Pfannebecker Jens, Fröhlich Jürgen
Institute of Microbiology and Wine Research, Johannes Gutenberg-University of Mainz, Becherweg 15, D-55099 Mainz, Germany.
Int J Food Microbiol. 2008 Dec 10;128(2):288-96. doi: 10.1016/j.ijfoodmicro.2008.08.019. Epub 2008 Sep 7.
In this study, the 23S rRNA genes of nine different Pediococcus type strains were sequenced. By using a multiple sequence alignment with 23S rDNA sequences of related lactic acid bacteria two primer pairs were constructed, one for the general identification of the genus Pediococcus and one for the identification of the atypical species, P. dextrinicus. Furthermore, a primer set for a rapid multiplex PCR identification of the eight typical Pediococcus species was developed. With this technique, the species P. damnosus, P. parvulus, P. inopinatus, P. cellicola, P. pentosaceus, P. acidilactici, P. claussenii, and P. stilesii could be discriminated simultaneously in a single PCR. Experiments with inoculated grape musts showed that the detection limit was 10 cells ml(-1). The multiplex PCR assay was tested by the usage of 62 Pediococcus strains from different culture collections and 47 strains recently isolated from German wines and musts. In addition, contaminations with P. parvulus and P. damnosus could be detected after purification of DNA from spoilt wine samples. The method demonstrates a rapid and easy to handle tool for the species affiliation of pediococci in beverages and food samples.
在本研究中，对9种不同的片球菌模式菌株的23S rRNA基因进行了测序。通过将相关乳酸菌的23S rDNA序列进行多序列比对，构建了两对引物，一对用于片球菌属的通用鉴定，另一对用于鉴定非典型种——糊精片球菌。此外，还开发了一套用于快速多重PCR鉴定8种典型片球菌的引物组。利用该技术，可在一次PCR中同时鉴别出有害片球菌、渺小片球菌、意外片球菌、栖细胞片球菌、戊糖片球菌、嗜酸片球菌、克劳森片球菌和斯氏片球菌。对接种葡萄汁的实验表明，检测限为10个细胞/毫升。通过使用来自不同菌种保藏中心的62株片球菌菌株以及最近从德国葡萄酒和葡萄汁中分离出的47株菌株，对多重PCR检测方法进行了测试。此外，从变质葡萄酒样品中提取DNA后，可检测到渺小片球菌和有害片球菌的污染。该方法为饮料和食品样品中片球菌的种属归属提供了一种快速且易于操作的工具。
