PCR-RFLP技术快速鉴定八种致病丝状病原真菌的实验研究

目的 建立能快速鉴定深部丝状真菌感染病原菌的PCR-RFLP方法。方法 用真菌通用引物扩增烟曲霉、黄曲霉、土曲霉、黑曲霉、杂色曲霉、构巢曲霉、尖端赛多孢和串珠镰刀菌的ITS区,分别用HhaⅠ、HaeⅢ、HinfⅠ、TaqⅠ和MspⅠ 5种限制性核酸内切酶对PCR产物进行酶切,建立以PCR为基础的RFLP方法,然后对22株临床株和2株环境分离株进行PCR-RFLP图谱分析。结果 对PCR产物进行RFLP分析可以准确鉴定8种深部致病丝状真菌,从DNA提取到酶切分析可以在1个工作日完成。22株临床株和2株环境分离株PCR-RFLP鉴定结果与传统的形态学鉴定结果一致。结论 PCR-RFLP技术是一种能够快速鉴定丝状真菌的有效方法。     

临床分离阿萨希毛孢子菌IGS1区克隆及RFLP分析

目的 明确临床分离的5株阿萨希毛孢子菌（T. asahii）基因间隔区（IGS1）的序列,分析T. asahii种内基因多态性。方法 用玻璃珠法分别提取临床分离5株T. asahi及标准对照株总DNA,用ITS及IGS1区特异引物,PCR扩增目的区域,IGS1区扩增产物纯化后进行克隆、测序,测序结果分别提交到基因库,获取登录号。用BLAST和CLUSTAL X 1.83软件对序列进行比对分析。分别用限制性内切酶HapⅡ、MboⅠ、AluⅠ、HhaⅠ对ITS及IGS1区PCR产物进行酶切及RFLP分析。结果 6株菌均成功扩增出长度约为540 bp的ITS区及643 bp的IGS1区基因片段,IGS1区序列相似性为97% ～ 99%。RFLP分析发现,菌株BZP07003 IGS1区HapⅡ酶切结果及菌株BZP07004 IGS1区 HhaⅠ酶切结果与其他菌株不同,MboⅠ及AluⅠ酶切6株菌结果无差异,ITS区6株菌酶切结果无差异。结论 T. asahii IGS1区种内存在差异。HhaⅠ及HapⅡ可用于T. asahii种内多态性的分析研究,临床分离5株菌存在3种酶切结果,初步提示T. asahii临床分离株可能具有种内多态性及遗传差异。     

PCR-RFLP鉴别临床常见的皮肤癣菌

目的 建立一种快速而简便的分子水平上鉴定常见皮肤癣菌的方法。方法 采用通用引物ITS1（5‘-TCCGTAGGTGAACCTGCGG-3‘)、ITS4(5‘-TCCTCCGCTTATTGATATGC-3‘)对7种常见皮肤癣菌的核糖体的ITS（internal transcribed spacer)区（包含5.8SrDNA)进行PCR扩增，PCR产物分别应用限制性内切酶MvaI、TaqI、HinfI酶切。结果 7种皮肤癣菌的ITS区扩增产物条带大小不同；以MvaI、TaqI、HinfI三种酶分别酶切PCR-ITS区产物，7种皮肤癣菌均产生独特且易于区分的酶切图，尤其是MvaI酶产生的酶切图在种间差异最为明显。可将7种菌明确区分开。结论 核糖体ITS区PCR-RFLP分析是区分常见皮肤癣菌的有价值的方法。     

应用PCR-RFLP进行申克孢子丝菌的分子生物学鉴定

目的　探索一种简单、快速的申克孢子丝菌的鉴定方法。方法　应用真菌通用引物ITS1和ITS4对来源于不同地区及不同临床型别孢子丝菌病的 2 8株申克孢子丝菌以及 9种其他临床上重要的真菌进行PCR扩增 ,利用限制性内切酶HaeⅢ对PCR产物进行酶切分析鉴定。结果　所有 2 8株申克孢子丝菌和其他 9种真菌均扩增出一条约 3 5 0bp的片段 ,其中 2 8株申克孢子丝菌RFLP带型一致 ,与 9种其他临床上重要的真菌RFLP带型差异较明显。 结论　PCR RFLP可以为建立一种简单、快速鉴定申克孢子丝菌的方法提供依据。     

马拉色菌致儿童甲真菌病的临床与实验室观察

目的对3例马拉色菌所致儿童甲真菌病进行菌种鉴定及临床分析。方法刮取病甲甲屑,经真菌镜检和培养确定为马拉色菌属。生化鉴定后提取DNA,利用通用引物内转录间隔区1和4(ITS1、ITS4)进行PCR扩增,扩增产物测序后在NCBI数据库进行同源性比对。结果仅有例3在沙堡培养基上长出少量不明菌落,根据孢子和菌丝形态,不能判断菌种类型。在含菜籽油培养基上,第3天开始均有白色菌落生长,第10天有乳白色酵母样菌落,荧光染色后可见圆形或卵圆形出芽孢子。生化鉴定结果显示:例1、2为糠秕马拉色菌,例3疑似球形马拉色菌。DNA扩增产物测序比对结果与生化鉴定结果一致,其中例3为混合菌种。结论 3例儿童甲真菌病致病菌均有马拉色菌,其中2例为糠秕马拉色菌,1例为含有球形马拉色菌的混合菌种,且生化鉴定和分子生物学鉴定结果一致。     

PCR法扩增角鲨烯环氧化酶鉴定白念珠菌的研究

目的：通过扩增角鲨烯环氧化酶基因的开放读框,进行PCR来鉴定白念珠菌。方法：根据白念珠菌角鲨烯环氧化酶基因的开放读框中编码1MSSVKY^6的序列设计上游引物5‘－ATGAGTTCAGTTAAGTATG－3‘,编码^492NEIVR^496的序列设计下游引物5‘-CTATCTTACAATCTCGTTC-3‘,对白念珠菌ATCC11006,22株临床分离株,7株其它致病性念珠菌,8株致病性丝状真菌,1株新生隐球菌及1份人的基因组DNA进行了PCR扩增,并对PCR产物用酶切方法鉴定,结果：所有23株白念菌均可获得约1．5kb大小的PCR产物,16株其它致病性真菌及人的DNA标本均无扩增产物,PCR产物经BamHI酶切,产生两个大小分别约为1．0kb和500bp的片段。结论：利用设计的引物,扩增角鲨烯环氧化酶基因的开放读框,可以特异地鉴定白念珠菌。     

PCR结合基因扫描分析技术对几种深部真菌病致病菌的快速检测及鉴定

目的 用PCR法快速鉴定22种(23株)深部真菌病致病菌种。方法 应用荧光标记的真菌通用引物ITS4与ITS86对22种(23株)深部真菌病致病菌种的菌悬液进行PCR扩增，扩增产物经ABI PRISM^TM377测序仪及基因扫描分析软件精确测定片段大小，与对照组——相应菌种采用传统方法抽提DNA后扩增片段的扫描分析结果相对照。结果 ①17种致病菌种菌悬液经ITS4、ITS86扩增出单一的片段(除了构巢曲霉和黑曲霉、白念珠菌和类星形念珠菌、裴氏着色真菌和皮炎外瓶霉片段长度相同)。②菌悬液扩增片段的扫描分析结果与其DNA扩增结果相近，差异无显著性。③整个检定过程仅需6h。结论 采用ITS通用引物及菌悬液PCR检测法，结合基因扫描分析技术可准确、特异、快速、敏感地检测并鉴定出22种深部真菌病致病菌种，这将有望成为快速诊断深部真菌病的一种方法。     

菌落PCR在快速鉴定头癣病原菌中的临床应用北大核心CSCD

目的评价菌落PCR在快速鉴定头癣病原菌中的可靠性及临床实用性。方法于2016年1月至2017年3月在无锡市第二人民医院皮肤科门诊收集儿童头癣病例17例,采用菌落PCR技术检测病原菌,同时与常规PCR及形态学鉴定结果进行比较,评价菌落PCR应用于头癣病原菌鉴定的可靠性。结果17例儿童头癣患者临床标本经培养收集菌种、菌落PCR均扩增成功,从断发或皮屑标本中取材至DNA模板制备成功耗时（3．82±0．50）d,较传统形态学鉴定（14d）明显缩短。以常规PCR鉴定结果为标准,菌落PCR菌种鉴定正确率为100％,优于传统的形态学鉴定（正确率88．2％）。结论菌落PCR可用于临床头癣病原菌的检测,是一种快速、经济、可靠的分子检测技术。     

致病性外瓶霉的PCR-RFLP分类

应用PCR方法对致病性外瓶霉进行分类鉴定。以ITS3和ITS4为引物 ,对常见的7种致病性外瓶霉的模式株核糖体DNA转录间隔区(ITSⅡ)进行扩增 ,4种内切酶(HinFI、MspI、BsuRI和RsaI)酶切。各种间多态性显著 ,常规方法难以鉴别的皮炎外瓶霉和甄氏外瓶霉较易区分。PCR RFLP准确可靠 ,可用于形态及其他方法难以确定的致病菌种的鉴别。     

拓扑异构酶Ⅱ基因区PCR-RFLP法鉴别常见皮肤癣菌

目的：探讨拓扑异构酶Ⅱ基因区PCR～RFLP法鉴别常见皮肤癣菌的可行性。方法：用真菌拓扑异构酶Ⅱ基因区通用引物dPsD1、dPsD2，先后对6种34株临床常见皮肤癣菌和2株白念珠菌以及4株曲霉的DNA进行巢式PCR（nested PER）扩增，扩增后的阳性产物分别用限制性内切酶HincⅡ和HinfⅠ酶切，进行限制性片段长度多态性（RFLP）分析，根据结果来鉴定皮肤癣菌并在种的水平上区分这6种常见皮肤癣菌。结果：通用引物dPsD1对6种皮肤癣菌均能扩增出3390bp、dPsD2能扩增出2380bp的片段，而白念珠菌和曲霉则未见阳性扩增条带。扩增出的产物分别经HincⅡ和HinfⅠ酶切后表现为58—1670bp长短不等的片段组合，根据这些特异性条带谱可以区分这6种临床常见皮肤癣菌。结论：拓扑异构酶Ⅱ基因区的巢式PCR—RFLP方法，是鉴定和区分常见皮肤癣菌的有效方法。     

半巢式PCR-RFLP法快速鉴定临床标本中的皮肤癣菌

目的探索快速鉴定皮肤癣菌病临床标本中致病菌的分子生物学方法。方法以皮肤癣菌的基因组DNA为模板,采用一对半真菌通用引物(NS5、ITS1、ITS4)行半巢式PCR扩增rDNA上的ITS段,用限制性内切酶BciT130Ⅰ及DdeⅠ酶切目的片段,凝胶电泳。结果7种65株常见皮肤癣菌培养菌株和17例临床标本的半巢式PCR-RFLP图谱特异;且临床标本与相应的培养菌株酶切图谱一致。结论半巢式PCR-RFLP方法适合培养菌株和临床标本病原菌的快速准确鉴定。     

APPLICATION OF POLYMERASE CHAIN REACTION (PCR) AND PCR BASED RESTRICTION FRAGMENT LENGTH POLYMORPHISM FOR DETECTION AND IDENTIFICATION OF DERMATOPHYTES FROM DERMATOLOGICAL SPECIMENS

Objective:

To develop and optimize polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) targeting 18S rDNA and internal transcribed spacer (ITS) region of fungi for rapid detection and identification of dermatophytes.

Materials and Methods:

Two PCR-RFLP methods targeting 18S rDNA and ITS regions of fungi were optimized using standard and laboratory isolates of dermatophytes and other fungi. Sixty-eight dermatological clinical specimens (nail clippings (56), material obtained from blisters (8), hair root (2), scraping from scaly plaque of foot (1) and skin scraping (1) collected by the dermatologist were subjected to both the optimized PCR-RFLP and conventional mycological (smear and culture) methods.

Results:

PCRs targeting 18S rDNA and the ITS region were sensitive to detect 10 picograms and 1 femtogram of T. rubrum DNA, respectively. PCR targeting 18S rDNA was specific for dermatophytes and subsequent RFLP identified them to species level. PCR-RFLP targeting the ITS region differentiated dermatophytes from other fungi with identification to species level. Among the 68 clinical specimens tested, both PCR-RFLP methods revealed the presence of dermatophytes in 27 cases (39.7%), whereas culture revealed the same only in 2 cases (7.40%), increasing the clinical sensitivity by 32.3%. Among 20 smear positive specimens, both PCR-RFLP methods detected dermatophytes in 12 (17.6%). Both the methods detected the presence of dermatophytes in 13 (19.11%) smear and culture negative specimens, increasing the clinical sensitivity by 36.1%.

Conclusion:

PCR-RFLP methods targeting 18S rDNA and the ITS regions of fungi were specific and highly sensitive for detection and speciation of dermatophytes.     

二次PCR用于临床标本真菌感染快速分子诊断的研究

目的 探讨二次PCR技术诊断疑似真菌感染临床标本的敏感性。方法 收集临床疑似真菌感染的深部位痰及肺泡灌洗液标本共29份,分别进行真菌直接镜检、真菌培养、真菌通用引物单次与二次PCR扩增rDNA的ITS区,并对真菌检出阳性率和多种真菌菌种检出率进行比较分析。结果 临床疑似病例痰及肺泡灌洗液标本真菌镜检、真菌培养、单次PCR和二次PCR真菌检出阳性率分别为20.7%（6/29）、37.9%（11/29）、17.2%（5/29）和48.3%（14/29）。真菌培养、单次PCR和二次PCR提示二种以上真菌菌种检出的比例分别为6.9%（2/29）、3.4%（1/29）和24.1%（7/29）。二次PCR与单次PCR的真菌检出率差异有统计学意义（χ2 = 6.34,P < 0.05）。在两种以上菌种检出率方面,二次PCR与真菌培养和单次PCR间差异均有统计学意义（χ2 = 4.09,6.30,P值均 < 0.05）。结论 二次PCR技术有助于提高临床标本真菌分子诊断的敏感性。     

Molecular diagnosis of cutaneous leishmaniasis and species identification: analysis of 122 biopsies with varied parasite index

Background: Cutaneous leishmaniasis is endemic in the Middle East and North Africa. Confirming the diagnosis histologically depends on amastigote identification, which varies significantly depending on the inoculum, strain type, host response and disease stage. Accurate histological diagnosis is mandatory for appropriate therapy. Methods: Skin biopsies from 122 patients from Lebanon, Syria and Saudi Arabia with clinical diagnosis of untreated leishmaniasis were reviewed and clinical data extracted. Cases were classified according to the modified Ridley's parasitic index. DNA was extracted from formalin‐fixed paraffin‐embedded blocks. Polymerase chain reaction (PCR) was performed using Leishmania‐specific ribosomal internal transcribed spacer 1 (ITS1‐PCR). Nested ITS1‐PCR was performed on cases negative for conventional ITS1‐PCR. ITS1‐PCR amplicons were digested with HaeIII for subsequent restriction fragment length polymorphism (RFLP) subspeciation. Results: Of 122 cases, 54 (44.3%) showed a parasitic index of 0–1+ (no unequivocal amastigotes). ITS1‐PCR (conventional and nested) was positive for all cases as compared with negative control tissue. RFLP identified Leishmania tropica in all cases. Patients with clinically suspected leishmaniasis, whose skin biopsies failed to detect amastigotes represented 44.3% of our cases. Conclusions: In this study, we describe a rapid and optimized protocol from DNA extraction to leishmaniasis subspeciation. ITS1‐PCR showed high sensitivity and specificity in confirming clinically suspected cases. Yehia L, Adib‐Houreih M, Raslan WF, Kibbi A‐G, Loya A, Firooz A, Satti M, El‐Sabban M, Khalifeh I. Molecular diagnosis of cutaneous leishmaniasis and species identification: analysis of 122 biopsies with varied parasite index.     

PCR and PCR-RFLP techniques targeting the DNA topoisomerase II gene for rapid clinical diagnosis of the etiologic agent of dermatophytosis

BACKGROUND: We have focused on the DNA topoisomerase II genes of several pathogenic fungi, and developed polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) methods targeting this gene for identification of dermatophytes. OBJECTIVE: To assess the availability of the PCR-based identification for an etiologic study of dermatophytosis, by testing these PCR and PCR-RFLP methods for stability and reproducibility. METHODS: Three hundred and fifty-six dermatophyte strains were isolated from 305 patients with tinea, and their genomic DNAs were used as templates for the PCR using primer mixes (PsT, PsME, dPsD1 or dPsD2) composed of gene-specific primers for identification of dermatophytes to the species level. The genomic DNAs of Trichophyton rubrum were further subjected to subrepeat element analysis of the nontranscribed spacer (NTS) of ribosomal DNA (rDNA). RESULTS: In this study, six dermatophyte species (T. rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum, and Epidermophyton floccosum) were obtained. In all cases, the identifications obtained from the PCR and PCR-RFLP targeting the DNA topoisomerase II gene coincided with those from the conventional morphological features-based identification technique. The sensitivity of the PCR-based identification was found to be a colony of approximately 3mm in diameter. Furthermore, T. rubrum was divided into three groups (17 types) on the basis of the sizes and numbers of the products generated from the TRS-1 region, and three types from the TRS-2 region. CONCLUSION: The PCR and PCR-RFLP targeting the DNA topoisomerase II gene were rapid, stable, and reproducible for species identification of dermatophytes, and thus are convenient tools for an etiologic study of dermatophytosis.     

Signature polymorphisms in the internal transcribed spacer region relevant for the differentiation of zoophilic and anthropophilic strains of Trichophyton interdigitale and other species of T. mentagrophytes sensu lato

Background Dermatophytes are the main cause of superficial mycoses in humans and animals. Molecular research has given useful insights into the phylogeny and taxonomy of the dermatophytes to overcome the difficulties with conventional diagnostics. Objectives The Trichophyton mentagrophytes complex consists of anthropophilic as well as zoophilic species. Although several molecular markers have been developed for the differentiation of strains belonging to T. mentagrophytes sensu lato, correct identification still remains problematic, especially concerning the delineation of anthropophilic and zoophilic strains of T. interdigitale. This differentiation is not academic but is essential for selection of the correct antimycotic therapy to treat infected patients. Methods One hundred and thirty isolates identified by morphological characteristics as T. mentagrophytes sensu lato were investigated using restriction fragment length polymorphism (RFLP) and sequence analysis of the polymerase chain reaction‐amplified internal transcribed spacer (ITS) region of the rDNA. Results Species of this complex produced individual RFLP patterns obtained by the restriction enzyme MvaI. Subsequent sequence analysis of the ITS1, 5.8S and ITS2 region of all strains, but of T. interdigitale in particular, revealed single unique polymorphisms in anthropophilic and zoophilic strains. Conclusions Signature polymorphisms were observed to be useful for the differentiation of these strains and epidemiological data showed a host specificity among zoophilic strains of T. interdigitale/Arthroderma vanbreuseghemii compared with A. benhamiae as well as characteristic clinical pictures in humans when caused by zoophilic or anthropophilic strains. The delineation is relevant because it helps in determining the correct treatment and provides clues regarding the source of the infection.