Department of Immunology and Gnotobiology, Institute of Microbiology ASCR, v.v.i., Doly 183, Nový Hrádek, 549 22, Czech Republic.
J Microbiol Methods. 2011 Oct;87(1):10-6. doi: 10.1016/j.mimet.2011.06.014. Epub 2011 Jul 2.
Bifidobacterium longum is considered to play an important role in health maintenance of the human gastrointestinal tract. Probiotic properties of bifidobacterial isolates are strictly strain-dependent and reliable methods for the identification and discrimination of this species at both subspecies and strain levels are thus required. Differentiation between B. longum ssp. longum and B. longum ssp. infantis is difficult due to high genomic similarities. In this study, four molecular-biological methods (species- and subspecies-specific PCRs, random amplified polymorphic DNA (RAPD) method using 5 primers, repetitive sequence-based (rep)-PCR with BOXA1R and (GTG)(5) primers and amplified ribosomal DNA restriction analysis (ARDRA)) and biochemical analysis, were compared for the classification of 30 B. longum strains (28 isolates and 2 collection strains) on subspecies level. Strains originally isolated from the faeces of breast-fed healthy infants (25) and healthy adults (3) showed a high degree of genetic homogeneity by PCR with subspecies-specific primers and rep-PCR. When analysed by RAPD, the strains formed many separate clusters without any potential for subspecies discrimination. These methods together with arabionose/melezitose fermentation analysis clearly differentiated only the collection strains into B. longum ssp. longum and B. longum ssp. infantis at the subspecies level. On the other hand, ARDRA analysis differentiated the strains into the B. longum/infantis subspecies using the cleavage analysis of genus-specific amplicon with just one enzyme, Sau3AI. According to our results the majority of the strains belong to the B. longum ssp. infantis (75%). Therefore we suggest ARDRA using Sau3AI restriction enzyme as the first method of choice for distinguishing between B. longum ssp. longum and B. longum ssp. infantis.
长双歧杆菌被认为在维持人类胃肠道健康方面发挥着重要作用。双歧杆菌分离株的益生菌特性严格依赖于菌株，因此需要可靠的方法来鉴定和区分该物种的亚种和菌株水平。由于基因组相似度高，长双歧杆菌亚种之间的区分是困难的。在这项研究中，比较了四种分子生物学方法（种和亚种特异性 PCR、使用 5 个引物的随机扩增多态性 DNA（RAPD）方法、用 BOXA1R 和（GTG）（5）引物的重复序列基于（rep）-PCR 和扩增核糖体 DNA 限制分析（ARDRA））和生化分析，用于对 30 株长双歧杆菌菌株（28 株分离株和 2 株收集株）进行亚种水平分类。用亚种特异性引物和 rep-PCR 进行 PCR 分析时，最初从母乳喂养的健康婴儿（25 株）和健康成人（3 株）粪便中分离的菌株显示出高度的遗传同质性。当用 RAPD 分析时，这些菌株形成了许多独立的聚类，没有亚种区分的潜力。这些方法与阿拉伯糖/密二糖发酵分析相结合，仅能将收集株清晰地区分成长双歧杆菌亚种和长双歧杆菌亚种。另一方面，ARDRA 分析使用Sau3AI 酶对属特异性扩增子进行切割分析，将菌株分为长双歧杆菌/婴儿亚种。根据我们的结果，大多数菌株属于长双歧杆菌亚种。因此，我们建议使用 Sau3AI 限制酶进行 ARDRA，作为区分长双歧杆菌亚种和长双歧杆菌亚种的首选方法。
