环介导等温扩增技术检测化妆品中铜绿假单胞菌的研究

本文建立了环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)快速检测化妆品中铜绿假单胞菌的方法。采用铜绿假单胞菌外膜蛋白oprI基因保守序列引物,评价检测铜绿假单胞菌灵敏度和特异性,并与普通PCR相比,检测人工污染样品中的铜绿假单胞菌。结果显示,LAMP检测铜绿假单胞菌的灵敏度为62. 5 pg/μL,而且特异性高,人工污染样品中的检出限为102cfu/m L,比PCR检测灵敏度高10倍。该方法具有灵敏度高、特异性好、操作简便、耗时短,可用于化妆品中铜绿假单胞菌的快速检测。     

基于聚合酶螺旋反应技术快速检测铜绿假单胞菌

【背景】铜绿假单胞菌是一种重要的水源和食源性致病菌,可引起急性肠道炎、脑膜炎、败血症和皮肤炎症等疾病。加强铜绿假单胞菌的快速检测,对保障食品安全具有重要的意义。【目的】建立聚合酶螺旋反应(Polymerasespiralreaction,PSR)方法快速检测铜绿假单胞菌。【方法】针对铜绿假单胞菌外毒素A调控基因——ETA基因(toxA)设计引物,通过引入加速引物、优化反应条件和筛选颜色指示剂,建立快速检测铜绿假单胞菌的PSR方法,并研究方法的特异性、敏感性和可靠性。【结果】建立的方法在等温65°C条件下,40 min内可完成PSR反应,且可通过钙黄绿素和羟基萘酚蓝直接判读结果。方法特异性强、灵敏度高,最低检出限分别为20 CFU/mL细菌和1.011 5 pg/μL基因组DNA。可视化PSR方法检测包装饮用水来源的分离菌株与传统生化方法检测结果一致。【结论】研究建立的可视化PSR方法为铜绿假单胞菌DNA快速检测提供了一种可行的有效手段。     

铜绿假单胞菌脂多糖对白念珠菌生物膜形成的影响

目的研究铜绿假单胞菌脂多糖(lipopolysaccharide,LPS)对白念珠菌生物膜形成不同时期的影响。方法甲基四氮盐(XTT)减低法用于检测铜绿假单胞菌LPS对白念珠菌生物膜形成不同阶段生成量的影响,利用倒置显微镜观察生物膜形态学改变。结果铜绿假单胞菌对白念珠菌生物膜形成的影响具有阶段差异性和浓度差异性。结论铜绿假单胞菌LPS抑制白念珠菌生物膜菌丝的形成。     

包装饮用水中铜绿假单胞菌快速检测试剂盒的研制与评价

【背景】在包装饮用水企业生产活动中,铜绿假单胞菌是被重点监测的致病菌之一。随着分子检测相关技术的不断发展,研制用于包装饮用水中铜绿假单胞菌简便、高效的快速检测产品至关重要。【目的】评价基于环介导恒温扩增(loop-mediated isothermal amplification,LAMP)技术的铜绿假单胞菌快速检测试剂盒在包装饮用水铜绿假单胞菌检测中的实效性。【方法】优化该LAMP反应体系,反应试剂采用冻干工艺,确定试剂盒组成,并评价其特异性、灵敏度、重复性、保质期等性能指标。【结果】铜绿假单胞菌标准菌株和分离菌株均检测为阳性,非铜绿假单胞菌标准菌株和分离菌株均检测为阴性,未发现有交叉反应;试剂盒最低检验限为18 CFU/mL;该试剂盒的特异性、灵敏度及准确度与传统方法相比具有较高的一致性;批内、批间检测重复率均为100%,可在4℃保存12个月以上,并且可在42℃环境中储存72 h以上。【结论】该试剂盒性能好,检测结果稳定、可靠,适用于包装饮用水中铜绿假单胞菌的快速检测。     

银染法观察铜绿假单胞菌生物被膜

目的 评估银染法鉴定铜绿假单胞菌生物被膜的效果.方法 体外平板法制备铜绿假单胞菌生物被膜模型,用银染法观察鉴定.结果 银染后普通光学显微镜和扫描电镜观察铜绿假单胞菌生物被膜.结论 银染法鉴定铜绿假单胞菌生物被膜简单可靠.     

5-甲基间苯二酚对铜绿假单胞菌及其生物膜形成的影响

探讨5-甲基间苯二酚对铜绿假单胞菌(Pseudomonas aureginosa)及其生物膜形成的影响。通过微量肉汤稀释法检测铜绿假单胞菌对5-甲基间苯二酚的敏感性并绘制时间-杀菌曲线;通过微孔板培养生物膜结合结晶紫染色法检测5-甲基间苯二酚对铜绿假单胞菌生物膜形成和分散的影响。当5-甲基间苯二酚的浓度为512μg/mL时,可显著抑制铜绿假单胞菌PAO1生物膜的形成,而5-甲基间苯二酚对铜绿假单胞菌PA47生物膜的形成无影响。32μg/mL的5-甲基间苯二酚还能显著分散铜绿假单胞菌PAO1成熟生物膜,但无明显的剂量依赖性。不同临床菌株生物膜对5-甲基间苯二酚的敏感性各异。结果表明,5-甲基间苯二酚能抑制铜绿假单胞菌生物膜的形成并能分散已形成的生物膜。     

甲型链球菌与铜绿假单胞菌对白假丝酵母菌生长及粘附影响的体外观察

目的:观察甲型链球菌与铜绿假单胞菌对白假丝酵母菌生长及粘附的影响.方法:建立白假丝酵母菌的生长梯度模型,并引入甲型链球菌与铜绿假单胞菌或其培养后上清;建立人脐周静脉内皮细胞EVC-304的六孔板生长模型,与上述白假丝酵母菌生长梯度(引入及未引入甲型链球菌与铜绿假单胞菌)共同孵育.计数粘附数.结果:引入甲型链球菌与铜绿假单胞菌的白假丝酵母菌生长缓慢,对人脐周静脉内皮细胞的粘附数减少;而引入甲型链球菌与铜绿假单胞菌培养后上清的白假丝酵母菌生长良好.结论:甲型链球菌与铜绿假单胞菌对白假丝酵母菌生长及其对人脐周静脉内皮细胞的粘附有明显的抑制作用,且此种抑制作用与甲型链球菌与铜绿假单胞菌的分泌作用无关.     

植物发酵液提取物对铜绿假单胞菌生物膜的作用

【目的】探讨植物发酵液提取物(plant fermentation extract,PFE)对铜绿假单胞菌生物膜的抑制作用,为临床上铜绿假单胞菌感染相关疾病的治疗提供参考。【方法】通过划线法分离临床标本中的铜绿假单胞菌并进行鉴定,通过报告菌株测定铜绿假单胞菌的毒力因子,采用试管法和激光共聚焦扫描显微镜测定生物膜的形成。【结果】在分离出的16株铜绿假单胞菌中,PFE对PA007菌株的作用效果最好,1%PFE显著降低PA007菌株生物膜、绿脓菌素和N-(3-oxododecanoyl)-HSL(3-oxo-C12-HSL)的产量(P0.05)。同时,也显著降低Las A蛋白酶的活性以及持留菌存活率(P0.05)。荧光定量PCR实验结果表明PFE能显著抑制las I和pqs A基因的表达(P0.05)。【结论】PFE具有抗铜绿假单胞菌感染能力,在临床上铜绿假单胞菌感染疾病的治疗中具有巨大的潜在价值。     

铜绿假单胞菌环介导等温扩增技术检测方法在实验小鼠微生物控制中的应用

目的建立铜绿假单胞菌Pseudomonas aeruginosa环介导等温扩增技术(LAMP)检测方法并初步应用于实验小鼠微生物控制。方法根据铜绿假单胞菌oprL基因设计LAMP特异性引物,优化反应条件,确立LAMP的检测体系;再通过对小鼠血清样本的检测,与《GB/T 14926. 17-2001实验动物绿脓杆菌检测方法》对比,阳性结果再用PCR方法验证。结果新建立的LAMP方法特异性强,灵敏度比普通PCR高10~3倍;当反应温度为66℃,内引物和环引物的浓度分别为70μmol·L^-1和30μmol·L^-1时,LAMP反应体系最佳;利用建立的LAMP方法检测87份小鼠血清样本,铜绿假单胞菌检出率为11. 5%(10/87),比《GB/T 14926. 17-2001实验动物绿脓杆菌检测方法》的高(0/87),阳性结果与PCR方法一致。结论本研究建立的LAMP方法特异性强、灵敏度高、可重复率高、稳定性好,为检测铜绿假单胞菌提供了新的研究手段。     

口腔细菌对铜绿假单胞菌黏附及侵入肺上皮细胞能力的影响

目的 通过比较铜绿假单胞菌和口腔细菌单独或共同作用于肺上皮细胞时,细菌黏附和侵入细胞的能力,探讨细菌间相互作用在呼吸道感染的最初阶段的作用机制.方法 应用培养法和抗生素保护法检测铜绿假单胞菌和口腔细菌单独或共同作用于肺上皮细胞时,细菌黏附和侵入肺上皮细胞的能力.结果 铜绿假单胞菌与口腔细菌共同作用于肺上皮细胞,牙龈卟啉单胞菌和伴放线放线杆菌降低了铜绿假单胞菌的黏附能力,却增强了其侵入能力;而铜绿假单胞菌能够影响口腔细菌对肺上皮细胞的黏附,同时增强口腔细菌侵入肺上皮细胞的能力.结论 口腔细菌,尤其是牙周可疑致病菌主要通过增强铜绿假单胞菌对肺上皮细胞的侵入而影响呼吸道感染过程.     

光声光谱技术用于绿脓杆菌的鉴定

【目的】探索一种无创、快速、可靠、经济地鉴定绿脓杆菌的新方法。【方法】利用光声光谱技术分别对大肠杆菌、绿脓杆菌在35°C下培养24 h后产生的挥发性代谢产物(Bacterial volatile compounds,BVCs)进行连续检测,获得各细菌挥发性气体的光声光谱图谱,并用“逆向思维”的方法对其检测结果进行分析。【结果】利用光声光谱技术对绿脓杆菌、大肠杆菌的挥发性代谢产物进行检测分析,发现绿脓杆菌产生了较高浓度的氰化氢(HCN),而大肠杆菌并未检测出HCN,据此可以对绿脓杆菌进行初步鉴定。【结论】光声光谱技术为绿脓杆菌的鉴定提供了一种简单、快速、经济的方法。为加速其在临床中的应用,提出了“三步走”方案:建立“大数据”、完善“比对算法”、创建“自动检测-比对-校对-鉴定”系统。     

MALDI-TOF-MS方法快速鉴定食品中空肠弯曲菌的技术研究

运用基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of-flight mass spectrometry,MALDI-TOF-MS)技术快速鉴定食品中空肠弯曲菌.通过对该方法的样品前处理的选择、稳定性、特异性等方面进行研究,确定了方法...     

Functionalized quantum dots with dopamine dithiocarbamate as the matrix for the quantification of efavirenz in human plasma and as affinity probes for rapid identification of microwave tryptic digested proteins in MALDI-TOF-MS

Functionalized quantum dots with dopamine dithiocarbamate (QDs-DDTC) were utilized for the first time as an efficient material for the quantification of efavirenz in human plasma of HIV infected patients and rapid identification of microwave tryptic digest proteins (cytochrome c, lysozyme and BSA) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The synthesized QDs-DDTC was characterized by using spectroscopic (UV-visible, FT-IR and (1)H NMR) and microscopic (SEM and TEM) techniques. Functionalized QDs-DDTC exhibited a high desorption/ionization efficiency for the rapid quantification of small molecules (efavirenz, tobramycin and aspartame) at low-mass region. QDs-DDTC has well ability to trap target species, and capable to transfer laser energy for efficient desorption/ionization of analytes with background-free detection. The use of QDs-DDTC as a matrix provided good linearity for the quantification of small molecules (R(2)=~0.9983), with good reproducibility (RSD<10%), in the analysis of efavirenz in the plasma of HIV infected patients by the standard addition method. We also demonstrated that the use of functionalized QDs-DDTC as affinity probes for the rapid identification of microwave tryptic digested proteins (cytochrome c, lysozyme and BSA) by MALDI-TOF-MS. QDs-DDTC-based MALDI-TOF-MS approach provides simplicity, rapidity, accuracy, and precision for the determination of efavirenz in human plasma of HIV infected patients and rapid identification of microwave tryptic digested proteins. This new material presents a marked advance in the development of matrix-free mass spectrometric methods for the rapid and precise quantitative determination of a variety of molecules. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Characterization of bacteria in ballast water using MALDI-TOF mass spectrometry

To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time.     

多重PCR快速检测化妆品中三种致病菌的研究

利用多重PCR技术建立快速检测化妆品中三种致病菌的方法。根据已报道的大肠杆菌phoA基因、铜绿假单胞菌外膜蛋白基因oprL和金黄色葡萄球菌特异性序列SmaI选择特异性引物,对人工染菌化妆品进行多重PCR检测。结果显示,三种致病菌的基因组DNA均可与各自引物特异性结合,扩增产物大小分别为622 bp、504 bp和426 bp。该方法用于人工污染的化妆品中,大肠杆菌的检出限浓度为103 CFU/mL,铜绿假单胞菌和金黄色葡萄球菌的检出限浓度为105 CFU/mL。作者建立的多重PCR方法可同时快速、特异地对化妆品中三种致病菌进行检测,在化妆品行业具有较大的应用价值。     

Applications de la spectrométrie de masse de type MALDI-TOF en microbiologie

In the last few years, matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) has been introduced in clinical laboratories for routine identification. Initially used for research, MALDI-TOF-MS allows rapid and accurate identification of the main microorganisms isolated from clinical samples, by intact cell mass spectrometry or directly from positive blood culture or urines. Other applications are being in processed as bacterial typing, research and detection of antibiotic resistance or particularly virulent strains.     

Rapid detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water using peptide nucleic acid probes

A new chemiluminescent in situ hybridization (CISH) method that provides simultaneous detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water within 1 working day has been developed. Individual micro-colonies of P. aeruginosa were detected directly on membrane filters following 5 h of growth by use of soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeted to a species-specific sequence in P. aeruginosa rRNA. Within each micro-colony, reaction of the peroxidase with a chemiluminescent substrate generated light that was subsequently captured by film or with a digital camera system. Each spot of light represented one micro-colony of P. aeruginosa. Sensitivity and specificity for the identification of P. aeruginosa were 100% as determined by testing 28 P. aeruginosa strains and 17 other bacterial species that included closely related Pseudomonas species. Furthermore, the number of micro-colonies of P. aeruginosa represented by light spots correlated with counts of visible colonies following sustained growth. We conclude that PNA CISH speeds up traditional membrane filtration techniques and adds the specificity of PNA probe technology to generate fast and definitive results.     

Protein separation and characterization by np-RP-HPLC followed by intact MALDI-TOF mass spectrometry and peptide mass mapping analyses

Because of their complexity, the separation of intact proteins from complex mixtures is an important step to comparative proteomics and the identification and characterization of the proteins by mass spectrometry (MS). In the study reported, we evaluated the use of nonporous-reversed-phase (np-RP)-HPLC for intact protein separation prior to MS analyses. The separation system was characterized and compared to 1D-SDS-PAGE electrophoresis in terms of resolution and sensitivity. We demonstrate that np-RP-HPLC protein separation is highly reproducible and provides intact protein fractions which can be directly analyzed by MALDI-TOF-MS for intact molecular weight determination. An in-well digestion protocol was developed, allowing for rapid protein identification by peptide mass fingerprinting (PMF) and resulted in comparable or improved peptide recovery compared with in-gel digestion. The np-RP sensitivity of detection by UV absorbance at 214 nm for intact proteins was at the low ng level and the sensitivity of peptide analysis by MALDI-TOF-MS was in the 10-50 fmol level. A membrane protein fraction was characterized to demonstrate application of this methodology. Among the identified proteins, multiple forms of vimentin were observed. Overall, we demonstrate that np-RP-HPLC followed by MALDI-TOF-MS allows for rapid, sensitive, and reproducible protein fractionation and very specific protein characterization by integration of PMF analysis with MS intact molecular weight information.     

MALDI-TOF质谱技术分析与鉴定病原细菌研究

本文通过基于基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术分析病原细菌的方法进行研究, 阐明影响分析结果的重要因素, 并建立了MALDI-TOF-MS 分析病原细菌的标准方法。对不同属、种和亚种的12株植物病原细菌进行全细胞分析结果表明：MALDI-TOF-MS能快速而准确的区分和鉴定病原细菌, 分析过程简单、灵敏度高。此法在细菌属、种、亚种和菌株水平上, 可快速、准确地区分和鉴定。     

Detection of Pseudomonas aeruginosa from clinical and environmental samples by amplification of the exotoxin A gene using PCR.

PCR was used to detect Pseudomonas aeruginosa from water samples by amplifying a 396-bp region of the exotoxin A (ETA) structural gene sequence. The identify of the amplified 396-bp fragment was confirmed by digesting it with PvuI restriction endonuclease, which produced the predicted 246- and 150-bp fragments. Specific primers amplified ETA-positive P. aeruginosa DNA, whereas other species of Pseudomonas and GC-rich bacteria did not yield any 396-bp fragment. The specificity and sensitivity of the assay were 100 and 96%, respectively, which confirms the assay's reliability for diagnostic and epidemiological studies. The assay can detect as few as 5 to 10 cells in a 10-ml water sample or 0.1 pg of P. aeruginosa DNA per reaction mixture (5 microliters) by ethidium bromide staining of an agarose gel. Ten-times-lower concentrations were detected by hybridization with a digoxigenin-labeled oligonucleotide probe internal to the PCR product. With this PCR method, ETA-positive P. aeruginosa was detected in animal cage water samples at a level of 40 cells per ml. This method is rapid and less cumbersome than other diagnostic methods for the identification of P. aeruginosa strains. The method described can be used to detect a low level of P. aeruginosa from environmental and clinical samples without the use of selective media or additional biochemical tests.