种特异性引物鉴定申克孢子丝菌

目的 研究申克孢子丝菌的分子鉴定方法,为孢子丝菌感染的分子诊断奠定基础.方法 对22株申克孢子丝菌和12种12株暗色真菌临床分离株的核糖体DNA(rDNA)内转录间隔区(ITS)进行聚合酶链反应扩增.对10株来源于中国不同地区及1株来源于美国的申克孢子丝菌的扩增产物测序并进行分析,以获得的ITS2区序列为靶序列设计出申克孢子丝菌的种特异性引物(SSP),并用该引物扩增全部受试菌株.结果 序列分析显示,申克孢子丝菌rDNA的ITS2区相当保守,特异性引物对22株申克孢子丝菌可扩增出一条300bp的片段,其他受试菌株均为阴性.结论 应用设计出的种特异性引物,结合PCR方法,鉴定申克孢子丝菌特异、敏感、可靠,可用于临床诊断.     

四对孢子丝菌引物的特异性和敏感性评价

目的 筛选对孢子丝菌特异且敏感的引物。方法 以50株不同地区来源的孢子丝菌临床分离株及标准株的基因组DNA作为研究样本,以毛霉、烟曲霉、念珠菌临床分离菌株基因组DNA作为对照,通过特异引物PCR扩增的方法筛选国内外文献中报道的4对孢子丝菌引物。所有受试孢子丝菌菌株均出现同一扩增产物,而对照菌株未出现者记为特异性引物,随后将基因组DNA模板倍比稀释后依次行PCR扩增,记录各引物出现阳性扩增结果时的最小模板浓度,检测敏感性。结果 在相同PCR条件下,引物S2-R2、SSHF31-SSHR97及ITS3-SSP具有较好特异性,而引物SS3-SS4对孢子丝菌及念珠菌菌株均可扩增出同样大小的PCR产物。引物S2-R2的敏感性最好,基因组DNA模板浓度为5 pg/μL时即可被检出。结论 针对几丁质合成酶1基因的引物S2-R2为孢子丝菌特异且敏感的引物。     

阴囊孢子丝菌病1例及申克孢子丝菌基因型检测

孢子丝菌病是由申克孢子丝菌引发的一种皮肤、皮下组织和附近淋巴管的慢性感染性疾病,多发于四肢等容易暴露受伤的部位.本文报道1例发生于阴囊的固定型皮肤孢子丝菌病,并对申克孢子丝菌进行了形态学和分子生物学水平的鉴定.     

一株引起皮肤播散性孢子丝菌病的孢子丝菌临床分离株的基因型鉴定

孢子丝菌病是由申克孢子丝菌引起的常见深部真菌病,临床多见于淋巴管型及固定型,播散性孢子丝菌病少见.孢子丝菌进入人体后引起不同临床类型的孢子丝菌病与机体免疫状态有关^[1],但是否与菌型有关尚无定论.为此,我们从一皮肤播散性孢子丝菌病患者皮损中分离1株孢子丝菌菌株,利用常规真菌学和分子生物学方法对其进行鉴定,并探讨该菌株与皮肤淋巴管型孢子丝菌在基因水平上的异同.     

球形孢子丝菌致面部孢子丝菌病

正患者女,56岁,农民。主诉:右面部红色丘疹、斑块1年。现病史:患者于1年前无明显诱因右颊部出现一个针尖大小红色丘疹,后其周围出现类似几个丘疹,并逐渐增大融合成斑块,部分上覆少量黄色和暗红色痂,无明显瘙痒及疼痛。患者曾于外院就诊,具体诊断不详,未予治疗。后类似皮疹逐渐增多,向下蔓延至颏部     

应用PCR技术快速鉴定申克孢子丝菌的实验研究

采用Biospin真菌基因组DNA提取试剂盒提取基因组DNA,用申克孢子丝菌种特异性引物分别对21株申克孢子丝菌的基因组DNA进行PCR扩增.成功提取21株申克孢子丝菌基因组DNA,并用申克孢子丝菌种特异性引物分别从21株申克孢子丝菌基因组DNA中获得长度为320 bp的扩增产物.以申克孢子丝菌特异引物为基础的聚合酶链反应法鉴定申克孢子丝菌简便、快捷、特异,可用于临床诊断.     

申克孢子丝菌基因差异、致病力与孢子丝菌病临床型别的关系

目的 探讨申克孢子丝菌基因差异、致病力与孢子丝菌病不同临床型别的关系。方法 ①收集不同临床型别孢子丝菌病的申克孢子丝菌分离株并提取DNA,进行随机扩增多态DNA(RAPD)扩增。②BALB/c小鼠接种不同临床型别孢子丝菌病的分离株菌悬液,观察实验动物发病及病变情况。③发病小鼠皮肤及内脏组织病理学检查,观察接种不同临床型别孢子丝菌病的分离株菌悬液后小鼠病变内申克孢子丝菌孢子数量及分布。结果 ①不同临床型别孢子丝菌病的申克孢子丝菌分离株聚合酶链反应产物电泳带型差异较明显:播散型分离株可见1800bp、850bp、500bp、180bp,皮肤淋巴管型分离株见1400bp、800bp、700bp、500bp,皮肤固定型分离株见2500bp、1400bp、1000bp、700bp。②注射播散型孢子丝菌病分离株菌悬液的BALB/c小鼠比注射皮肤淋巴管型分离株小鼠发病早、病变部位广且死亡率高;注射皮肤淋巴管型分离株的小鼠较注射固定型孢子丝菌病分离株小鼠皮损出现早、病变范围广且严重。③实验BALB/c小鼠病变皮肤及内脏组织病理学检查显示:注射播散型孢子丝菌病分离株的小鼠病变内孢子数量明显多于注射皮肤淋巴管型分离株小鼠病变内孢子数量,而后者较注射固定型孢子丝菌病分离株的小鼠病变内孢子数量多。结论 不同临床型别孢子丝菌病的申克孢子丝菌的基因差异、致病力与孢子丝菌病不同临床型别的关系密切。     

孢子丝菌病研究进展

【摘要】 世界卫生组织将孢子丝菌病归为“被忽视的热带病”,近年来对孢子丝菌病相关的研究逐渐增多,本文从病原体、流行病学、诊断、治疗、疫苗这五个方面综述孢子丝菌病的近期研究进展。     

以多发溃疡为表现的孢子丝菌病1例

患者男,65岁.因左小腿伸侧近踝部溃疡1年,于2008年10月来我科就诊.患者1年前左小腿伸侧近踝部被铁丝刺伤后形成溃疡,伤口难以愈合,曾在我院外科予以清创处理,溃疡渐愈合,但原溃疡部位渐出现红斑、水疱、渗出,于多家医院按湿疹外用糖皮质激素治疗.效果不佳.皮损反复,并出现溃疡、结痂,遂收住我院治疗.既往有原发性高血压病10年,20年前曾行阑尾切除术,否认药物过敏史.     

Rapid identification of Sporothrix schenckii in biopsy tissue by PCR

Background The dimorphic fungus Sporothrix schenckii is the etiological agent of sporotrichosis, an important cutaneous mycosis with a worldwide distribution. At present, it is challenging to rapidly discover and identify Sporothrix schenckii in biopsy tissues nowadays. Aims To explore new methods for rapid diagnosis of sporotrichosis. Materials and Methods We screened specific primers for Sporothrix schenckii using 50 clinical isolates from patients with sporotrichosis. DNA was extracted from the lesions of 30 cases of clinically suspected sporotrichosis using the Graham s method of CTAB and amplified by PCR using the screened specific primers. Results The primer S2‐R2 was applicable for the identification of S. schenckii from different geographic areas and clinical types with high specificity and sensitivity. Twenty‐five out of the thirty cases (83.3%) amplified using the primer S2‐R2 showed positive bands. Further positive bands were observed in 95.6% of cases tested positive by fungal culture. Conclusions Using the PCR technique and specific primers, we developed a new diagnostic method that can rapidly diagnose sporotrichosis with tissues obtained from clinical biopsies.     

无毛小鼠接种申克孢子丝菌检测抗申克孢子丝菌细胞外蛋白酶抗体研究

通过申克孢子丝菌接种无毛小鼠（Ｈａｉｒｌｅｓｍｉｃｅ）来检测两种细胞外蛋白酶的血清抗体。在小鼠皮内注射申克孢子丝菌并形成结节，镜下示真菌消失，并在每周通过酶联免疫吸附试验（ＥＩＡ）检测蛋白酶Ⅰ和Ⅱ的血清抗体。结果显示抗体滴度与实验小鼠孢子丝菌病肉眼和镜下观察的变化趋势相一致。本试验证明了申克孢子丝菌在体内能产生蛋白酶Ⅰ和Ⅱ，而且蛋白酶的抗体可用于孢子丝菌病的血清学诊断。     

In vitro activity of itraconazole in combination with terbinafine against clinical strains of itraconazole-insensitive Sporothrix schenckii

Four strains of Sporothrix schenckii were isolated from patients with cutaneous sporotrichosis who failed itraconazole (ITC) treatment. To investigate the susceptibility of these strains to ITC and terbinafine (TRB) alone and in combination in 2 growth phases in vitro, a checkerboard microdilution method was used in accordance with the recommendations of the Clinical Laboratory Standards Institute (CLSI) was used in our study. The drug interaction was evaluated by assessing the fractional inhibitory concentration index (FICI). The geometric means (GMs) of the minimum inhibitory concentrations (MICs) of 4 insensitive strains were obviously higher than those of the sensitive isolates used for comparison. The FICI analysis revealed that only 2 isolates (25%) were synergistic in yeast form. Our results indicate the failure of clinical treatments might be caused by the insensitivity to ITC of these strains.     

Immunoperoxidase localization of Sporothrix schenckii and Cryptococcus neoformans. Staining of tissue sections fixed in 4% formaldehyde solution and embedded in paraffin.

An indirect immunoperoxidase staining method has been successfully applied to 4% formaldehyde solution tissue sections fixed in and embedded in paraffin for the localization of Sporothrix schenckil and Cryptococcus neoformans without prior trypsinization of tissue sections. A comparison of this method with an analogous immunofluorescence staining technique has been made.     

32株孢子丝菌临床株两种基因分型方法比较

目的 确定哪种孢子丝菌基因分型的方法分辨力更高,所分基因型与临床表现及地域分布相关性更好。方法 以32株不同地区及临床类型的孢子丝菌菌株为研究样本,分别进行mtDNA RFLP分型,基因组DNA RAPD分型和rDNA RFLP-Southern blotting杂交分型,并进行比较。结果 mtDNA-RFLP方法将受试菌株分为5型,分别对应Ishizaki等命名的1,4,6,7,20五种基因型;RAPD方法将受试菌株分为7种基因型（I-VII型）; rDNA RFLP-Southern blotting杂交方法将受试菌株分为15种基因型（A-O型）,且其所分基因型与菌株的南北方分布及临床表现有一定相关性。结论 三种孢子丝菌基因分型方法中,rDNA RFLP-Southern blotting杂交方法分辨力较高,所分基因型与临床表现及地域分布相关性更好。     

Detection of mycobacterial DNA in cervical granulomatous lymphadenopathy from formalin-fixed, paraffin-embedded tissue by PCR

Cervical tuberculous lymphadenitis is the most common form of inflammatory neck mass in Korea. The diagnosis of tuberculosis requires proof of the presence of Mycobacterium tuberculosis by acid-fast staining or bacterial growth in culture. However, these are often difficult in cervical tuberculous lymphadenitis. The aim of this study was to investigate the value of the polymerase chain reaction (PCR) technique for detection of mycobacteria in routinely processed tissue sections of cervical granulomatous lymphadenopathy. In this retrospective study, twenty formalin-fixed, paraffin-embedded biopsy specimens from clinically and/or histopathologically diagnosed cervical granulomatous lymphadenopathy were analyzed for mycobacterial DNA by PCR. Two different primers to amplify mycobacterial-common 383-base pair (bp) DNA and Mycobacterium tuberculosis-complex-specific 123-bp DNA were used. Positive PCR products were sequenced directly. Mycobacterial-common DNA (383-bp positive) was found in 10 of the 20 cases. Among them, 7 cases were PCR positive with both primer sets. These seven cases can be considered as tuberculosis. The other three cases indicated possible atypical mycobacteriosis. PCR is a useful technique for the demonstration of mycobacterial DNA fragments in patients with clinically suspected cervical tuberculous lymphadenitis who have acid fast-negative histology and/or unsuccessful mycobacterial cultures.     

寻常型银屑病患者血清S100A13的检测及意义

目的:检测寻常型银屑病患者血清钙结合蛋白S100A13水平.方法:酶联免疫吸附法(ELISA)测定寻常型银屑病患者和健康人群血清S100A13水平.结果:轻症20例寻常型银屑病患者组血清水平为(66.172±22.076)μg/L,重症20例寻常型银屑病患者组为(168.492±101.127)μg/L,正常对照组血清水平为(40.075±10.338)μg/L,组间差异有统计学意义(P＜0.01).结论:寻常型银屑病皮损的过度增殖和异常分化可能与S100A13的大量上调及分泌有关.