PCR法检测正常女性宫颈中的溶脲脲原体生物群

目的了解正常妇女宫颈中溶脲脲原体两个生物群的分布及药物敏感性。方法采用PCR技术对妇科门诊的正常妇女宫颈的溶脲脲原体两个生物群进行检测。结果50例正常妇女经PCR-CE检测,21例(42.0%)溶脲脲原体阳性其中溶脲脲原体生物群1阳性16例(76.2%),生物群2阳性5例(23.8%)。结论溶脲脲原体生物群1可能是女性生殖道一种正常菌群。而溶脲脲原体生物群2能引起妇科疾病从而导致不孕。     

应用Real-time PCR检测溶脲脲原体生物群的实验研究

目的建立一种检测溶脲脲原体两个生物群的快速简便方法。方法采用Real-time PCR技术对50例男性科门诊的尿道炎患者中的溶脲脲原体的两个生物群进行了检测。结果50例男性患者经Real-time PCR技术检测,溶脲脲原体阳性23例（36.9%）,在这23例溶脲脲原体阳性男性患者中,溶脲脲原体生物群1所占的比例为14%,溶脲脲原体生物群2所占的比例是32%,溶脲脲原体生物群1,2均阳性所占的比例为2%。结论Real-time PCR方法是检测溶脲脲原体两个生物群的一个快速,简便的方法。     

解脲脲原体生物群聚合酶链反应-毛细管电泳检测方法的建立及应用

目的 建立一种检测解脲脲原体生物群1、2的聚合酶链反应-毛细管电泳(PCR-CE)方法,研究解脲脲原体生物群与男性非淋菌性尿道炎(NGU)的关系。方法 合成解脲脲原体生物群1、2的通用引物,进行PCR,然后使用CE检测解脲脲原体生物群1、2的扩增产物。对该方法进行敏感性、特异性的评价。使用该方法检测不同男性人群尿样中解脲脲原体2个生物群。结果 PCR-CE法的敏感性为10拷贝/50μl。该方法仅特异扩增解脲脲原体生物群1、2。解脲脲原体生物群2在NGU组、非沙眼衣原体引起的NGU(NCNGU)组中的检出率高于对照组(P<0.05),解脲脲原体生物群1在NGU及NCNGU组中的检出率和对照组无差异(P>0.05)。结论 建立了一种敏感性高、特异性强、分辨率高的检测解脲脲原体2个生物群的PCR-CE方法。解脲脲原体生物群2与男性NGU有一定的关系。解脲脲原体生物群1不能引起男性NGU。     

PCR-CE检测溶脲脲原体生物群的实验研究

建立了一种通用引物-PCR-CE检测溶脲脲原体2个生物群的方法,对这个方法进行了敏感性、特异性及重复性的研究,并与通用引物-PCR-琼脂糖凝胶电泳法进行了比较。研究表明,通用引物-PCR-CE法敏感性高,特异性强,重复性好,在区分溶脲脲原体2个生物群能力上要优于通用引物-PCR-琼脂糖电泳法。     

解脲脲原体的生物群、血清型与致病

解脲脲原体可以寄居于人泌尿生殖道,在性成熟无症状妇女40%～80%子宫颈或阴道内携带解脲脲原体,感染多见于年青、多性伴及口服避孕药者.作为一种条件致病微生物,女性子宫颈处的解脲脲原体不一定引起临床上可见的炎症改变,其致病性与多种因素有关,如宿主免疫力、性伴数、血清型别等,如果无明显的临床体征,要考虑这些因素的影响,本文主要论述其致病性与可能血清型别的关系.     

解脲脲原体生物学特性的研究

本文研究了4株解脲脲原体的生物学特性,发现解脲脲原体在不含血清的液体培养基中加有青霉素3.1×10³—1.2×10⁴u/ml即可生长2—4代;在半固体培养基培养时,除指示剂变色外无肉眼可见生长现象。解脲脲原体不同株对醋酸铊敏感性不一,当浓度<0.1mg/ml时,无抑制生长作用。     

对不同浓度解脲脲原体的药敏结果分析

目的 了解不同浓度解脲脲原体＜10^4CFU/ml和≥10^4CFU/ml耐药率情况.方法 采用珠海黑马生物工程有限公司集分离、培养、计数、药敏于一体的试剂盒,按说明进行操作.结果 交沙霉素、美满霉素、强力霉素的总耐药率较低,左旋氧氟沙星、司帕沙星、罗红霉素、阿奇霉素、克林霉素对不同浓度解脲脲原体的耐药率差异有高度显著性.结论 交沙霉素、美满霉素、强力霉素可作为治疗解脲脲原体的首选药物,克林霉素总耐药率已高达57.7%（164/284）,不适宜用于解脲脲原体的治疗.珠海黑马产品质量优于其它同类产品.     

慢性阴道炎患者生殖道解脲脲原体和沙眼衣原体感染分析

目的通过荧光定量聚合酶链反应(PCR)技术检测慢性阴道炎患者阴道内解脲脲原体(Uu)和沙眼衣原体(Ct)感染状况,用以指导临床正确治疗。方法应用荧光定量聚合酶链反应对380例慢性阴道炎患者的阴道分泌物进行解脲脲原体和沙眼衣原体PCR检测。结果解脲脲原体阳性率为42.9%,其阳性标本的平均拷贝数为3.4×105copies/ml,沙眼衣原体阳性率为16.6%,其阳性标本的平均拷贝数为5.8×104copies/ml,解脲脲原体和沙眼衣原体合并感染的阳性率为12.6%。结论PCR技术具有简便、快捷、准确的优点,是目前快速诊断慢性阴道炎患者Uu、Ct感染状况的可靠诊断方法之一。     

反相斑点杂交法对解脲脲原体分型的研究

目的研究以聚合酶链反应为基础的快速检测与鉴定解脲脲原体基因型的方法。方法选择2003年11月至2005年11月在中山大学附属第二医院门诊就诊的有外阴阴道炎症状和体征的患者601例,设为病例组,同期无自觉症状的正常体检人群306例,设为对照组,分别取宫颈分泌物待检测。将解脲脲原体不同基因型的特异探针固定在硝酸纤维素膜上,临床标本按常规方法提取解脲脲原体DNA,采用生物素标记的解脲脲原体特异通用引物PCR扩增DNA,然后分别与解脲脲原体不同基因型特异探针杂交、显色。结果病例组解脲脲原体阳性421例占70.0%,对照组解脲脲原体阳性126例占41.2%。病例组中单型别感染的U.parvum占65.4%,其中1型、3型、6型和14型分别占28.8%、43.3%、26.0%和1.9%,U.urealyticum占18.4%;对照组中单型别感染的U.parvum占79.3%,其中1型、3型、6型和14型分别占63.2%、21.1%、15.7%和0.0%,U.urealyticum占13.8%。18例阳性标本随机DNA测序鉴定,均为相应的解脲脲原体基因型。结论U.parvum群,尤其是其中的1、3、6型别是正常人群携带的可能性较大,U.urealyticum则有可能和1型起协同作用或独自导致疾病。用反相斑点杂交进行解脲脲原体基因分型,方法简单、实用,适用于临床。     

棋盘稀释法在解脲脲原体药敏试验中的应用

解脲脲原体是引起泌尿生殖道感染的重要病原体。目前使用的药敏试验方法即药敏卡法没有考虑样本中脲原体的实际含量而存在局限性。本研究将棋盘稀释法应用于解脲脲原体的药敏试验,可获得脲原体的准确耐药情况,有助于临床治疗中抗生素的正确选择;同时,有利于控制耐药菌株的产生和在人群中的扩散。     

甲苯胺蓝介导的超声疗法对牙菌斑中细菌灭活效果的研究

目的：验证甲苯胺蓝介导超声对龈上牙菌斑中细菌的抑制作用。方法：从门诊20例牙周炎病人下前牙区取龈上菌斑一份,每个患者的菌斑于实验室培养后,在同一甲苯胺蓝浓度下与不同超声强度联合作用,利用涂板计数法分析其对菌斑中细菌的抑制作用。实验分组：空白组、单独甲苯胺蓝组、单独超声组、甲苯胺蓝＋超声组。结果：单独超声作用时没有灭菌效果,细菌数目反而有所增长（P〈0.05）,超声联合甲苯胺蓝作用时取得明显的灭菌效果,超声强度2W/CM2灭菌率可达到79%。结论：低强度超声能够促进细菌生长,甲苯胺蓝介导一定强度的超声疗法对龈上牙菌斑中的细菌有良好的抑制作用。     

溶脲脲原体及其两个生物群与非淋菌性尿道炎相关性的研究

目的 阐明溶脲脲原体及其2个生物群与非淋菌性尿道炎的关系。方法 使用通用引物-PCR-毛细管电泳法对淋菌性尿道炎组,非淋菌性尿道炎组和对照组中的溶脲脲原体的2个生物群进行检测。结果 溶脲脲原体生物群2在非淋菌性尿道炎中的检出率高于对照组（P〈0．05）,溶脲脲原体生物群1在淋菌性尿道炎中的检出率低于对照组（P〈0．05）,而在非淋菌性尿道炎和对照组中,溶脲脲原体生物群1的检出率差异无显著性（P〉0．05）。结论 溶脲脲原体生物群2是和非淋菌性尿道炎有一定关系的,溶脲脲原体生物群2可能才是引起非淋尊性尿道炎的病原体之一,而生物群1不引起非淋菌性尿道炎,淋球菌的增殖有可能抑制尿道中的溶脲脲原体生物群1的生长。     

Level of colonization by Ureaplasma urealyticum of definite biovars in a group of women with different clinical symptoms

To evaluate the level of U. urealyticum colonization of female urogenital tract, the method of the multiplex polymerase chain reaction (PCR) in the presence of two pairs of primers, corresponding to genes controlling U. urealyticum 16S rRNA and unique human osteopontin was used. The study of 93 clinical specimens showed no correlation between high colonization level and the presence of definite clinical manifestations of U. urealyticum infection. The determination of ureaplasmic biovars was carried out by the method of PCR in the presence of 3 primers corresponding to the multiple-banded antigen (MBA) gene. Biovar parvo was detected in 85% of the specimens, biovar T960 in 11% and both biovars were detected in 4% of the specimens. The biovar distribution in the groups of women with different clinical symptoms was approximately similar. U. urealyticum of biovar T960 occurred more frequently (33% of the specimens) only in a group of women with vaginal discharge characteristic of inflammation.     

The photodynamic effect of methylene blue and toluidine blue on <Emphasis Type="Italic">Candida albicans</Emphasis> is dependent on medium conditions

Due to the increased number of immunocompromised patients, the infections associated with the pathogen of the genus Candida and other fungi have increased dramatically. Photodynamic antimicrobial chemotherapy (PACT) has been presented as a potential antimicrobial therapy, in a process that combines light and a photosensitizing drug, which promotes a phototoxic response by the treated cells. In this work, we studied the effects of the different medium conditions during PACT, using either methylene blue (MB) or toluidine blue (TB) on Candida albicans. The inhibition of the growth produced by PACT was decreased for different pH values (6.0, 7.0, and 8.0) in a buffered medium. The phototoxic effects were observed only in the presence of saline (not buffered medium). PACT was modulated by calcium in a different manner using either MB or TB. Also when using MB both verapamil or sodium azide were able to decrease the phototoxic effects on the C. albicans. These results show that PACT is presented as a new and promising antifungal therapy, however, new studies are necessary to understand the mechanism by which this event occurs.     

Gene responsible for protecting Escherichia coli from sodium dodecyl sulfate and toluidine blue plus light.

Escherichia coli cells were killed by visible light irradiation in the presence of the photosensitizing dye, toluidine blue. Two uvrB mutant strains of E. coli K-12 (AB1885 and N3-1) were much more sensitive than the isogenic uvrA and uvrC strains to treatment with toluidine blue plus light, suggesting that the uvrB+ gene product was involved in repair of DNA damage induced by the treatment. The uvrB+ gene cloned in a high- or low-copy-number plasmid was transformed into the uvrB strain (AB1885). Although all the transformants showed the same resistance as its wild-type strain (AB1157) to UV irradiation, they were as sensitive as AB1885 was to treatment with toluidine blue plus light. The two uvrB strains were more sensitive to sodium dodecyl sulfate than the other strains, suggesting that these strains had a defect in the cell surface. A sodium dodecyl sulfate-resistant revertant obtained from AB1885 was more resistant than AB1885 was to treatment with toluidine blue plus light. The two uvrB strains (AB1885 and N3-1) appear to have a defective gene (tentatively called dvl) different from uvrB. Its map position was around 7 min on the E. coli map.     

解脲脲原体耐药性研究进展

解脲脲原体是条件致病菌。目前对其耐药性的研究主要包括对喹诺酮类、大环内酯类和四环素类3种抗菌药物相关耐药突变位点的检测,以及生物膜对病原体相关药物敏感性的影响,研究方法和检验手段仍较为传统、局限,研究方案也仅停留在对前人实验的重复。近年来,有学者将多位点序列分型技术用于解脲脲原体耐药序列类型的研究。在完善耐药机制研究的基础上,如何实现对耐药株的快速鉴定,从而指导抗菌药物的合理选择等仍需进一步研究。     

Mutagenic potential of toluidine blue evaluated in the Ames test.

Toluidine blue is a vital, metachromatic thiazine dye which is used as an adjunct in clinical examination for the early detection of asymptomatic recurrent or secondary primary carcinoma in individuals who are at high risk for developing oral cancer. Because available data on the mutagenicity of toluidine blue was limited and contradictory, this study was conducted to evaluate the mutagenic potential of toluidine blue in the in vitro Ames Salmonella test. Tester strains TA97a, TA98, TA100 and TA102 were used. Toluidine blue was tested at concentrations of 0.1, 1.0, 10, 50, 100, 250 and 500 micrograms/plate, with and without S9 microsomal activation, and positive and negative controls were included. Results from tests without S9 showed a significant increase (p less than 0.05) in number of revertants in TA102 and in TA97a with 50 and 100 micrograms toluidine blue/plate, respectively. In tests with S9 activation, doses of toluidine blue ranging from 10 to 250 micrograms/plate induced dose-related increases in the number of revertants in all 4 strains. The results of this study indicate that toluidine blue has a mutagenic effect in the Ames test.     

Attaching NorA efflux pump inhibitors to methylene blue enhances antimicrobial photodynamic inactivation of Escherichia coli and Acinetobacter baumannii in vitro and in vivo

Resistance of bacteria to antibiotics is a public health concern worldwide due to the increasing failure of standard antibiotic therapies. Antimicrobial photodynamic inactivation (aPDI) is a promising non-antibiotic alternative for treating localized bacterial infections that uses non-toxic photosensitizers and harmless visible light to produce reactive oxygen species and kill microbes. Phenothiazinium photosensitizers like methylene blue (MB) and toluidine blue O are hydrophobic cations that are naturally expelled from bacterial cells by multidrug efflux pumps, which reduces their effectiveness. We recently reported the discovery of a NorA efflux pump inhibitor-methylene blue (EPI-MB) hybrid compound INF55-(Ac)en–MB that shows enhanced photodynamic inactivation of the Gram-positive bacterium methicillin-resistant Staphylococcus aureus (MRSA) relative to MB, both in vitro and in vivo. Here, we report the surprising observation that INF55-(Ac)en–MB and two related hybrids bearing the NorA efflux pump inhibitors INF55 and INF271 also show enhanced aPDI activity in vitro (relative to MB) against the Gram-negative bacteria Escherichia coli and Acinetobacter baumannii, despite neither species expressing the NorA pump. Two of the hybrids showed superior effects to MB in murine aPDI infection models. The findings motivate wider exploration of aPDI with EPI-MB hybrids against Gram-negative pathogens and more detailed studies into the molecular mechanisms underpinning their activity.     

Detection of double-stranded RNA-protein interactions by methylene blue-mediated photo-crosslinking.

Double-stranded(ds) RNA-binding proteins have diverse functions in the cell. An obstacle to investigating the interactions between these proteins and dsRNA is the relative inefficiency of traditional UV-crosslinking methods for extended regions of dsRNA. We have therefore developed an alternative procedure for RNA-protein photo-crosslinking that efficiently induces RNA-protein crosslinks in double-stranded regions of RNA. We show that dsRNA-protein crosslinks can be induced by visible light in the presence of the dye methylene blue, which most likely mediates crosslinking by intercalating in the dsRNA helix. A recombinant dsRNA binding domain from the Drosophila staufen protein and human protein kinase R were crosslinked by UV or methylene blue to a series of dsRNAs. In each case, the degree of crosslinking was greater with methylene blue, particularly with RNAs with few single-stranded loops. Methylene blue-mediated crosslinking therefore complements and extends the existing repertoire of crosslinking methods for detecting RNA-protein interactions.