Dorn-In Samart, Bassitta Rupert, Schwaiger Karin, Bauer Johann, Hölzel Christina S
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.
Chair of Animal Hygiene, WZW, TUM, Weihenstephaner Berg 3, 85354 Freising, Germany.
J Microbiol Methods. 2015 Jun;113:50-6. doi: 10.1016/j.mimet.2015.04.001. Epub 2015 Apr 9.
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.
靶向细菌16S - rRNA基因的通用引物可通过定量聚合酶链反应（qPCR）对多种类型样本中的细菌总载量进行定量。然而，许多通用引物对也会扩增植物甚至古细菌及其他真核细胞的DNA。当样本中含有大量非靶标DNA时，使用这些引物可能会错误评估细菌总载量。因此，本研究旨在提供适用于定量和鉴定饲料、香料及消化器样本材料等样本中细菌DNA的引物对。针对42种引物，评估了其与植物叶绿体和线粒体序列的错配情况。进一步分析了6对引物是否能与古细菌、动物组织和真菌的DNA退火。随后，用植物、饲料、粪便、土壤和环境样本等样本基质对其进行测试。为此，通过qPCR对样本中的靶标DNA进行定量。对植物和饲料样本的聚合酶链反应（PCR）产物进一步进行单链构象多态性分析，随后进行序列分析。测序结果表明，引物对335F/769R仅能扩增植物和动物饲料等样本中的细菌DNA，其中植物DNA占主导。
