双歧杆菌体外对Caco-2的黏附及其表面性质分析

【目的】体外测定双歧杆菌的黏附能力并对其表面性质进行分析。【方法】利用Caco-2细胞作为黏附模型体外测定七株菌的黏附能力,同时分析其自动聚集能力和表面疏水性,通过采用不同酶及化学物质处理双歧杆菌菌体细胞表面初步确定双歧杆菌细胞表面黏附相关化合物的类型,并对双歧杆菌表面蛋白进行电泳分析。【结果】自动聚集能力和表面疏水性均高的双歧杆菌菌株,其黏附能力高于自动聚集能力和表面疏水性均低的菌株,表现出明显的正相关。此外,受试菌株的黏附能力对蛋白酶和高碘酸钠敏感,利用LiCl对菌体表面蛋白进行提取后,其黏附能力明显下降,SDS-PAGE结果表明LiCl提取物中含有分子量大小不等的多个蛋白。【结论】双歧杆菌体外对Caco-2细胞的黏附具有菌株特异性,其黏附能力与表面疏水性质和自动聚集能力相关,此外,推测双歧杆菌表面可能含有能调节其黏附的糖蛋白类物质。     

双歧杆菌属特异性定量引物的设计及验证

【目的】旨在设计一对双歧杆菌属特异性引物以检测不同样品中低丰度双歧杆菌的含量。【方法】在NCBI中下载57株双歧杆菌全基因组序列,以其共有单拷贝核心基因为目的片段设计双歧杆菌属特异性引物;并对引物进行PCR初筛和特异性复筛;之后借助ddPCR(Droplet Digital PCR,微滴式数字PCR)依次对筛选出的引物进行特异性、灵敏度和实用性验证。【结果】引物Bif-D-9特异性最好,可扩增出4株双歧杆菌而不能扩增20株非双歧杆菌中的任何一株菌;同时通过ddPCR仪定量稀释后的DNA,其扩增结果呈线性下降趋势,证明其灵敏度较好;另外,Bif-D-9结合ddPCR定量出婴儿粪便中双歧杆菌的拷贝数为71 copies/μL,母亲粪便中双歧杆菌的拷贝数为2.7 copies/μL,证明了该方法的实用性。【结论】引物Bif-D-9具有双歧杆菌属特异性,且灵敏度较高、实用性较好,适用于复杂样品中双歧杆菌属定量。     

转基因棉花MON88701品系特异性实时荧光PCR检测方法的建立

【目的】建立转基因棉花MON88701品系特异性实时荧光聚合酶链反应(polymerase chain reaction,PCR)检测方法。【方法】利用实时荧光PCR技术,根据转基因棉花MON88701品系特异性序列设计引物和探针,建立转基因棉花MON88701实时荧光PCR检测方法,并测定本方法的灵敏度、特异性及可重复性。【结果】建立的检测方法特异于转基因棉花MON88701成分的检测,灵敏度测试表明其定量下限为34拷贝;重复性试验显示其相对标准偏差在可接受范围内。【结论】本研究建立的转基因棉花MON88701品系特异性实时荧光PCR检测方法具有良好的特异性和高灵敏度,适合对转基因棉花MON88701品系进行检测。     

实时荧光定量TaqMan-MGB探针法检测杆菌样巴尔通体

【目的】应用Taq Man探针实时荧光定量PCR技术建立特异性强、敏感性高和稳定性好的快速杆菌样巴尔通体检测方法。【方法】应用生物信息学方法查找杆菌样巴尔通体特有基因,从中筛选出一段特有的基因序列为模板设计探针和引物。通过比较Ct值和荧光强度确定扩增反应的最佳退火温度、探针和引物浓度;将扩增产物连接到p EASY-T载体上制备标准品,绘制标准曲线,分析扩增效率和线性关系;评估方法的特异性、敏感性及重复性。【结果】优化后退火温度为60°C,探针和引物浓度均为200 nmol/L,反应体系20μL。特异性实验显示只有杆菌样巴尔通体扩增出荧光信号,其他种属细菌均未见荧光信号;标准曲线线性关系良好(R2=1),扩增效率E=98.18%;最低检出限为每个PCR反应3个拷贝;组内和组间的变异系数CV值分别为0.21%–0.42%和0.29%–0.59%,在允许范围内。【结论】研究建立的实时荧光定量Taq Man-MGB探针法特异性强、灵敏度高、稳定性好,可快速检测鉴定杆菌样巴尔通体,为这种巴尔通体所引起的一系列疾病的早期快速诊断、监测和流行病学调查等研究提供有效手段。     

烟草环斑病毒的定量检测技术研究

目的:建立特异、灵敏、快速的TaqMam实时荧光定量PCR方法,用于烟草环斑病毒(TRSV)的定量检测。方法:用纳米磁珠法提取病毒RNA,构建包含烟草环斑病毒全CP序列的质粒标准品。根据CP保守序列设计特异性的引物和TaqMam荧光探针,构建标准曲线,建立TRSV的实时荧光绝对定量PCR方法,并对该方法的特异性、灵敏度和重复性进行评估。结果:建立的方法特异性好,与南芥菜花叶病毒、马铃薯X病毒和马铃薯Y病毒均无交叉反应;至少能检测到767个病毒拷贝,灵敏度比普通PCR高100倍;同一样品试验内及试验间重复性实验的变异系数均小于3%,重复性好;检测结果准确可靠,构建的标准曲线有较好的线性关系(R2=0.997)。结论:建立的TRSV TaqMan实时荧光定量PCR检测方法可满足口岸高通量、快速、准确的检验检疫要求。     

双重荧光定量PCR检测志贺毒素stx1和stx2基因

目的建立一种双重荧光定量PCR检测志贺毒素stx1和stx2基因的方法。方法根据不同细菌来源的stx1和stx2序列,设计PCR引物和TaqMan探针,建立双重实时荧光定量PCR检测体系,进行灵敏度、特异性和重复性评价,并对腹泻患者粪便样本进行检测分析。结果双重实时荧光定量PCR检测含志贺毒素基因重组质粒的最低检测下限为102 copies/mL;该法对12种常见肠道病原菌均无特异性扩增,对不同浓度的标准质粒检测重复性高,Ct值变异系数均小于10%;对急性腹泻粪便标本的检测阳性率高于细菌分离培养。结论建立的双重实时荧光定量PCR可作为不同细菌来源的志贺毒素基因的快速鉴定方法,亦可用于人感染性腹泻标本的快速筛查。     

叠氮溴化丙锭-荧光定量PCR法实时快速检测5种乳杆菌活菌数方法的建立与应用

【背景】乳杆菌属是发酵食品中最常见的微生物之一,与食品的品质和安全密切相关,定量检测乳杆菌活菌数、解析乳杆菌群落组成对发酵乃至肠道微生物等具有重要意义。【目的】建立一种在种水平上定量检测5种乳杆菌活菌数的叠氮溴化丙锭-荧光定量PCR (propidium monoazide-quantitative PCR,PMA-qPCR)检测方法并探讨其适用性。【方法】以植物乳杆菌、发酵乳杆菌、短乳杆菌、嗜酸乳杆菌和干酪乳杆菌等发酵食品中常见的5种乳杆菌为目标菌株,查找并筛选特异性引物用于荧光定量PCR (qPCR)检测,优化叠氮溴化丙锭(PMA)处理条件,测定PMA-qPCR检测法的特异性、灵敏度及可靠性。最后利用PMA-qPCR法检测黄酒酿造过程中5种乳杆菌的活菌数。【结果】PMA最佳处理条件为：浓度20 μmol/L下暗处理15 min后曝光15 min,此时可抑制样品中99.89%的死菌DNA扩增。该方法特异性高,能够准确识别5种乳杆菌;线性关系强,R2>0.98;灵敏度高,检测限为101.8?103.2 CFU/mL;重复性好,Cq值变异系数小于1%;与平板计数相比差异不显著(统计学上),p>0.05。利用该方法检测黄酒中5种乳杆菌的活菌数,发现发酵乳杆菌、干酪乳杆菌和短乳杆菌是主要的乳杆菌(总计占比59%?89%),与已知黄酒酿造中乳杆菌群落组成相符。【结论】建立的PMA-qPCR法能够快速、准确地检测5种乳杆菌的活菌数,为解析样品中乳杆菌的实时组成及检测具有活性但不可培养(viable but nonculturable,VBNC)状态的乳杆菌提供了可靠的手段。     

适用于分离人肠道中双歧杆菌的选择性培养基

【目的】比较并评价5种双歧杆菌选择性培养基对人源双歧杆菌的分离效果,试图筛选出一种适用于人肠道中双歧杆菌分离培养的选择性培养基。【方法】采集6份健康人粪便样品稀释涂布于5种选择性培养基上,厌氧培养后计数菌落并挑选单菌落进行鉴定。同时提取样品中细菌宏基因组DNA,应用变性梯度凝胶电泳技术(Denaturing Gel Gradient Electrophoresis,DGGE)和荧光定量PCR技术(Quantitative Polymerase Chain Reaction,q-PCR)揭示样品中双歧杆菌种类和数量,并以此为依据,客观评价上述5种选择性培养基的分离效果。【结果】BSM培养基和BLM培养基上双歧杆菌的计数结果与q-PCR的定量结果最为接近,并显著高于其它3种培养基。BLM培养基上分离到双歧杆菌的种类与DGGE图谱多样性分析的结果最为接近。【结论】BLM培养基是一种适用于人肠道中双歧杆菌分离培养的选择性培养基。     

荧光定量PCR法定量白酒固态发酵过程中产土味素的链霉菌

【目的】荧光定量PCR法具有特异性高、灵敏度高等优点,在定量复杂环境中微生物数量方面得到广泛应用。本文采用荧光定量PCR方法对固态白酒发酵过程产土味素链霉菌进行定量分析并验证其准确性。【方法】通过优化大曲和酒醅中微生物基因组提取方法,并建立相应的大曲和酒醅两种基质条件下的荧光定量PCR标准曲线,并对方法的精度和准确度进行验证分析。采用荧光定量PCR方法对大曲和酒醅中产土味素链霉菌进行定量分析。【结果】结果表明大曲中产土味素链霉菌数量在105数量级,并且清茬曲中此类链霉菌数量最高。酒醅发酵起始阶段产土味素链霉菌数量在104数量级,而后随着发酵的不断进行,酒醅中此类链霉菌数量有所减少。【结论】荧光定量PCR方法能够对白酒固态发酵过程中产土味素链霉菌准确进行定量分析,对采用此方法定量其他微生物具有借鉴意义。     

马铃薯立枯丝核菌拮抗菌QHZ11的实时荧光定量PCR快速检测与应用

【背景】植物根际促生菌(plant growth-promoting rhizobacteria,PGPR)在根际的定殖是其发挥作用的基础,直观有效的跟踪技术和定量方法是研究PGPR在根际原位分布规律的重要工具。【目的】建立一种马铃薯黑痣病病原菌——立枯丝核菌拮抗菌QHZ11的实时荧光定量PCR快速检测体系,并检测拮抗菌QHZ11在马铃薯根际的动态变化。【方法】根据GenBank中登录的类芽孢杆菌及近源菌株gyrB基因序列差异筛选特异性引物,优化反应条件;通过盆栽试验对马铃薯根际拮抗菌进行快速检测。盆栽试验设3个处理,T1：对照(无菌水,CK);T2：QHZ11菌悬液灌土(QHZ11);T3：将功能菌在有机肥中进行二次固体发酵制成生物有机肥(BOF11)。【结果】筛选出拮抗菌QHZ11的专用引物为gyrB-F/gyrB-R;建立的拮抗菌QHZ11实时荧光定量PCR检测方法特异性好、灵敏度高且重复性较好,线性相关系数为0.999 8,检测组内变异系数均在1%以内,扩增效率为0.9,可检测出1×103?1×1010 copies/g-soil的拮抗菌,具有检出限低和扩增效率高的特点。盆栽试验结果发现,T3处理马铃薯根际拮抗菌QHZ11的数量从接种的第10天即高出T2处理一个数量级,并于马铃薯盛花期(接种后第60天)达到峰值,说明二次固体发酵增加了拮抗菌在根际土壤中的存活和繁殖。【结论】建立的实时荧光定量PCR快速检测体系灵敏、高效,可为研究拮抗菌在马铃薯根际原位分布以及与病原菌的互作方面提供便捷有效的方法。     

嗜酸乳杆菌NCFM粘附宿主后对其粘附相关基因表达的影响

摘要：【目的】益生菌粘附于肠道上皮细胞上是它的一种益生作用。本研究通过体内外实验,分析嗜酸乳杆菌NCFM对粘附相关基因的影响。【方法】利用GO (Gene Ontolog) 分类筛选Human Genome U133 Plus 2.0 Array基因表达谱芯片中的粘附相关基因,通过体外Caco-2细胞培养模型和体内小鼠粘附模型,采用Real-time PCR方法对粘附相关基因进行验证分析。【结果】经NCFM作用后,12个粘附相关基因呈上调表达。利用Real-time PCR验证,12个基因在体内和体外经嗜酸乳杆菌NCFM作用后亦均同样为上调表达,其中CCL2基因上调表达最为明显。【结论】经体内外研究表明,嗜酸乳杆菌NCFM粘附肠上皮细胞后能够引起宿主粘附相关基因出现特定表达变化,为今后深入揭示其粘附作用提供必要基础。     

Formation of diacylglycerol by a phospholipase D-phosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells

The conversion of phosphatidylcholine (PC) to diacylglycerol (DAG) was studied in sonicated endothelial cells and in subcellular fractions in the presence of 0.05% Triton X-100 and 2 mM EDTA. DAG formation occurred predominantly in an organelle fraction that sedimented at 15,000 x g. In parallel reactions with exogenous 1-oleoyl-2-[3H]oleoyl-PC (sn-2-[3H]DOPC) and phosphatidyl[3H]choline ([choline-3H]PC), [3H]DAG was formed by a reaction pathway in which [3H]choline was the only product derived from [choline-3H]PC. [3H]Choline was not formed secondarily from [3H]glycerophosphocholine or [3H]phosphocholine. Small amounts of [3H]phosphatidate ([3H]PA) were isolated from reactions with sn-2-[3H]DOPC at short incubation times, and substantial PA phosphatase activity was demonstrated. These data, taken together, supported a phospholipase D-PA phosphatase pathway of DAG formation. Kinetic data established that the low ratio of [3H]PA/[3H]DAG formed in reactions with sn-2-[3H]DOPC was due to a 15-fold higher Vmax and 7-fold lower apparent Km of the PA phosphatase. The [3H]PA/[3H]DAG product ratio was increased by addition of unlabeled PA or by selective extraction of phospholipase D with Triton X-100. The characteristics of the phospholipase D indicated a unique enzyme. Activity was optimal in the presence of EDTA and was almost totally dependent upon Triton X-100. The pH profile displayed a peak at 7.0. Of particular significance was the stringent substrate specificity. Phosphatidylinositol was not hydrolyzed, and activities towards phosphatidylethanolamine and sphingomyelin were at most 30- to 50-fold lower than those towards PC. Phospholipase D and PA phosphatase were identified in a number of rat tissues and other cells. The highest activities of phospholipase D were present in lung and endothelial cells. Phospholipase D was partially purified from rat lung by Triton X-100 extraction and anion exchange chromatography. When linked with PA phosphatase, the phospholipase D could initiate a pathway of DAG formation that is highly specific for PC.     

Evaluation of surface hydrophobicity of immobilized protein with a surface plasmon resonance sensor

Immobilization is widely used to isolate agglutinative and associative proteins with large hydrophobic surfaces. Surface hydrophobicities of immobilized proteins were quantified by measuring the adsorption amounts of Triton X-100 as a hydrophobic probe with a biosensor that utilizes the phenomena of surface plasmon resonance (SPR). We measured SPR signal changes derived from adsorption of Triton X-100 to five kinds proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller equation, partly modified with a term for correcting an influence of the net charge of immobilized protein. SPR signal changes obtained by this method correlated with the values of surface hydrophobicities obtained by conventional assay using a hydrophobic probe. Thus this measuring method using an SPR sensor and Triton X-100 is expected to be a tool for quantifying surface hydrophobicities of immobilized proteins.     

Effect of Triton X-100 on properties of acetylcholinesterase from human erythrocytes

The effect of Triton X-100 on catalytic properties of acetylcholinesterase from human erythrocytes under acetylcholine hydrolysis, on sensitivity of acetylcholinesterase to specific phosphoorganic inhibitors and eserine, and on the mobility and isoenyme spectrum under analytical electrophoresis in polyacrylamide gel is investigated. Triton X-100, independently on its concentration within 0.05-1.0%, slightly changes V and [S]opt values and increases Km value in 2-3 times. The inhibitory effect of Triton X-100 is mainly competitive, 0.5% Triton X-100 decreases bimolecular constant (kII) of the interaction of acetylcholinesterase with phosphoorganic inhibitor and eserine in 2.5-4 times. In the presence of phosphoorganic inhibitor, kII sharply decreased when 0.02% Triton X-100 was added, and then it did not change under the increase of Triton X-100 concentration up to 1.0%. On the basis of these data, an analytical method of estimating Triton X-100 content in protein solution is proposed. The introduction of 0.1% Triton X-100 into polyacrylamide gel results in considerable quantitative redistribution of acetylcholinesterase isoenzyme fractions and in the change of the mobility of one fraction under electrophoresis.     

Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions.

Thirteen human bifidobacterial strains were tested for their abilities to adhere to human enterocyte-like Caco-2 cells in culture. The adhering strains were also tested for binding to the mucus produced by the human mucus-secreting HT29-MTX cell line in culture. A high level of calcium-independent adherence was observed for Bifidobacterium breve 4, for Bifidobacterium infantis 1, and for three fresh human isolates from adults. As observed by scanning electron microscopy, adhesion occurs to the apical brush border of the enterocytic Caco-2 cells and to the mucus secreted by the HT29-MTX mucus-secreting cells. The bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage. The adhesion to Caco-2 cells of bifidobacteria did not require calcium and was mediated by a proteinaceous adhesion-promoting factor which was present both in the bacterial whole cells and in the spent supernatant of bifidobacterium culture. This adhesion-promoting factor appeared species specific, as are the adhesion-promoting factors of lactobacilli. We investigated the inhibitory effect of adhering human bifidobacterial strains against intestinal cell monolayer colonization by a variety of diarrheagenic bacteria. B. breve 4, B. infantis 1, and fresh human isolates were shown to inhibit cell association of enterotoxigenic, enteropathogenic, diffusely adhering Escherichia coli and Salmonella typhimurium strains to enterocytic Caco-2 cells in a concentration-dependent manner. Moreover, B. breve 4 and B. infantis 1 strains inhibited, dose dependently, Caco-2 cell invasion by enteropathogenic E. coli, Yersinia pseudotuberculosis, and S. typhimurium strains.     

Carnitine palmitoyltransferase: separation of enzyme activity and malonyl-CoA binding in rat liver mitochondria

Carnitine palmitoyltransferase activity and malonyl-CoA binding capacity have been studied in Triton X-100 extracts and membrane residues of rat liver mitochondria. Rat liver mitochondria extracted twice with 0.5% Triton X-100 in a salt-free medium showed increased specific binding of [2-14C]malonyl-CoA when compared with intact mitochondria. High malonyl-CoA binding required the presence of salts and was inhibited by albumin. Further solubilization of the membrane residues in the Triton/KCl medium and subsequent hydroxylapatite chromatography gave a complete separation of carnitine palmitoyltransferase and malonyl-CoA binding. The results show that malonyl-CoA binds to mitochondrial component(s) which is different from and more difficult to extract from the mitochondrial membrane than most of the carnitine palmitoyltransferase.     

Detergents inhibit exocytosis in PC 12 cells: evidence for an effect on ion fluxes

Membrane events in exocytosis were studied by examining the effect of different detergents on the K+-stimulated release of noradrenaline in the secretory cell line PC 12. The nonionic detergent Triton X-100 and the cationic detergent cetyltrimethylammonium bromide (CTAB) inhibit the noradrenaline release evoked by 55 mM K+ by 50% at very low concentrations (30 microM and 10 microM, respectively). These values are tenfold lower than the critical micellar concentrations (CMC). No such effect was seen with the anionic detergent sodium dodecyl sulphate (NaDodSO4). The inhibitory effect of 30 microM Triton X-100 is reversible, and the recovery from inhibition correlates with the loss of detergent from the cells as demonstrated by binding studies using [3H]Triton X-100. The possible relationship between this inhibition of secretion and the structural properties of the detergent was investigated. The inhibition in the presence of purified Triton X-100 subfractions turned out to be a function of the length of the oligometric ethyleneglycol chain (C6 to C26). The maximal effect was observed for Triton X-100 molecules having a chain length of 16 carbon atoms, which can penetrate just half of the lipid bilayer of the membrane. Additionally, the phase transition at 13-14 degrees C observed in an Arrhenius plot of noradrenaline release in stimulated cells was abolished. In the presence of 30 microM Triton X-100, 22Na+ uptake, 86Rb+ release, and 45Ca2+ uptake were reduced by 50-60%. These data suggest that the site of action of Triton X-100 is at the level of altering the movement of ions in PC 12 cells during the stimulatory phase of secretion.     

Characterization of acetylcholinesterase in Caco-2 cells

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was solubilized from cultured Caco-2 cells. It was established that this enzyme activity is acetylcholinesterase by substrate specificity (acetylthiocholine, acetyl-beta-methylthiocholine>propionylthiocholine>butyrylthiocholine), substrate inhibition, and specificity of inhibitors (BW284c51>iso-OMPA). The acetylcholinesterase activity increased proportional to the degree of differentiation of the cells. Most of the enzyme was membrane bound, requiring detergent for solubilization, and the active site faced the external fluid. Only one peak of activity, which corresponded to a monomeric form, could be detected on linear sucrose density gradients. The sedimentation of this form of the enzyme was shifted depending on whether Triton X-100 or Brij 96 detergent was used. These results indicate that the epithelial-derived Caco-2 cells produce predominantly an amphiphilic, monomeric form of acetylcholinesterase that is bound to the plasma membrane and whose catalytic center faces the extracellular fluid.