多重RT-PCR诊断甲真菌病的体外模拟研究

目的:拟建立多重RT-PCR诊断甲真菌病的方法。方法:采用Trizol提取真菌的RNA;选择3对引物(真菌通用引物,皮肤癣菌特异性引物和酵母特异性引物)建立3重RT-PCR体系,采用红色毛癣菌、白念珠菌和短帚霉摸索反应条件和验证体系的适用性;并用红色毛癣菌来测定反应的灵敏度。结果:(1)所采用的真菌通用引物28srDNA、皮肤癣菌特异性引物ACT和酵母特异性引物ACT1有较好的通用性和特异性;(2)在适宜的反应条件下,无论对单模板,还是多模板,该反应体系均能扩增出目的片段,未见明显非特异片段的干扰;(3)该反应体系对模拟临床标本的检测灵敏度为102cfu/mL。结论:多重RT-PCR技术可在较短时间内检测出皮肤癣菌、酵母和霉菌等,并可判断菌的活力,将为临床快速诊断和及时、合理地治疗甲真菌病和其他真菌性疾病提供参考。     

巢式PCR诊断甲真菌病中红色毛癣菌和须癣毛癣菌

目的：评价巢式PCR诊断甲真菌病中红色毛癣菌和须癣毛癣菌的敏感性和特异性。方法：液氮冷冻甲标本,微量法提取DNA,应用特异性引物,巢式PCR方法扩增DNA。结果：36例直接镜检和培养均为阳性的甲标本,培养显示24例为红色毛癣菌,12例为须癣毛癣菌。巢式PCR中FirstPCR34例阳性,34例标本均产生了650bp目的片段;NestPCR32例阳性,32例中有22例标本产生了137bp的片段,为红色毛癣菌;10例标本产生了102bp的片段,为须癣毛癣菌。PCR敏感性为88．9％,特异性为100％。结论：巢式PCR是一种快速、特异和敏感的诊断甲真菌病中红色毛癣菌和须癣毛癣菌的方法。     

皮肤癣菌的分子生物学研究进展

皮肤癣菌是浅部真菌病的致病菌 ,对其分子生物学研究主要集中于基因结构的分析、基因诊断、基因分类等方面。目前 ,已在红色毛癣菌等某些癣菌的线粒体DNA上发现了重要的呼吸链酶 ,如细胞色素氧化酶、NADH脱氢酶的结构基因 ,以及诸多氨基酸的tRNA基因簇 ;建立了用真菌特异性通用引物或癣菌特异性引物聚合酶链反应诊断皮肤癣菌病的方法 ;应用限制性酶切片段多态性研究、随机扩增多态DNA分析、聚合酶链反应等方法进行皮肤癣菌的基因分类 ,分析癣菌的种间差异 ,对一些重要的皮肤癣菌 (如红色毛癣菌、须癣毛癣菌 )还进行了种内的分型研究。     

临床疑诊甲真菌病1036例真菌学分析

目的　了解近 5年本院甲真菌病病原菌的种类和构成 ,观察流行病学特点。方法　对近 5年临床疑诊的10 3 6例甲真菌病患者的真菌学实验室检查情况进行系统分析、总结。结果　共培养出真菌 63 1株 ,其中酵母菌占49.60 % ,以克柔念珠菌、红酵母、近平滑念珠菌为主 ;皮肤癣菌占 2 1.71% ,主要菌种为须癣毛癣菌和红色毛癣菌 ;其他丝状真菌占 19.81% ,主要菌种为曲霉和青霉 ;污染真菌占 8.87%。结论　本院近 5年甲真菌病患者病原菌依次为酵母菌、皮肤癣菌、其他丝状真菌 ,排前 5位的真菌分别是须癣毛癣菌 (11.2 5 % ) ,克柔念珠菌 (10 .14 % ) ,红酵母 (9.98% ) ,红色毛癣菌 (9.5 1% ) ,曲霉 (8.87% )。     

甲真菌病患者多部位红色毛癣菌感染分离株DNA同源性分析

目的 探讨甲真菌病患者多部位红色毛癣菌感染分离株基因型的差异。方法 采用PCR扩增红色毛癣菌rDNA非转录间隔区（NTS）中TRS1片段,并用随机引物OPAA11随机扩增DNA,检测基因多态性。比较同一患者不同感染部位分离菌株的基因型差异。结果 共分离30株红色毛癣菌,按照PCR扩增TRS1区指纹图分为5型,随机引物OPAA11扩增指纹图分为11型,基因型分布与感染部位关系不大。在10例受试患者中,PCR扩增TRS1区显示,7例不同感染部位分离得到的菌株基因型有差异;随机引物OPAA11扩增显示,8例不同感染部位分离得到的菌株基因型有差异。结论 甲真菌病患者不同部位红色毛癣菌感染可能为不同菌株引起,提示部分甲真菌病患者不同部位皮肤癣菌感染可能存在不同菌株的感染。     

杭州地区973例甲真菌病分析

目的了解杭州地区甲真菌病病原菌的种类和构成情况,获取流行病学资料.方法我们于2004年6月-11月对直接镜检阳性的973例甲真菌病患者进行了真菌分离培养及流行病学调查.结果共培养出722株病原菌,皮肤癣菌、酵母、非皮肤癣菌霉菌(NDM)所占比例分别为43.63%、38.37%、18.01%.皮肤癣菌中红色毛癣菌占89.52%(282/315),酵母中近平滑念珠菌居首,占21.66%,非皮肤癣菌霉菌中以曲霉和青霉为主.结论杭州地区的甲真菌病病原菌为皮肤癣菌、酵母菌和非皮肤癣菌霉菌,其中以红色毛癣菌和近平滑念珠菌为主.要注意非皮肤癣菌的污染或寄生问题.     

皮肤癣菌的分子生物学研究进展

皮肤癣菌是浅部真菌病的致病菌，对其分子生物学研究主要集中于基因结构的分析、基因诊断、基因分类等方面。目前，已在红色毛癣菌等某些癣菌的线粒体ＤＮＡ上发现了重要的呼吸链酶，如细胞色素氧化酶、ＮＡＤＨ脱氢酶的结构基因，以及诸多氨基酸的ｔＲＮＡ基因簇；建立了用真菌特异性通用引物或癣菌特异性引物聚合酶链反应诊断皮肤癣菌病的方法；应用限制性酶切片段多态性研究、随机扩增多态ＤＮＡ分析、聚合酶链反应等方法进行皮肤     

基于28S rRNA基因序列的巢式PCR方法快速检测甲真菌病病原菌种

目的应用基于28S rRNA基因序列的巢式PCR的方法快速诊断甲真菌病。方法液氮冷冻甲标本,微量法提取DNA,应用特异性引物,巢式PCR方法扩增DNA。结果本组共收集180例直接镜检和培养均为阳性的甲标本,First PCR 144例阳性,Nest PCR 168例阳性。其中127例标本经PCR扩增可产生约137bp目的片段,为红色毛癣菌;26例标本经PCR扩增可产生约102bp目的片段,为须癣毛癣菌;10例标本经PCR扩增可产生约445bp目的片段,为白念珠菌;5例标本经PCR扩增可产生约170bp目的片段,为曲霉。巢式PCR敏感性为93.33%(168/180),特异性为100%(168/168)。结论此方法是一种快速、敏感、特异的诊断甲真菌病的方法。     

甲真菌病400例致病真菌分析

目的:确定上海地区甲真菌病的致病菌种。方法:对本院皮肤科门诊就诊的直接镜检阳性的400例甲真菌病患者的甲标本做真菌分离培养和分析。结果:分离出致病真菌233株,其中皮肤癣菌120株(红色毛癣菌104株,须癣毛癣菌10株,犬小孢子菌3株,絮状表皮癣菌3株),酵母菌68株,非皮肤癣菌11株(曲霉6株,青霉5株),其余为丝状真菌。结论:上海地区甲真菌病的致病真菌以皮肤癣菌为主,酵母菌中非白念珠菌占有一定比例。     

深圳地区甲真菌病病原菌流行病学的多中心研究

目的 了解深圳地区最近4年甲真菌病病原菌的种类和构成,发现流行病学特点。方法 以多中心研究方式选取深圳地区6家不同区域的主要医院,选取临床表现典型、真菌镜检阳性的1162例甲真菌病患者进行真菌分离培养。结果共培养出致病真菌553株。皮肤癣菌占68.53％,以红色毛癣菌为主占52.26％,其次是须癣毛癣菌占11.21％;酵母菌占25.68％,其中以白念珠菌最多占9.22％;霉菌占5.79％,主要是曲霉属和青霉属;混合感染占4.30％。结论深圳地区最近4年甲真菌病病原菌为皮肤癣菌、酵母菌和霉菌。红色毛癣菌和白念珠菌所占比例最高。     

四种分枝杆菌快速检测方法的研究

目的 建立敏感、特异的快速检测4种分枝杆菌的方法。方法 用特异性引物对结核分枝杆菌、鸟分枝杆菌、胞内分枝杆菌和堪萨斯分枝杆菌菌悬液DNA进行PCR扩增,验证其敏感性和特异性。将上述4种分枝杆菌的特异性引物同时放入PCR扩增反应体系中,以此反应体系分别对该4种分枝杆菌菌悬液DNA、及其两两组合或特定的三重组合进行扩增,同时验证其敏感性。结果 4种分枝杆菌的特异性引物分别放入各自的PCR扩增反应体系中,可分别特异性地扩增出该4种分枝杆菌对应的DNA片段,敏感性达1×10¹～1×10²个菌细胞/mL。4种分枝杆菌的特异性引物同时放入同一PCR扩增反应体系中可分别特异性地扩增出对应的4种分枝杆菌单菌及其两两组合或三种分枝杆菌组合的DNA片段,敏感性达1×10²～1×10³个菌细胞/mL;4种分枝杆菌的特异性引物对其他分枝杆菌进行扩增,结果均为阴性。结论 多重PCR方法能敏感、特异地快速检测4种分枝杆菌。     

Direct detection and differentiation of causative fungi of onychomycosis by multiplex polymerase chain reaction-based assay

A rapid and reliable triplex PCR procedure was developed to detect pathogenic fungi directly from specimens of onychomycosis. One hundred and four patients were included in this study. Of them, forty-five (43.3%) were finally diagnosed with onychomycosis according to the diagnostic criteria. The sensitivity of PCR, microscopy and culture were 93.3%, 100% and 64.4%, respectively; the specificities were 100%, 86.4% and 100%, respectively; the positive predictive values were 100%, 84.9% and 100%, respectively; the negative predictive values were 95.2%, 100% and 78.7%, respectively. This molecular diagnostic process could distinguish the 3 groups of pathogens in onychomycosis (dermatophyte, yeast and mold) and could be completed within 8?h. This multiplex PCR assay could used in laboratories with no mycological specialization for rapid etiologic diagnosis and treatment selection, especially in suspected fungus cases if they can not be detected by conventional methods or if a rapid diagnosis of onychomycosis is needed.     

Detection and differentiation of causative fungi of onychomycosis using PCR amplification and restriction enzyme analysis

Background Onychomycosis, a fungal nail infection, has become one of the most important dermatophytoses. Unfortunately, a predictably successful diagnostic approach to onychomycosis does not yet exist. Objective The purpose of this study was to develop a deoxyribonucleic acid (DNA)-based diagnostic method to improve the sensitivity and specificity of the detection and differentiation of the pathogenic fungi of onychomycosis. Methods We attempted to detect fungi in the nail using polymerase chain reaction (PCR) primer systems that were designed in conserved sequences of the small ribosomal subunit 18S-rRNA genes shared by most fungi, and differentiated between species by restriction enzyme analysis of the amplified product. Results Fragments of the gene coding for 18S-rRNA were amplified successfully from medically important fungi species, but not from normal nails. Restriction fragment length polymorphism patterns using HaeIII endonuclease were sufficiently different to allow the recognition of individual species. Conclusions The PCR–restriction enzyme analysis method appears to be a more sensitive detection and identification technique for onychomycosis than conventional methods, and has considerable diagnostic value.     

Development of a novel polymerase chain reaction–enzyme‐linked immunosorbent assay for the diagnosis of Trichophyton rubrum onychomycosis

Background  The prevalence of onychomycosis has increased steadily in the past decade. An accurate diagnosis at the outset is important for successful and cost‐effective treatment of patients. However, current diagnostic tests for onychomycosis are not rapid, sensitive or specific. Objectives  To develop a microsatellite‐based polymerase chain reaction (PCR)‐enzyme‐linked immunosorbent assay (MS‐ELISA) for the detection of Trichophyton rubrum, which is the most common aetiological agent of onychomycosis. Methods  An archival set of 434 nail and skin specimens from 217 patients was included as the test sample in this study. We also compared MS‐ELISA with an earlier published topoisomerase PCR‐ELISA (TI‐ELISA) using template DNA extracted by another method. Results  The MS‐ELISA detected the highest number of positive samples (69%) followed by direct microscopy (56%), TI‐ELISA (44%) and fungal culture (30%). When an identical DNA extraction method was applied to 120 specimens, the MS‐ELISA proved to be twice as sensitive as the TI‐ELISA. Conclusions  We have optimized a target gene and DNA extraction method for rapid detection of T. rubrum onychomycosis.     

Molecular detection of dermatophytes and nondermatophytes in onychomycosis by nested polymerase chain reaction based on 28S ribosomal RNA gene sequences

Background  Onychomycosis is often caused by dermatophytes, but the role of nondermatophytes is underestimated due to the difficulty of identifying them by conventional direct microscopy and culture.
Objectives  This study aims to detect nondermatophytes, as well as dermatophytes, in the nail samples of patients with onychomycosis using a polymerase chain reaction (PCR)-based culture-independent method.
Materials and methods  The nested PCR assay targeting the sequence of the 28S ribosomal RNA gene was used to amplify fungal DNAs from 50 microscopy-positive nail specimens. Newly designed primer sets for dermatophyte universal, Trichophyton rubrum , T. mentagrophytes , Aspergillus spp., Scopulariopsis brevicaulis , Fusarium solani , F. oxysporum , F. verticillioides , Candida albicans and C. tropicalis were used after confirmation of their specificity.
Results  Forty-seven cases (94%) were positive for fungal DNA, among which dermatophytes were detected in 39 cases (83·0%): T. rubrum in 35 cases (74·5%) and T. mentagrophytes in eight cases (17·0%). Surprisingly, nondermatophytes were detected in 18 cases (38·3%), both dermatophytes and nondermatophytes in 10 cases (21·3%) and nondermatophytes alone in eight cases (17·0%). Aspergillus spp. alone was observed in five cases (10·6%).
Conclusions  This study indicates that most of the affected nail plates of patients with onychomycosis were positive for specific fungal DNAs, and suggests that nondermatophytes detected at high rates may be involved in the pathogenesis of onychomycosis.     

荧光多重PCR与血清型特异性抗体检测HSV感染的比较

目的：比较荧光多重PCR和血清型特异性抗体测定在生殖器疱疹临床诊断中的应用价值及评价各自的优缺点。方法：以细胞培养法作为“金标准”对照，分别用荧光多重PCR和血清型特异性抗体检测法对121例临床诊断为生殖器疱疹的标本进行检测。结果：以培养法作标准，并通过结果的差异性分析，荧光多重PCR的敏感性为100％，特异性为88．89％；血清型特异性抗体测定则分别为77．68％和77．78％，荧光多重PCR的敏感性和特异性均显高于血清型特异性抗体测（P＜0．05），但前不能检测出无皮损患HSV的DNA，而后可检测出无皮损患中的HSV抗体。结论：荧光多重PCR和血清型特异性抗体检测各有其自身的优缺点，单独用PCR和其它病毒分离的方法和单独使用血清特异性抗体检测的方法来诊断生殖器疱疹都是不完整的，均可造成漏诊。临床上将两种方法有机的结合起来应用能发挥各自的优势，取长补短，对早期、准确、快速地诊断生殖器疱疹及进行流行病学调查有着十分重要的意义和使用价值。     

复合PCR同时检测淋球菌沙眼衣原体和解脲支原体

目的：建立复合ＰＣＲ同时检测淋球菌（ＮＧ）、沙眼衣原体（ＣＴ）和解脲支原体（ＵＵ）。方法：用复合ＰＣＲ检测了１１６例尿道（宫颈）拭子，与传统方法及单对引物ＰＣＲ对照。结果：与ＮＧ涂片或培养、ＣＴ直接免疫荧光检查、ＵＵ培养比较，复合ＰＣＲ检测ＮＧ、ＣＴ、ＵＵ敏感性分别为１００％、９６．９％和３３．３％，特异性分别为９４．３％、９２．９％和９４．４％，与ＮＧ－ＰＣＲ、ＣＴ－ＰＣＲ、ＵＵ－ＰＣＲ的符合率分别为１００％、９７．４％和４１．３％。结论：复合ＰＣＲ可快速、敏感、特异地一次性检出ＮＧ、ＣＴ、ＵＵ泌尿生殖道感染，并可初步区分ＵＵ正常寄居抑或致病二种不同状态。     

自身淬灭探针荧光定量PCR检测淋病奈瑟菌

目的利用自身淬灭探针技术建立敏感、特异、快速、价廉且能广泛应用的淋病奈瑟菌荧光定量PCR检测方法。方法构建重组质粒pGEM-11Zf-CPPB作为标准品,自行设计自身淬灭探针,建立、优化定量PCR体系,并进行方法学评价及临床应用。结果所建立方法的线性检测范围为101～109拷贝/μL,灵敏度为10拷贝/μL,特异性为100%。天间变异系数(CV)为2.38%,批内CV为1.32%,批间CV为2.75%。该方法比培养方法更快速、灵敏。结论以自身淬灭探针技术为平台的淋病奈瑟菌荧光定量PCR方法灵敏度高、特异性好,对淋病的早期快速诊断有较高价值。