Microarray analysis of microbiota of gingival lesions in noma patients

We used phylogenetic low-density microarrays targeting the 16S rRNA gene to characterize the gingival flora of acute noma and acute necrotizing gingivitis lesions, and compared them to healthy control subjects of the same geographical and social background. Various types of samples were collected (column characteristics); patients from the same hospital without mouth infection (H), matched control populations (T), patients suffering gengivitis (Gengivitis), patient suffering NOMA (noma), patient suffering NOMA receiving antimicrobials (N-ATB). Sampled from patients were retrieved from both sides (column Description); healthy- or lesion-side of the mouth. All controls are matched with specific patients (see column patient category and number) We designed low-density 16S rDNA arrays representing 339 different phylotypes. We used an arbitrary cutoff of 1% of overall abundance to select from this dataset the most abundant sequences for probe design. Using this cutoff, the 132 most abundant 16S rRNA gene sequences were scanned for probes respecting defined physico-chemical properties (Tm = 65±5°C; probe length = 23–50 nt; < -5.0 kcal/mol for hairpins; < -8.0 kcal/mol for self-dimers; and dinucleotide repeats shorter than 5 bp) using a commercial software (Array Designer TM 2.0 by Premier Biosoft). The 335 oligonucleotide probes were synthesized with a C6-linker with free primary amine (Sigma-Aldrich) and spotted on ArrayStrips microarrays (Clondiag GmbH, Jena, Germany).

本研究采用靶向16S核糖体RNA（16S rRNA）基因的系统发育低密度微阵列，对急性坏疽性口炎（acute noma）与急性坏死性龈炎病变的牙龈菌群进行特征分析，并与相同地理与社会背景的健康对照受试者开展对比。本研究收集了多种类型的样本（详见列特征）：同院无口腔感染患者（H）、匹配对照人群（T）、牙龈炎患者、坏疽性口炎患者（NOMA）以及接受抗菌药物治疗的坏疽性口炎患者（N-ATB）。所有样本均取自患者口腔的双侧（详见描述列），分为健康侧与病变侧。所有对照均与特定患者一一匹配，具体信息详见患者分类与编号列。本研究设计了覆盖339个不同系统型的低密度16S rDNA阵列。我们以总丰度1%作为任意截断阈值，从该数据集中筛选丰度最高的序列用于探针设计。基于该截断阈值，我们针对132条丰度最高的16S rRNA基因序列，使用商业软件（Premier Biosoft公司的Array Designer™ 2.0）筛选符合预设理化性质的探针：解链温度（Tm）为65±5℃；探针长度为23~50 nt；发夹结构自由能≤-5.0 kcal/mol；自身二聚体自由能≤-8.0 kcal/mol；二核苷酸重复序列长度小于5 bp。本研究合成了335条带有游离伯胺C6接头的寡核苷酸探针（Sigma-Aldrich公司），并将其点样于ArrayStrips微阵列芯片（德国耶拿Clondiag GmbH公司）。