红色毛癣菌的基因分型研究

目的 探讨探针与DNA印迹杂交法对红色毛癣菌的基因分型并分析其基因型与来源地区的相关性。方法 用溴化十六烷基三甲铵（CTAB）法提取DNA，以皮肤癣菌的特异性引物NS5[5′-AACTAAAGGAATTGACGGAAG-3′]与ITS4[5′-TCCTCCGCTTATTGATATGC-3′]为引物，以红色毛癣菌的标准菌株为模板，用PCR法扩增出其rDNA部分18S区，ITSI区，5.8S区和ITSⅡ区为探针，用随机引物法将探针标记^32P，用EcoRI酶切基因组DNA，采用DNA印迹的标准流程将酶切的基因组DNA与探针杂交；显示的不同带型，以此作为红色毛癣菌基因分型的依据。结果 所试49株红色毛癣菌（南京21株，大连26株，北京2株）分为20型（A-T型），其中A-C型占48.98%。南京株绝大多数为3条带，大连株大部分为4条带。结论 用ITS区域探针与DNA印迹杂交法对红色毛癣菌基因组分型敏感性强，分辨力高；南京与大连两地区红色毛癣菌DNA分型具有明显差异，DNA分型对红色毛癣菌病的流行病学，临床疗效的判定以及指导用药均具有重要价值。     

用聚合酶链反应进行红色毛癣菌种内分型

目的 建立一种快速且可重复的方法应用于红色毛癣菌的菌株区分。方法 PCR扩增20株红色毛癣菌核糖体基因的非转录间隔区内的两个串联重复亚单位trs-1,trs-2，克隆两段PCR产物，用PGEM-T载体构建重组质粒载体进行目的基因测序。结果 特异性扩增trs-1和trs-2区产生菌株特征的条带图，两个串联重复亚单位的基因测序结果说明了PCR指纹图差异的原因。结论 这种PCR方法产生的特征性的指纹图提供了一种快速，稳定的分子分型方法，可应用于红色毛癣菌感染的流行病学和致病性的进一步研究。     

红色毛癣菌和须癣毛癣菌的PCR指纹分析

红色毛癣菌和须癣毛癣菌的传统鉴别主要依据培养形态、生理生化等方法,但红色毛癣菌的菌落产红现象受培养基类型、 pH、培养温度等因素的影响 [1],我们应用 PCR方法,以微小卫星 DNA引物,对这 2种癣菌的基因组 DNA进行扩增,可以在很短时间内把它们区分开来。 泳道 1为 Markerλ DNA/EcoRⅠ＋ HindⅢ,泳道 2、 7、 8、 9为红色毛癣菌,泳道 3～ 6为须癣毛癣菌 图 1 (GACA)4作引物的 PCR扩增结果 一、材料与方法 (一 )实验菌株：本研究采用红色毛癣菌临床分离株 23株 ,须癣毛癣菌临床分离株 11株。红色毛癣菌中 11株来自上海…     

红色毛癣菌随机扩增DNA多态性分型研究

红色毛癣菌(Trichophyton rubrum)是皮肤癣菌中最常见的病原菌,我国大多数地区的检出率在30%以上^[1],传统的表型分型法将其分为五型,但是,由于红色毛癣菌大多数为不典型株,而且传代过程中表型特征可发生转变或丧失,因此,表型分型的不稳定性导致它不能准确反映红色毛癣菌的遗传特征。我们应用随机扩增DNA多态性(RAPD)分析法对46株分别从广州、香港、雅加达(印尼)3个城市浅部真菌感染患者获得的红色毛癣菌进行DNA分型,对三地红色毛癣菌的表型特征与基因型别关系进行了初步探讨。     

须癣毛癣菌随机扩增DNA多态性分型研究

研究须癣毛癣菌的ＤＮＡ分型，了解ＤＮＡ型别与形态学分型，有性型型别，以及与菌种来源地区，与人体感染部位之间的关系。用氯化苄法提取ＤＮＡ，以随机扩增ＤＮＡ多态性方法对临床分离的须癣毛癣功４２株以及部分其它皮肤癣菌和我科保藏的菌种Ａｒｔｈｒｏｄｅｒｍａ８株进行分型。     

甲真菌病患者多部位红色毛癣菌分离株的DNA分型

目的探讨甲真菌病患者多部位红色毛癣菌感染不同部位分离菌株基因型的差异。方法采用PCR扩增红色毛癣菌rDNA非转录间隔区(NTS)中Trs-1片段,检测基因多态性并比较。结果30株受试菌株按照PCR指纹图共分为3型,基因型分布与感染部位无相关。10例受试患者中,5例不同感染部位分离出的菌株基因型存在差异。结论甲真菌病患者多部位红色毛癣菌感染可能为多菌株引起,部分患者可能存在多菌株的混合感染。     

红色毛癣菌菌落表型和基因型的分型研究

目的:确定红色毛癣菌菌落表型和基因型的相关性以及与感染部位的关系。方法:收集红色毛癣菌临床分离株138株,采用传统培养方法对红色毛癣菌进行表型分型,利用红色毛癣菌特异性引物扩增TRS-1区重复序列进行种内基因分型,并分析检测结果与感染部位的相关性。结果:138株红色毛癣菌共分离出3种菌落表型:绒毛型、沟纹型和粉末型;分离出5型基因型,其中Type1 64株,Type2 18株,Type3 19株,Type4 12株,Type5 25株。红色毛癣菌的表型和基因型与感染部位均无相关性(均P0.05),红色毛癣菌的菌落表型与基因型有一定的相关性(P0.05)。结论:红色毛癣菌基因型与菌株来源有关而与感染部位无关,可为判断复发和再感染提供依据。     

红色毛癣菌表型稳定性研究

为探讨红色毛癣菌表型的稳定性,所有菌株采用沙堡琼脂试管培养基(SDA)培养。菌株鉴定及分型依据菌落的特征及色素,大小分生孢子的形态,尿素酶试验和毛发穿孔试验。对近期临床分离的10株红色毛癣菌和1株标准株,间隔4周传代1次,共传4次。207株红色毛癣菌共分离出4个表型,其中绒毛型占首位(45.4%),沟纹型(24.1%),羊毛型(20.8%),粉末型(9.7%),未见颗粒型。保存1年后207株有54株发生形态变异,变异率为26.1%,沟纹型变异最小,相对稳定。11株红色毛癣菌传代后菌落形态或色素发生变异。红色毛癣菌表型不稳定,保存和传代后的菌落形态或产色均易发生变异;沟纹型变异率最低。     

须癣毛癣菌基因分型的研究

目的探讨ITS区域的探针与DNA印迹杂交法对须癣毛癣菌的基因分型,并分析基因型与来源地区的相关性。方法用溴化十六烷基三甲铵(CTAB)法提取DNA,以皮肤癣菌的特异性引物NS5[5-′AACTTAAAGGAATT-GACGGAAG-3′]与ITS4[5-′TCCTCCGCTTATTGATATGC-3′]为引物,以须癣毛癣菌的标准菌株为模板,用PCR法扩增rD-NA部分18S区、ITSⅠ区、5.8 s区和ITSⅡ区为探针,随机引物法将探针标记,EcoR1酶切基因组DNA,按ECL试剂盒标准流程将酶切的基因组DNA与探针杂交;根据杂交的不同带型而分型。结果所试35株须癣毛癣菌分为14型(A~N型),A,B,C,D四型占62.86%。南方株以A和C型为主,北方株以B和D型为主。结论用ITS区域探针与DNA印迹杂交法对须癣毛癣菌基因组分型敏感性强、分辨力较高;南北方须癣毛癣菌DNA分型存在一定差异;DNA分型对须癣毛癣菌病的流行病学调查和菌学的生物学研究具有重要价值。     

两足一手型手足癣致病菌种红色毛癣菌的基因型分析

目的对两足一手型手足癣患者致病菌种红色毛癣菌进行菌株水平的基因型分析,通过手足间菌株基因型异同推测手足间菌株传染途径。方法扩增红色毛癣菌NTS区的串联重复系列TRS-1区,对临床分离菌株进行基因分型。结果共分析了229株红色毛癣菌,包括从63例两足一手型手足癣患者手足部位分离到173株红色毛癣菌和15例两足两手型手足癣患者手足部获得56株红色毛癣菌。两组中,80%以上的患者手足部位菌株基因型相同,且两组患者基因型无统计学差异。结论两足一手型手足癣患者手部菌株主要来自其感染的足部。     

多部位红色毛癣菌感染分离菌株DNA分型研究

目的 探讨多部位红色毛癣菌感染不同部位分离菌株基因型的差异。方法 采用巢式PCR扩增真菌rDNA非转录间隔区(NTS)中Trs-1片段,检测基因多态性。并比较同一患者不同感染部位分离菌株的基因型差异。结果 受试36株菌株按照PCR指纹图共分为10型,基因型分布与感染部位关系不大。在17例受试患者中,8例不同感染部位分离得到的菌株基因型有差异,且与不同感染部位的病程差异无关。结论 多部位红色毛癣菌感染可能为多菌株引起,提示部分多部位皮肤癣菌感染患者不同部位皮肤癣菌感梁的传染源可能来源于外界,而与机体原已存在的感梁灶关系可能不大。     

大连、沈阳两地须癣毛癣菌复合体的分子鉴定及DNA分型

目的探讨大连和沈阳两地须癣毛癣菌复合体的主要组成菌种及主要组成菌种肉芽肿株和体癣株DNA分型有无差异。方法选取大连和沈阳地区已行表型鉴定的须癣毛癣菌16株(12株体癣株,4株肉芽肿株),通过PCR扩增核糖体DNA(rDNA)的内转录间隔区(ITS),测序后利用数据库进行序列比对,对须癣毛癣菌复合体进行鉴定;PCR扩增rDNA非转录间隔区(NTS)的3个串联重复亚单位S0,S1和S2区,进行种内分型,比较菌株间型别的差异性。结果大连、沈阳两地的16株须癣毛癣菌中,2株鉴定为断发毛癣菌,1株鉴定为苯海姆节皮菌,13株鉴定为万博节皮菌;3对不同引物扩增13株万博节皮菌NTS区,共产生8种特征性带型;万博节皮菌的体癣株和肉芽肿株的带型除S2区外,在S0,S1区及总区间均有差异。结论大连、沈阳两地须癣毛癣菌复合体的主要组成菌种为万博节皮菌;万博节皮菌肉芽肿株和体癣株NTS区的带型有差异。     

Specific detection of Trichophyton rubrum and Trichophyton interdigitale based on loop‐mediated isothermal amplification (LAMP) from onychomycosis specimens

In diagnosing onychomycosis, diseases with similar features must be excluded by demonstrating the presence of fungal infection and identifying the fungal species. However, fungal culture of onychomycosis‐derived samples usually takes many weeks to yield species identification results, and is associated with a low successful culture rate. Loop‐mediated isothermal amplification (LAMP) is a highly sensitive and specific molecular biological method that can amplify DNA at a constant temperature, allowing for a simpler testing procedure, shorter detection time and less cost than conventional techniques including quantitative polymerase chain reaction. We have developed a new LAMP method specifically to detect Trichophyton rubrum (T. rubrum) and Trichophyton interdigitale (T. interdigitale), major causative dermatophytes for onychomycosis, and analyzed the correlation between LAMP results and those of the existing fungal culture method for the detection and identification of Trichophyton species from onychomycosis‐derived samples. The results showed that all 59 specimens in which T. rubrum or T. interdigitale was identified by fungal culture also tested positive by LAMP, giving a 100% positivity concordance rate between the two methods. Moreover, all 55 and four specimens in which T. rubrum and T. interdigitale were identified by fungal culture, respectively, also tested positive for each species by LAMP, again giving a 100% species‐identification concordance rate. The high correlation demonstrated between LAMP and fungal culture results in detection and identification of Trichophyton species from onychomycosis‐derived samples suggests high reliability of LAMP as a promising, alternative mycological detection and identification technique which can serve as an alternative to the fungal culture method.     

成人及儿童皮肤癣菌病患者红色毛癣菌的基因分型与药物敏感性分析

目的 探讨成人和儿童皮肤癣菌病临床分离菌株中红色毛癣菌基因型的差异，基因型与发病部位、基因型与药物敏感性的关系。方法 利用随机引物OPAA11 5′-ACCCGACCTC-3′，OPD18 5′-GAGAGCCAAC-3′分别对来自成人及儿童皮肤癣菌病的红色毛癣菌菌株进行任意引物PCR，根据产物电泳带型进行基因分型，采用微量液体稀释法对氟康唑、伊曲康唑、特比萘芬、酮康唑、利拉萘酯、布替萘芬、益康唑、联苯苄唑进行体外敏感性分析。结果 两组的主要致病菌都为红色毛癣菌，两种随机引物扩增出的红色毛癣菌不同株带型均稳定清楚，按随机引物 OPAA11的扩增结果，其基因型分为4型。成人中Ⅰa、Ⅲa型分别占41.94%，儿童中Ⅰa型为65.96%，基因型构成在两组人群中的差异有统计学意义（P < 0.05）；体癣、足癣中基因型的分布在两组间差异均有统计学意义（P < 0.01；P < 0.05）。甲癣和股癣中基因型的分布差异无统计学意义（P > 0.05）；各基因型对8种抗真菌药物的MIC值均较低，特比萘芬的MIC最低，氟康唑的MIC值相对较高，酮康唑和氟康唑对Ⅰa、联苯苄唑对Ⅱa的活性高于其他基因型，伊曲康唑对Ⅲa型的活性略低于其他基因型，差异有统计学意义（P < 0.01或P < 0.05）。结论 红色毛癣菌为南京及周边地区儿童及成人皮肤癣菌病的主要致病菌，成人感染以Ⅰa、Ⅲa型为主，儿童感染以Ⅰa型为主。8种抗真菌药物对各基因型均有较好的抗菌活性，但酮康唑、氟康唑、联苯苄唑、伊曲康唑、特比萘芬对各基因型的敏感性有差异。     

Antifungal susceptibility and genetic similarity of sequential isolates of Trichophyton rubrum from an immunocompetent patient with chronic dermatophytosis

Chronic cutaneous dermatophytoses caused by Trichophyton rubrum are common in immunocompromised patients. In immunocompetent indivuals, the disease is more often associated with onychomycosis and tinea pedis. The aim of this study was to perform antifungal susceptibility tests and genetic analysis of sequential isolates of T. rubrum from an immunocompetent patient with chronic dermatophytosis. Antifungal susceptibility tests against griseofulvin, ketoconazole, itraconazole and fluconazole were performed with sequential isolates of T. rubrum. Genetic relationship among the isolates was analysed by the random amplification of polymorphic DNA (RAPD) method. The results revealed that treatment failure was not related to the development of drug resistance, as all of the sequential T. rubrum isolates were sensitive to antifungals tested in vitro. The RAPD data demonstrated that this disease was caused by identical isolates, with no genetic differences among them, representing a single T. rubrum strain. Treatment failure and chronicity of infection do not seem to be related to antifungal resistance.     

Detection and identification of Trichophyton tonsurans from clinical isolates and hairbrush samples by loop‐mediated isothermal amplification system

Since the 1990s, there have been reports of the spread of dermatophytosis caused by Trichophyton tonsurans among contact sports athletes in several countries, including Japan. This study was performed to develop a loop‐mediated isothermal amplification (LAMP) system for rapid and accurate detection and identification of T. tonsurans from clinical isolates or hairbrush samples for diagnosis and to prevent the spread of infection. A specific primer set was prepared by comparing the whole genome sequence of T. tonsurans with those of six other closely related dermatophytes. After confirming the sensitivity and specificity of this system, LAMP assay was performed using 37 clinical samples obtained from three healthy volunteers and 24 judo athletes. A total of 155 fungal isolates (56 strains of various standard fungi, 96 identified T. tonsurans isolates, three hairbrush‐cultured isolates from judo athletes) and 37 hairbrush samples (34 samples from 24 judo athletes, and three samples from three healthy volunteers) were used for culture and LAMP assay, respectively. The assay showed no cross‐reactivity to standard strains other than T. tonsurans. The detection limit was 100 copies of DNA template per tube. All of the 96 T. tonsurans isolates were amplified, and all samples from healthy volunteers showed negative results. Four of the 34 hairbrush samples obtained from judo athletes showed positive results in LAMP assay, and two of the four were positive in both culture and LAMP assay. We developed a rapid LAMP system with high specificity and sensitivity for diagnosis of T. tonsurans infection.