【作者】 郑龙；

【导师】 刘福英；

【作者基本信息】 河北医科大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 目的：皮肤真菌包括三个属，即毛癣菌属（Trichophyton sp）、小孢子菌属(Microsporum sp)和表皮癣菌属（Epidermophyton sp），多数皮肤真菌为人兽共患致病真菌，它们可以侵犯皮肤、毛发和指（趾）甲,寄生或腐生于表皮角质、毛发和甲板的角蛋白组织中，引起浅部真菌病。引起实验动物皮肤真菌病的病原菌主要是须毛癣菌(Trichonphyton mentagrophyton),石膏样小孢子菌（Microsporum gypseum），犬小孢子菌（Microsporum canis）。这三种菌是实验动物应排除的病原菌，不论是从动物皮肤病灶处或从非病灶处分离到这三种菌均视为异常，因为非病灶处的真菌很可能随后引起皮肤病，同时造成饲养和实验人员的感染。对皮肤病原真菌检测主要是依靠真菌培养物的菌落形态及真菌菌丝的显微镜观察。镜检虽然快速，但需要有多年的实践经验,并很难鉴别到种。实际上，真菌的表型特征很容易受到外界因素的影响，例如温度变化，培养基成份和化学治疗，这些都影响真菌代谢过程，从而影响结果的观察。而且要想得到典型的形态学和镜下特征一般需要2~3周的培养时间，对一些缺少特异的形态及镜下特征的菌种还需要进行特殊培养基的第二次培养。PCR技术以其特异性强、敏感性高、快速的特点已被用于皮肤真菌病检测的研究。目前大<WP=4>多是选择真菌通用引物扩增皮肤真菌DNA的CHS基因，18S、25S和28S r RNA和 r RNA的ITS区，然后对扩增产物进行测序或使用限制性内切酶做RFLP进行菌种的鉴别（PCR—RFLP），一些报道使用随机扩增DNA多态性(RAPD)对皮肤真菌直接进行种水平的鉴别。但与医学有关的真菌种类就有百种多，皮肤真菌也有几十种，目前尚无一种分子生物学方法能够快速、简便、准确用于皮肤真菌种水平的鉴别。得到完整的核酸是应用PCR方法的前提，因真菌细胞壁坚固性，通常的提取方法得不到真菌DNA。目前已有多种真菌DNA提取方法的报道，如使用破壁酶Novobiocin、玻璃珠法、使用研钵研磨等，这些方法或所用材料较昂贵，或对得到的DNA的质量有影响；同时常规的DNA抽提过程步骤繁琐、耗时，常使工作人员疲劳厌烦。本试验目的是设计一种快速简便的提取皮肤病原真菌DNA的方法及建立一种快速、敏感和特异的核酸鉴定方法来检测实验动物皮肤病原真菌。方法：取标准菌株须毛癣菌、石膏样小孢子菌、犬小孢子菌传种至沙氏斜面固体培养基，27℃培养7天。无菌取少量须毛癣菌、石膏样小孢子菌接种于沙氏液体培养基，置27℃恒温摇床80转/min振荡培养10天。将培养基倒入100ml离心管内，离心沉淀装入1.5ml Ep 管中，-70℃或液氮保存。取液氮保存的须毛癣菌、石膏样小孢子菌少量菌丝（约10mg）或使用破壁酶或在研钵内精细研磨，或用无菌牙签破坏菌丝壁，分别使用酚氯仿和醋酸钾-异丙醇抽提DNA；从犬小孢子菌沙氏斜面固体培养基刮取少量菌丝，使用破壁酶及醋酸钾-异丙醇抽提DNA。选择皮肤真菌通用引物(CHS1 1S, CHS1 1R)和随机引物(FM1)通过PCR扩增鉴别三种皮肤真<WP=5>菌。结果1 将石膏样小孢子菌分别接种到几个沙氏斜面固体培养基上，培养2周后，一些培养基上生长的菌落已变为淡黄色粉末状菌落，而另一些培养基上仍为白色菌落。2 DNA电泳结果表明四种获取DNA的方法(方法1~4)都能够得到完整、适量的DNA。根据电泳图谱显示(图1~4)，方法1，使用研杵在研钵内研磨的方法有时会造成基因组DNA断裂；方法2，使用无菌牙签破坏菌丝壁的方法整个过程耗时最短，但获得的DNA量较少；方法3，使用破壁酶的方法可以得到较多的基因组DNA，而且很少发生DNA降解；方法4，直接从培养基刮取小量犬小孢子菌菌丝，使用酶破壁的方法可以提取到足量的DNA。3 方法5，直接从固体培养基刮取真菌菌丝，使用无菌牙签破坏细胞壁然后抽提，没有得到真菌DNA。4 使用醋酸钾抽提核酸DNA用于PCR得到清晰的扩增产物电泳图谱（图2~4）。5 测量DNA浓度约为90ng/μl，260nm/280nm吸光度比值>1.7。6 皮肤真菌通用引物(CHS1 1S, CHS1 1R)在三种真菌中均扩增出DNA片段，其中须毛癣菌约为350bp，石膏样小孢子菌约400bp，犬小孢子菌约为440bp（图5）。7随机引物FM1在三种真菌中扩增出不同大小的DNA片段。其中须毛癣菌主要扩增条带有两条，一条大小约500bp,另一条>1kb；石膏样小孢子菌主要扩增条带有一条，大小约为350bp；犬小孢子菌只有一条扩增条带，大小约为200bp<WP=6>（图6）。结论1 4种提取真菌基因组DNA的方法（方法1~4）均提取到完整适量的DNA, 其中直接从固体培养基刮取菌丝使用破壁酶提取DNA的方法（方法4）可以大大减少培养时间，缩短真菌检测时间，同时减少液体培养中过多的操作造成污染的可能性，适用于大批量样本检测，最适合实验动物皮肤病原真菌的检测及临床皮肤真菌快速检测。 2 醋酸钾快速抽提方法提取的DNA用于PCR，产生清晰的扩增条带，可满足PCR扩增实验。与酚氯仿法比较，此方法简单、快速，是一种产生高质量DNA用于PCR的DNA抽提方法。3 皮肤真菌通用引物(CHS1 1S, CHS1 1R)用于PCR扩增真菌样本，可初步鉴别出皮肤真菌。4 随机引物FM1用于真菌RAPD分析，可将三种常见的实验动物皮肤病原真菌须毛癣菌、石膏样小孢子菌、犬小孢子更多还原

【Abstract】 Objective: Dermatophytes have been classified into three genera, Trichophyton, Microsporum and Epidermophyton. They invade keratinized tissues of human and other animal, that is, superficial cornified skin layers, hair and nails, causing a superficial cutaneous infection called dermatophytosis. Dermatophytes which produced an infection to laboratory animal are mainly Trichophyton mentagrophytes, Microsporum gypseum and Microsporum canis. The three kinds of dermato- phytes should be banned to laborary animal, no matter they were isolated in infectious sites or uninfectious sites. Because the dermatophytes in uninfectious sites was probable causing dermaophytosis of laboratory animal and producing infection to laboratory man.The current diagnosis of dermatophytosis is based upon the status of colony and microscopic identification of hyphae directly from lesion materials followed by in-vitro culture. Although rapid and economical, microscopical examination is not species-specific and needing a lots of experience. In fact, the phenotypic characteristics of dermatophytes can be easily influenced by outside factors, such as temperature variations, <WP=8>chemotherapy and culture media, that may interfere with the metabolic process of the dermatophyte and affect the interpretation of in-vitro culture results. In-vitro culture is capable of providing typical characteristics of morphological and biochemistry in 10~15 days. However, for some unusual and atypical characteristic dermatophytes, identification may require a range of special culture media. These tests will consum several weeks. PCR technology which is high sensitivity, speciality and rapidity have been used for dermatophytes identification. Now these studies amplified DNA of dermato- phytes selecting universal primers which get from 18S, 25S ,28S ribosomal RNA genes and internal transcribed spacer (ITS) regions, as well as chitin synthase 1 gene, and these methods frequently require additional manipulation such as sequencing after amplification or restriction endonuclease digestion. Except that, random amplification and identification of DNA fragments unique to a certain strain or species in RAPD enables various dermatophytes to be differentiated rapidly and precisely from each other. But there are hundreds of various fungi related to medicine, and several decades of dermatophytes. Now there is no one molecular biology method which enable to identificate species of dermatophytes with rapidity, simplicity and accuracy.Dermatophytes have strong cell walls which are often resistant to traditional DNA extraction procedures. Now there are already some reports about extraction methods of dermato- phytes DNA, such as enzyme method (Novobiocin), glass beads <WP=9>method, ground with a pestle et al. These methods are either costing, or affecting the quality of DNA. And traditional DNA extraction procedures are steps-repeating and time-consuming, and making people feel tired.The test develop a method of nucleic acid identification with high sensitivity, speciality and rapidity to detect pathogenic dermatophytes in laboratory animals. And to establish a rapid and simple DNA isolation method from pathogenic dermato- phytes in laboratory animal by comparing several methods of extraction dermatophytes DNA.Methods: The standard strains (Trichophyton menta- grophytes, Microsporum gypseum and Microsporum canis) were inoculated on to Sabouraud dextrose agar and cultured at 27℃ for 7 days. Then Trichophyton mentagrophytes and Microsporum gypseum were cultured in Sabouraud liquid medium and incubated with shaking for 10 days at 27℃. The mycelial samples were collected by centrifugation and then put into 1.5ml Eppendorf tube in liquid nitrogen. The samples (about 10mg) were lysed with lysing enzyme, or grounded finely with a pestle in a prechilled mortar, or disrupted using a sterile toothpick. Then phenol-chloroform extraction method and potassium acetate extraction method were used to isolate DNA; A small lump of mycelia of Microsporum canis was scrapted from Sabourau更多还原