PCR技术在快速诊断艾滋病合并播散性马尔尼菲青霉菌病中的应用

目的探讨PCR技术在快速诊断艾滋病（AIDS）合并播散性马尔尼菲青霉病（PSM）中的应用价值。方法收集21例AIDS合并播散性PSM患者抗真菌治疗前的系列标本：未培养血液、阳性血液培养物和阳性骨髓培养物,以12例健康人血液为阴性对照。提取上述标本的基因组DNA,采用真菌通用引物ITS1和ITS4对基因组DNA进行PCR扩增,扩增产物测序后与GenBank核酸序列数据库进行比对分析。结果 21份阳性血液培养物、21份阳性骨髓培养物及2份未培养血液标本均扩增出目的条带,而12份健康人血液标本未扩增出条带。阳性PCR产物测序结果与广东、日本、泰国、印度尼西亚的马尔尼菲青霉菌（PM）rDNA ITS序列（登录号分别为AB298970、AB298950、L37406、AJ853738）一致性高达97%～100%,证实为PM。PM与新型隐球菌及烟曲霉菌rDNA ITS序列的遗传距离最远,与青霉菌属其他菌种绳状青霉菌和桔青霉菌rDNAITS序列的遗传距离最近。结论 PCR可敏感、特异的检测出培养阳性标本中PM,并能有效缩短PM检出时间,但直接采用患者血液进行PCR检测的阳性率较低,方法有待改进。     

10株青海湖盐单胞属菌株种间分子多样性分析

目的 对青海湖盐单胞属菌株进行种间分子多样性分析.方法 利用PCR扩增青海湖10株盐单胞菌16S rDNA序列和16S - 23S间隔序列,通过测序、Blast对比以及RFLP方法分析10株青海湖盐单胞菌的序列差异;并结合SDS-PAGE技术方法研究其全菌体蛋白的种间差异.结果 通过测定16S rDNA序列同源性、分析16S-23S rDNA ISR多态性和比较全菌体蛋白SDS -PAGE谱带,确定了10株青海湖盐单胞菌在种间具有相对的一致性.结论 初步验证了分子表型特性的多相研究对种单元初分类具有重要意义.     

Candidatus Neoehrlichia mikurensis infection identified in 2 hematooncologic patients: benefit of molecular techniques for rare pathogen detection

Hematooncologic patients often host rare or fastidious pathogens. Using 16S rDNA sequencing and transmission electron microscopy, we have identified 2 lymphoma patients infected with Candidatus Neoehrlichia mikurensis. In both individuals, the clinical presentation suggested ehrlichiosis-like syndrome. We believe that molecular techniques open new vistas in the field of pathogen detection.     

抗真菌活性放线菌P-127菌株的生物学特性及多相分类鉴定

目的：从土壤中分离到1株具有较强抗真菌活性的放线菌P－127菌株,对分离菌株的形态特征、培养特征、生理生化特征、16SrDNA序列进行了系统的研究。方法：采用琼脂扩散法研究P－127菌株的抗真菌活性;形态观察、培养特征、生理生化特征观察采用放线菌分类鉴定常规培养基;采用16SrDNA序列分析法构建系统发育树进行分子生物学分类鉴定。结果：该菌株对啤酒酵母、白色念珠菌以及小麦赤霉病菌等6种真菌具有显著的抑菌活性;形态特征、培养特征、生理生化特征表现出链霉菌属的典型特征,归于金色类群。在系统发育树中,该菌株的序列与Streptomyces padanus的序列完全相同（相似性100％）。结论：结合形态、培养特征、生理生化特征及16SrDNA序列比对分析将菌株P－127鉴定为Streptomyces padanus。     

Real-time PCR快速检测钩端螺旋体方法的建立

目的:采用新型TaqMan探针建立检测钩端螺旋体的实时荧光定量PCR。方法:根据钩端螺旋体16s rDNA设计的一对引物及探针,以钩体标准菌株的核酸为模板,在荧光定量PCR检测仪(ABI 7500)建立实时荧光定量PCR。结果:建立的定量标准曲线的循环CT值与模板拷贝数呈现良好的线性关系(r=0.998),与普通PCR相比,荧光PCR的灵敏度是其1000倍,检测其他细菌DNA,未检测到信号。结论:本研究中检测钩端螺旋体的实时荧光定量PCR具有高度的特异性和敏感性,特别适合检测样本中微量的钩端螺旋体。     

Comparative evaluation of prokaryotic 16S rDNA clone libraries and SSCP in groundwater samples

A comparison of ribosomal RNA sequence analysis methods based on clone libraries and single-strand conformational polymorphism technique (SSCP) was performed with groundwater samples obtained between 523-555 meters below surface. The coverage of analyzed clones by phylotype-richness estimates was between 88-100%, confirming that the clone libraries were adequately examined. Analysis of individual bands retrieved from SSCP gels identified 1-6 different taxonomic units per band, suggesting that a single SSCP band does often represent more than one single prokaryotic species. The prokaryotic diversity obtained by both methods showed an overall difference of 42-80%. In comparison to SSCP, clone libraries underestimated the phylogenetic diversity and only 36-66% of the phylotypes observed with SSCP were also detected with the clone libraries. An exception was a sample where the SSCP analysis of Archaea identified only half of the phylotypes retrieved by the clone library. Overall, this study suggests that the clone library and the SSCP approach do not provide an identical picture of the prokaryotic diversity in groundwater samples. The results clearly show that the SSCP method, although this approach is prone to generate methodological artifacts, was able to detect significantly more phylotypes than microbial community analysis based on clone libraries.     

Caco-2细胞与肠道菌共培养初建体外模拟肠道共生模型

 【目的】构建体外模拟肠道共生系统,用于评价食源性抗菌药及其耐药菌作用于肠道菌群时对人体健康造成的危害风险。【方法】21d体外培养构建Caco-2细胞三维肠上皮,分成无菌对照组、混合的正常菌群组、沙门氏菌组、混合菌群-沙门氏菌组4组,制备混悬液,各组分别单独和混合培养4h,荧光定量PCR法测试共培养前后各菌16SrDNA菌量变化,电阻仪测试跨膜上皮电阻(TEER)数量;免疫荧光染色观察紧密连接蛋白ZO-1变化,评价三维肠上皮屏障功能的改变。【结果】 在体外共培养模型内,4种肠道菌混合培养4h后同单独培养组间比较,细菌量增长无统计学差异（P＞0.05)）;需氧菌的增长速度明显高于厌氧菌(P＜0.01),厌氧菌:需氧菌浓度比接近100:1,厌氧菌仍为优势菌。TEER和ZO-1免疫荧光染色结果均提示混合肠道菌群对Caco-2细胞三维肠上皮无明显破坏,侵入性沙门氏菌可显著降低TEER值,并观察到模型的ZO-1结构受到破坏。这种破坏作用在混合肠道菌群加沙门氏菌群组较轻。【结论】 Caco-2细胞三维肠上皮同乳双歧杆菌、植物乳酸杆菌、大肠杆菌和粪肠球菌组成的混合菌群体外共培养能建立稳定的体外肠上皮模型,并具有生物学活性;正常肠道菌群对侵入性沙门氏菌有拮抗作用。该模型可进一步完善作为食源性抗菌药、耐药菌与肠道系统相互作用研究模型的基础。     

Identification of Dominant Bacteria in Feces and Colonic Mucosa from Healthy Spanish Adults by Culturing and by 16S rDNA Sequence Analysis

The aim of this work was to examine by culturing the changes in the total and indicator populations of the feces of two individuals over 1 year and to identify the dominant microbial components of a single sample of feces from each donor. Populations and dominant bacteria from a sample of colonic mucosa from a further individual were also assessed. The culture results were then compared to those obtained with the same samples by 16S rDNA cloning and sequencing. High interindividual variation in representative microbial populations of the gastrointestinal tract (GIT) was revealed by both the culture and the culture-independent techniques. Species belonging to Clostridium clusters (XIVa, IV, and XVIII) predominated in both the fecal and the mucosal samples (except in the mucose cultured isolates), members of Clostridium coccoides cluster XIVa being the most numerous microorganisms. Species of γ -proteobacteria (Escherichia coli and Shigella spp.), bifidobacteria, and actinobacteria appeared in lower numbers than those of clostridia. From the mucosal cultured sample, only facultative anaerobes and bifidobacteria were recovered, suggesting destruction of the anaerobe population during processing. In accordance with this, the microbial diversity revealed by 16S rDNA sequence analysis was greater than that revealed by culturing. Despite large interindividual differences, distinct human communities may have group-associated GIT microbiota characteristics, such as the low number of Bacteroides seen in the subjects in this study.     

Ⅲ型前列腺炎相关微生物的再认识

Ⅲ型前列腺炎是一种严重影响男性健康的疾病,虽然有很高的发病率,但其病因却迄今未明。伴随着各种微生物检测和鉴定技术的改进,特别是16S rDNA基因鉴定技术在微生物检测中的广泛应用,使得我们对于Ⅲ型前列腺炎相关微生物有了更加全面的认识。本综述就各种方法在检测Ⅲ型前列腺炎相关微生物中的应用进行介绍,以期能够对微生物在该疾病发生过程中所扮演的角色有更加深入的理解。     

Ribosomal DNA instability: An evolutionary conserved fuel for inflammaging

Across eukaryotes, ribosomal DNA (rDNA) loci are characterized by intrinsic genomic instability due to their repetitive nature and their base composition that facilitate DNA double strand breaks and RNA:DNA hybrids formation. In the yeast, ribosomal DNA instability affects lifespan via the formation of extrachromosomal rDNA circles (ERC) that accrue into aged cells. In humans, rDNA instability has long been reported in a variety of progeric syndromes caused by the dysfunction of DNA helicases, but its role in physiological aging and longevity still needs to be clarified. Here we propose that rDNA instability leads to the activation of innate immunity and inflammation via the interaction with the cytoplasmic DNA sensing machinery. Owing to the recent clarified role of cytoplasmic DNA in the pro-inflammatory phenotype of senescent cells, we hypothesize that the accrual of rDNA derived molecules (i.e. ERC and RNA:DNA hybrids) may have a role in aging by contributing to inflammaging i.e. the systemic pro-inflammatory drift that associates with the onset of geriatric syndromes and age related dysfunctions in humans.