart@ls.vetmed.uni-muenchen.de.
authors: Samart
authors: S
authors: Dorn-In
authors: Chair of Animal Hygiene, WZW, TUM, Weihenstephaner Berg 3, 85354 Freising, Germany.
authors: Rupert
authors: R
authors: Bassitta
authors: Chair of Food Safety, Faculty of Veterinary Medicine, LMU, Schönleutnerstr. 8, 85764 Oberschleißheim, Germany.
authors: Karin
authors: K
authors: Schwaiger
authors: Chair of Animal Hygiene, WZW, TUM, Weihenstephaner Berg 3, 85354 Freising, Germany.
authors: Johann
authors: J
authors: Bauer
authors: Chair of Animal Hygiene, WZW, TUM, Weihenstephaner Berg 3, 85354 Freising, Germany; Chair of Milk Hygiene, Faculty of Veterinary Medicine, LMU, Schönleutnerstr. 8, 85764 Oberschleißheim, Germany.
authors: Christina S
authors: CS
authors: Hölzel
mesh_terms: D000821:Animal Feed; D001105:Archaea / Q000235:genetics; D058491:Bacterial Load; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics*; D020459:Genes, rRNA; D018807:Polymorphism, Single-Stranded Conformational; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D004271:DNA, Fungal; D018744:DNA, Plant; D012336:RNA, Ribosomal, 16S
keywords: 16S rRNA; Archaea; Bacteria; PCR; Plant; Primer