【作者】 李晓红；

【导师】 陈世义；

【作者基本信息】 吉林大学 ， 皮肤病学与性病学， 2004， 硕士

【摘要】 孢子丝菌病(sporotrichosis)是由在世界范围内广泛分布的申克孢子丝菌(Sporothrix schenckii)所致的慢性深部真菌感染性疾病，申克孢子丝菌是一种土壤、木材及植物的腐生菌，它主要侵犯皮肤和皮下组织，引起非特异性肉芽肿,形成皮下结节,大多沿淋巴管分布。本菌可通过血流播散，在长期应用皮质类固醇激素、酗酒或HIV感染等免疫力低下的个体，可累及肺脏、脑膜及骨骼等多个器官，常因病势凶险，诊断和治疗困难而危机生命。近年来，本病的发病率有上升的趋势，艾滋病患者感染申克孢子丝菌的报道也明显增多，这对本病的早期诊断提出了更高的要求。本病的传统诊断包括真菌镜检、真菌培养、组织病理等方法，不仅费时费力，且诊断阳性率不高。尽管荧光性抗体试验或免疫组化技术也为孢子丝菌病提供了快速的诊断方法，但目前它们还不能被大多数临床实验室所采用。因此寻求一种简单、可靠、特异的实验方法来检测和鉴定申克孢子丝菌一直为国内外的学者所关注。最近的许多研究表明，迅速发展的分子生物学技术正在临床疾病的诊断中占据着越来越重要的位置，大有终将替代传统培养方法之趋势。目前国外市场上已有商品化的白念珠菌，烟曲霉等真菌PCR检测试剂盒销售，因此关于申克孢子丝菌的分子诊断方<WP=38>面的研究也成为人们关注的焦点。 本实验的研究目的在于建立一种申克孢子丝菌的种特异性引物聚合酶链反应鉴定方法，从而为临床孢子丝菌病的分子诊断奠定基础。我们采用根据申克孢子丝菌几丁质合成酶基因1序列设计合成的一对寡核苷酸引物CHS1（S2-R2）, 建立检测申克孢子丝菌CHS1基因的PCR法，CHS1（S2-R2）引物是以几丁质合成酶基因1的保守区为靶目标，扩增的是CHS1的保守顺序，以往的实验证明这对引物不能扩增细菌和人类基因的DNA，它们在CHS1基因序列的位置分别是174-199bp和468-492bp，针对引物扩增的序列长度为318bp。本研究共对26株申克孢子丝菌Sp0-25（包括1株标准株Sp0，7株实验室保存株Sp1-7，18株临床分离株Sp8-25）及6种6株普通真菌（分属于3个菌属）的基因组DNA进行了PCR扩增。申克孢子丝菌的临床分离株来自我院皮肤科首诊为孢子丝菌病的患者，经SDA斜面及平皿培养基初步培养，可疑申克孢子丝菌的菌株以划线法移种于BHIA斜面培养基上，经双相转化试验确证后编码登记，用于PCR。所有编码的菌株均经转种获得纯培养菌落。同时为保证真菌PCR技术及其相关技术的顺利开展，本研究还对来自于同一批培养基的4株申克孢子丝菌(Sp1,Sp2,Sp8,Sp9)及所有普通真菌菌株分别采用研磨-CTAB（十六烷基三甲基溴化胺）法及碱性异硫氰酸胍沸腾法抽提基因组DNA，并用紫外分光光度计测定基因组DNA样品OD260和OD280值，计算提取样品的浓<WP=39>度并估计核酸的纯度，然后采用配对资料t检验的统计学方法对这两种病原真菌基因组DNA提取方法的提取效果进行比较，为临床真菌标本的前期处理提供了有效参考。本研究的实验结果如下:(1)、CHS1（S2-R2）引物可选择性扩增26株申克孢子丝菌，其扩增产物是一大小为318bp的片段，而对念珠菌属、毛癣菌属、小孢子菌属的6株普通真菌基因组DNA包括白念珠菌(Candi A)、光滑念珠菌 (Candi G)、红色毛癣菌(Tr)、石膏样小孢子菌(Mg)、须癣毛癣菌(Tm)、犬小孢子菌(Mc),经相同方法扩增后均未见特异性条带。(2)、分别采用碱性异硫氰酸胍沸腾法和研磨－CTAB（十六烷基三甲基溴化胺）法提取各样本真菌基因组DNA，两者DNA样品的浓度差异有统计学意义(P<0.05)。 碱性异硫氰酸胍沸腾法提取的DNA浓度及纯度均高于研磨－CTAB（十六烷基三甲基溴化胺）法。通过实验我们认为：1、采用聚合酶链反应法鉴定申克孢子丝菌敏感、特异、简便、快捷，可用于临床诊断。2、申克孢子丝菌种特异性引物PCR法的建立，为孢子丝菌病的分子诊断奠定了基础。3、碱性异硫氰酸胍沸腾法提取病原性真菌基因组DNA具有简便、快速、价廉等特点，适用于病原真菌分子生物学研究的标本前期处理。更多还原

【Abstract】 Sporotrichosis is a chronic granulomatous fungal infection caused by Sporothrix schenckii with worldwide distribution. Sporothrix schenckii is a saprophyte in nature，is commonly found on vegetation or in soil.It is mainly encroached on the cutaneous and subcutaneous tissues,and presented a nonspecific granulomatous nodules. It usually spreads along the lymphatic vessels.The pulmonary and hematogenous disseminated forms of sporotrichosis，which are especially prevalent in immunocompromised persins such as HIV，can be crippling or fatal. Recently, the incidence of sporotrichosis is increasing and it needs a more effective method of early diagnosis of the disease. The conventional method for definitive diagnosis of sporotrichosis is based on time-consuming tissue culture，However，It frequently yield negative results.Although the fluorescent antibody or immunohistochemical techniques also provide a rapid diagnosis of sporotrichosis ,they are not available in most clinical laboratories.so the development of a easy, reliable ,specific and rapid detection system of Sporothrix <WP=41>schenckii would be very useful.In recent years, molecular techniques are starting to play an increasingly important role in fungal disease diagnosis. Molecular analyses such as PCR techniques have greatly improved the diagnosis of Candida albicans,However，a specific detection system of Sporothrix schenckii has not been applied in clinical laboratories.The aim of the present study was to establish a rapid method to detect and identify Sporothrix schenckii by using Species-specific oligonucleotide primer PCR techniques， and lay the fundation of molecular diagnosis for sporotrichosis. The Species-specific oligonucleotide primer pair S2-R2 was used in this study. It were designed from nucleotide sequences of chitin synthase 1 gene in Sporothrix schenckii. The departed essay proved that the primer pair S2-R2 did not amplify DNA from human cells and bacteria.Their location in Sporothrix schenckii CHS1 gene were 174-199bp and 468-492bp.Polymerase chain reaction analysis with the primer pair S2-R2 was able to amplified a 318bp fragment. For the examination of specificity, 26 strains of Sporothrix schenckii ,6 strains of clinical common fungi was <WP=42>amplified by PCR. 26 strains of Sporothrix schenckii include 1 strain of standard,7 strains of laboratory reserved and 18 strains of clinical isolated . 6 strains of clinical common fungi include Candida albicans , Candida glabrata , Trichophyton rubum , Microsporum gypseum , Trichophyton mentagrophyts and Microsporum canis . Clinical isolates of Sporothrix schenckii obtained from sporotrichosis,they were confirmed by tissue cultures.All strains of clinical isolated were grown on a Sabouraud medium at 25℃ for one or two weeks,then identified by double phase transformation assay.To guarantee the smoothly developing of fungal genomic DNA prepared in fungal study for PCR techniques,We used two method for extraction of fungal genomic DNA involving CTAB and alkaline guanidine thiocyanate boiling.At the same time，two methods were compared with each other.Result of test indicate: (1).All strains of standard and clinical isolated Sporothrix schenckii strains showed a specific fragment of 318bp with the species-specific primer pair.Using the same primer pairs， Candida albicans，Candida glabrata , Trichophyton rubum , Microsporum gypseum , Trichophyton mentagrophytes, Microsporum canis had no specific amplification.(2).To compare CTAB method with <WP=43>alkaline guanidine thiocyanate boiling method, genomic DNA consistence of the later was significantly higher than the former.Our conclusions: Species-specific primer PCR is a rapid，simple and feasible method for identifying Sporothrix schenckii,and alkaline guanidine thiocyanate boiling method for extraction of fungal genomic DNA is easy ,low cost and may be applicated in clinical laboratories.更多还原