Hertel Christian, Meroth Christiane B
Institut für Lebensmitteltechnologie, Universität Hohenheim.
Berl Munch Tierarztl Wochenschr. 2003 Nov-Dec;116(11-12):517-23.
The identification of lactic acid bacteria in a complex microbiota using bacteriological culture in combination with phenotypic and genotypic identification techniques is laborious and time-consuming. New molecular methods permit a fast and culture-independent characterisation of such microbiota. Denaturing gradient gel electrophoresis (DGGE) of PCR fragments of the 16S rRNA gene has been proven to be a suitable tool. Here the use of PCR-DGGE with group specific primers is described to investigate the dynamic of sourdough microbiota from addition of the starter until the microbiota remained stable. Species were identified by applying an identification ladder obtained from reference strains or by sequence analysis of the PCR fragments. Furthermore, a method for detection of strains in complex microbiota is described. A strain specific chromosomal DNA fragment of Lactobacillus paracasei LTH 2579 was isolated applying the subtraction hybridisation. Based on the acquired target sequence a specific PCR system was established and combined with a PCR system specific for the species L. paracasei. Use of this detection system permitted to identify and quantitatively detect L. paracasei LTH 2579 in fermented sausages and upon consumption in faecal samples.
利用细菌培养结合表型和基因型鉴定技术,在复杂微生物群中鉴定乳酸菌既费力又耗时。新的分子方法能够对这类微生物群进行快速且无需培养的表征。16S rRNA基因PCR片段的变性梯度凝胶电泳(DGGE)已被证明是一种合适的工具。本文描述了使用带有组特异性引物的PCR-DGGE来研究从添加发酵剂直至微生物群保持稳定期间酸面团微生物群的动态变化。通过应用从参考菌株获得的鉴定阶梯或对PCR片段进行序列分析来鉴定物种。此外,还描述了一种在复杂微生物群中检测菌株的方法。应用消减杂交分离出干酪乳杆菌LTH 2579的菌株特异性染色体DNA片段。基于获得的靶序列建立了特异性PCR系统,并与干酪乳杆菌物种特异性PCR系统相结合。使用该检测系统能够在发酵香肠中以及食用后在粪便样本中鉴定并定量检测干酪乳杆菌LTH 2579。