基于16S rRNA基因的不同基原、不同产地甘草微生物群落特征分析

目的 探究4批不同基原、不同产地的甘草微生物群落信息。方法 参照《中国药典》2020年版四部,对甘草药材中的需氧菌总数、霉菌和酵母菌总数、耐热菌总数以及控制菌进行检查,并对致病菌平板上的菌落进行革兰染色,同时进行甘草药材的16S rRNA基因测序及数据分析。结果 甘草药材中的需氧菌总数、霉菌和酵母菌总数、耐热菌总数对数值分别为4.15～4.95、2.40～4.85、1.67～3.15;经热处理(100℃、30 min)后发现其需氧菌总数对数值的下降百分比分别为100.00%、24.10%、40.20%、66.26%;各甘草中耐胆盐革兰阴性菌可能总数较高,但均未检测到大肠埃希菌和沙门菌。16S rRNA基因测序共获得19 phylum、40 class、68 order、137 family、236 genus,其中GC-2获得最多的phylum (18)、class (36)、order (58), GC-1和GC-3获得最多的family (121), GC-1获得最多的genus (162)。多样性分析结果表明,甘草药材中污染微生物主要分布于3个门、3个属,不同基原、不同产地的甘草...     

Monitoring the microbial population dynamics at the start-up stage of wastewater treatment reactor by terminal restriction fragment length polymorphism analysis based on 16S rDNA and rRNA gene sequences

The microbial population dynamics at the start-up stage of a wastewater treatment reactor was investigated using terminal restriction fragment length polymorphism (T-RFLP) analysis based on 16S rDNA and rRNA gene sequences. The results of fragment peaks suggested that the number and activity of nitrifying bacteria increased in association with the start of nitrification, and the relative ratios of 16S rRNA of these bacteria changed prior to those of the 16S rDNA. Furthermore, multidimensional scaling (MDS) analysis revealed that the 16S rRNA exhibited wider dispersion than the 16S rDNA at the start-up stage, indicating that the diversity of 16S rRNA in the microbial communities was strongly affected by environmental changes.     

基于16S rRNA基因与相关基因序列分析格氏乳球菌亲缘关系

通过PCR扩增7株分离自传统发酵乳制品中的格氏乳球菌的dnaA、rpoB、recA基因,将扩增产物测序,测得的序列构建系统发育树,进行分类鉴定.同时,比较了这三个基因位点与16S rRNA基因揭示的格氏乳球菌种内菌株间以及其与乳酸乳球菌种间的亲缘关系的远近.结果表明,dnaA、rpoB、recA基因能够有效的鉴定出格氏乳球菌,且比16S rRNA基因更清晰地揭示了格氏乳球菌菌株间以及其与乳酸乳球菌种间的亲缘关系,特别是将3个管家基因联合起来,亲缘关系更明确,验证了多个管家基因是研究细菌亲缘关系的有力工具.     

Identification of Acetobacter strains from Thai fermented rice products based on the 16S rRNA gene sequence and 16S-23S rRNA gene internal transcribed spacer restriction analyses

BACKGROUND: Fermented rice flour (khao‐khab, a non‐glutinous rice) and related products are Thai traditional products. The types of acetic acid bacteria (AAB) microflora in khao‐khab have not been reported. In this study, Acetobacter strains were isolated and identified based on the phenotypic and chemotaxonomic characteristics and molecular aspects. RESULTS: Twenty‐five acetic acid bacteria isolated from fermented rice products and a starter for sweetened rice in Thailand by an enrichment culture approach, were assigned to the genus Acetobacter by phenotypic and chemotaxonomic characterisations. On the basis of the 16S rRNA gene sequence and 16S–23S rRNA gene ITS restriction analyses, 25 isolates were divided into six groups and identified at the specific level: (1) Group 1 included five isolates, which were identified as A. indonesiensis; (2) Group 2 included two isolates, which were identified as A. lovaniensis; (3) Group 3 included one isolate, which was identified as A. orientalis; (4) Group 4 included eleven isolates, which were identified as A. pasteurianus; (5) Group 5 included three isolates, which were identified as A. syzygii and (6) Group 6 included three isolates, which were unidentified and considered to constitute a new species. CONCLUSION: Results revealed that various Acetobacter species were distributed in Thai fermented rice flour and related products. A novel Acetobacter species was isolated from the product. Copyright © 2011 Society of Chemical Industry     

16S rRNA基因序列、生化鉴定、质谱3类方法鉴定常见微生物的结果分析

目的比较16SrRNA基因序列、生化鉴定、质谱鉴定3类实验方法分析沙门氏菌、金黄色葡萄球菌、蜡样芽孢杆菌的鉴定结果的异同。方法挑选沙门氏菌、金黄色葡萄球菌、蜡样芽孢杆菌各50株,分别进行16S rRNA基因序列测定、VITEK COMPACT 2生化鉴定、质谱鉴定3类实验,并比较3类实验的鉴定结果。结果 3类实验方法对大部分沙门氏菌鉴定在属水平,生化鉴定方法对少数血清型鉴定到种水平;对金黄色葡萄球菌均鉴定到种水平; 16SrRNA基因序列、生化方法对蜡样芽孢杆菌鉴定到属水平,质谱鉴定到种水平。结论 3类实验方法对大部分沙门氏菌、所有金黄色葡萄球菌鉴定水平相同,质谱鉴定蜡样芽孢杆菌的结果更准确,且质谱鉴定时效性更高。     

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirão Preto, São Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L. monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTE™ QPCR SYBR^® Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fast and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method.