应用PCR技术快速鉴定申克孢子丝菌的实验研究

采用Biospin真菌基因组DNA提取试剂盒提取基因组DNA,用申克孢子丝菌种特异性引物分别对21株申克孢子丝菌的基因组DNA进行PCR扩增.成功提取21株申克孢子丝菌基因组DNA,并用申克孢子丝菌种特异性引物分别从21株申克孢子丝菌基因组DNA中获得长度为320 bp的扩增产物.以申克孢子丝菌特异引物为基础的聚合酶链反应法鉴定申克孢子丝菌简便、快捷、特异,可用于临床诊断.     

种特异性引物鉴定申克孢子丝菌

目的 研究申克孢子丝菌的分子鉴定方法,为孢子丝菌感染的分子诊断奠定基础.方法 对22株申克孢子丝菌和12种12株暗色真菌临床分离株的核糖体DNA(rDNA)内转录间隔区(ITS)进行聚合酶链反应扩增.对10株来源于中国不同地区及1株来源于美国的申克孢子丝菌的扩增产物测序并进行分析,以获得的ITS2区序列为靶序列设计出申克孢子丝菌的种特异性引物(SSP),并用该引物扩增全部受试菌株.结果 序列分析显示,申克孢子丝菌rDNA的ITS2区相当保守,特异性引物对22株申克孢子丝菌可扩增出一条300bp的片段,其他受试菌株均为阴性.结论 应用设计出的种特异性引物,结合PCR方法,鉴定申克孢子丝菌特异、敏感、可靠,可用于临床诊断.     

四对孢子丝菌引物的特异性和敏感性评价

目的 筛选对孢子丝菌特异且敏感的引物。方法 以50株不同地区来源的孢子丝菌临床分离株及标准株的基因组DNA作为研究样本,以毛霉、烟曲霉、念珠菌临床分离菌株基因组DNA作为对照,通过特异引物PCR扩增的方法筛选国内外文献中报道的4对孢子丝菌引物。所有受试孢子丝菌菌株均出现同一扩增产物,而对照菌株未出现者记为特异性引物,随后将基因组DNA模板倍比稀释后依次行PCR扩增,记录各引物出现阳性扩增结果时的最小模板浓度,检测敏感性。结果 在相同PCR条件下,引物S2-R2、SSHF31-SSHR97及ITS3-SSP具有较好特异性,而引物SS3-SS4对孢子丝菌及念珠菌菌株均可扩增出同样大小的PCR产物。引物S2-R2的敏感性最好,基因组DNA模板浓度为5 pg/μL时即可被检出。结论 针对几丁质合成酶1基因的引物S2-R2为孢子丝菌特异且敏感的引物。     

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。     

申克孢子丝菌基因型与临床表现型关系研究

目的了解申克孢子丝菌的基因学特征，探索其基因型特征与临床表现型的关系。方法收集不同临床型别孢子丝菌病的申克孢子丝菌分离株并提取DNA，再用随机扩增DNA多态性方法（RAPD）进行PCR扩增。结果①共选用10个随机引物进行扩增，筛选出4个具有较好扩增片段的引物。②不同菌株同-引物扩增均见-共有DNA片段。③不同临床型孢子丝菌病的申克孢子丝菌分离株带型有差异。结论随机扩增DNA多态性研究发现申克孢子丝菌具有一定的种内差异，其DNA带型与菌株的临床型有关。     

申克孢子丝菌基因型鉴定在临床诊断中的应用

目的：探讨申克孢子丝菌临床分离株的基因型鉴定在临床诊断中的应用。方法：使用PCR法扩增引起三种临床类型的申克孢子丝菌菌株核糖体保守区及内转录间隔区（5．8SrDNA，ITSII）的基因序列，并进行测序。结果：三株临床分离菌株核糖体保守区及ITSII区基因序列完全一致，经测定为申克孢子丝菌，该基因序列与基因库中申克孢子丝菌菌株（AB089138）相关序列的同源性达100％。结论：申克孢子丝菌的ITS区相对保守，通过对ITS段基因序列的测定可在基因水平上对孢子丝菌进行菌种鉴定，为临床快速、准确诊断和治疗提供确切依据。     

不同地区申克孢子丝菌基因分型研究

目的:探讨申克孢子丝菌基因分型特征与菌种来源的关系。方法:采用随机扩增DNA指纹方法(RAPD)对来源于不同地区的31株申克孢子丝菌临床分离株进行DNA多态性分析。结果:①筛选出4个具有较好扩增片段的引物。②31株菌的DNA带型不完全相同,具有一定的遗传变异性,与菌株的地区来源有关。③同一患者不同部位取材的菌株间DNA型可不同。结论:随机扩增DNA多态性方法可用于申克孢子丝菌的DNA分型,其DNA带型与菌株的地区来源有关。     

利用核糖体基因进行申克孢子丝菌基因分型

目的 利用探针与DNA印迹法对申克孢子丝菌进行种内分型,探讨其基因型特征与菌种来源及临床表现的关系.方法 CTAB法提取来源于不同地区31株孢子丝菌临床分离株及1株标准株的基因组DNA,以真菌通用引物ITS4、NS5扩增标准株的rDNA序列作为探针,与经限制性内切酶ApaI酶切后的基因组DNA进行印迹杂交.结果 杂交后形成多种清晰而稳定的带型,根据带型将31株孢子丝菌分为15种基因型(A-O型),其中A、B、C三型占51.61%.结论 探针与DNA印迹法是孢子丝菌种内分型较为敏感而可靠的方法,该方法所分基因型的不同与菌种的地区来源及临床表现有一定的相关性.     

申克孢子丝菌随机扩增 DNA多态性研究

目的 了解申克孢子丝菌的基因学特征,研究DNA分型与菌种来源和临床表现的关系。方法 以随机扩增DNA多态性方法对来源于不同地区及不同临床类型的24株申克孢子丝菌进行DNA多态性分析。结果 （1）筛选出3个具有较好扩增征段的引物,即引物OPAA11：5′-ACCCGACCTG-3′,OPD18：5′-GAGAGCCAAC-3′,OPBO7:5′-GGTGACGCAG-3′。（2）24株菌的DNA带型不完全相同,具一定遗传变异性。（3）不同菌株同一引物扩增均见一共有DNA片段,（4）播散型DNA带型与皮肤淋巴管型不同,结论 随机扩增DNA多态笥方法可用于申克孢子丝菌的DNA分型,其带型与菌株的地区来源和临床表现有关。     

申克孢子丝菌chs1基因生物信息分析

目的:对申克孢子丝菌几丁质合成酶基因(chs1)及其编码蛋白的结构和特性进行分析和预测。方法:利用NCBI网站、Ex PASy和CBS等工具预测申克孢子丝菌chs1基因及编码蛋白的结构和功能。结果:chs1 mRNA全长2 745 bp,编码914个氨基酸。BLAST分析显示,申克孢子丝菌与巴西孢子丝菌、蓝菌属真菌、足马杜拉分枝菌等真菌chs氨基酸序列一致性达到99%,且有保守域。预测chs1蛋白相对分子量为102 714.7,理论等电点为6.71。预测chs1编码蛋白ɑ螺旋、β折叠、β转角、无规则卷的比例分别是38.40%、17.94%、10.39%、33.26%。chs1蛋白是一种疏水蛋白,包含9个跨膜区,未发现信号肽。结论:成功预测了chs1基因及编码蛋白质的生化性质及结构特征,为下一步对其进行克隆、表达和敲除奠定基础。     

HPLC法检测申克孢子丝菌的辅酶Q系统

目的 探讨一种真菌化学分类的新方法,了解申克孢子丝菌不同临床株的辅酶Ｑ系统。方法以ＹＭＢ培养基获取菌体经高压灭菌、皂化、正己烷提取的等获取辅酶Ｑ,采用ＨＰＬＣ分析其Ｑ－６、Ｑ－１０的含量。结果 正己烷提取可获粗制的辅酶Ｑ,方法简便。７株申克孢子丝菌临床分离株皆含有Ｑ－１０,仅２株菌含有少量Ｑ－６。结论 正己烷提取真菌的辅酶Ｑ方法简便,ＨＰＬＣ分析法灵敏度较高,申克孢子丝菌主要含有辅酶Ｑ－１０。     

Rapid and specific identification of Sporothrix schenckii by PCR targeting the DNA topoisomerase II gene

BACKGROUND: In order to study the genotype variation of Sporothrix schenckii, we previously analyzed the genomic sequences of the DNA topoisomerase II genes of this fungus and S. schenckii-related species, such as S. schenckii var. luriei and Ceratocystis stenoceras. OBJECTIVE: To develop a PCR-based identification system that can distinguish S. schenckii from its related species for clinical diagnosis of sporotrichosis. METHODS: Based on the nucleotide sequences of the DNA topoisomerase II genes of S. schenckii, S. schenckii var. luriei and C. stenoceras, three gene-specific primers (SSHF31 and SSHR97 for S. schenckii, and SSLF64 for S. schenckii var. luriei) were designed and evaluated for their specificities in PCR amplifications. RESULTS: The primer set SSHF31/SSHR97 amplified PCR products of 663-817 bp from S. schenckii and approximately 660 bp from S. schenckii var. luriei, while SSLF64/SSHR97 exclusively amplified a major product of 305 bp from S. schenckii var. luriei. CONCLUSION: PCR targeting the DNA topoisomerase II gene specifically and rapidly distinguished S. schenckii from its related species.     

Rapid identification of Sporothrix schenckii in biopsy tissue by PCR

Background The dimorphic fungus Sporothrix schenckii is the etiological agent of sporotrichosis, an important cutaneous mycosis with a worldwide distribution. At present, it is challenging to rapidly discover and identify Sporothrix schenckii in biopsy tissues nowadays. Aims To explore new methods for rapid diagnosis of sporotrichosis. Materials and Methods We screened specific primers for Sporothrix schenckii using 50 clinical isolates from patients with sporotrichosis. DNA was extracted from the lesions of 30 cases of clinically suspected sporotrichosis using the Graham s method of CTAB and amplified by PCR using the screened specific primers. Results The primer S2‐R2 was applicable for the identification of S. schenckii from different geographic areas and clinical types with high specificity and sensitivity. Twenty‐five out of the thirty cases (83.3%) amplified using the primer S2‐R2 showed positive bands. Further positive bands were observed in 95.6% of cases tested positive by fungal culture. Conclusions Using the PCR technique and specific primers, we developed a new diagnostic method that can rapidly diagnose sporotrichosis with tissues obtained from clinical biopsies.     

应用聚合酶链反应检测某些机会致病真菌的初步探讨

目的 探讨快速检测某些机会致病性直菌的方法。方法 以源于医学机会致病性真菌１８ｓｒＲＮＡ保守区的一对寡核苷酸序列为引物,利用聚合酶链反应对医学上重要机会致病性真菌ＤＮＡ进行扩增。结果 ３属７种２９株重要机会致病性直菌,经扩增均出现一条１９７ｂｐ的ＤＮＡ片段,而对大肠埃希菌、金黄色葡萄球菌、绿脓假单胞菌、人白细胞则不扩增。以溴化乙啶染色,该ＰＣＲ方法对白念珠菌ＤＮＡ的最低检出限为１ｐｇ。３０份临床标     

Epidemiological data and molecular characterization (mtDNA) of Sporothrix schenckii in 13 cases from Mexico

BACKGROUND: Sporotrichosis is an endemic mycosis in Mexico and Central America. OBJECTIVES: To present epidemiological data and characterize molecular subtypes of 13 strains of Sporothrix schenckii, and to correlate clinical and epidemiologic presentation with subtypes. METHODOLOGY: Thirteen Mexican cases of sporotrichosis were identified, and mitochondrial DNA (mtDNA) from clinical isolates of S. schenckii were analyzed using RFLP Hae III. Molecular types 2, 3, 14, 28, and 29 were observed. RESULTS: Clinical presentations and molecular types were consistent with established epidemiologic patterns for S. schenckii in this region. CONCLUSIONS: This investigation provides further evidence of the strong regional specificity of molecular types of this organism.     

Rapid identification of Trichophyton tonsurans by specific PCR based on DNA sequences of nuclear ribosomal internal transcribed spacer (ITS) 1 region

BACKGROUND: Trichophyton tonsurans, a dermatophyte implicated in an international epidemic of tinea capitis, was also found to be responsible for infecting wrestling and Judo athletes nationwide in Japan since 2001. OBJECTIVE: A rapid and highly accurate means of identifying this pathogen has been required to control the infection. We have developed a T. tonsurans-specific PCR method based on the DNA sequences of the nuclear ribosomal internal transcribed spacer 1 region. SUBJECTS: Eighteen species of six genera of standard strains and 75 strains of clinically isolated Trichophyton species were used in this study. METHODS: A T. tonsurans-specific PCR primer pair (tonsF1 and tonsR1) was designed on the nuclear ribosomal internal transcribed spacer 1 region, located between 18S and 5.8S rDNA. Fungal DNA was extracted from the colonies grown on culture plates, and the specificity of the PCR primers was tested. RESULTS: The specific PCR product was amplified from the standard strain of T. tonsurans and from five strains isolated from black dot ringworms, but there was no band from the 70 clinical isolates of other Trichophyton species. This T. tonsurans-specific PCR method was able to detect 10 pg of T. tonsurans genomic DNA with ethidium bromide staining. CONCLUSIONS: A PCR identification system specific for T. tonsurans is rapid, sensitive, and specific.     

申克孢子丝菌酵母相对人急性单核细胞白血病细胞NF-κB信号通路及TNF-α的影响

目的:明确申克孢子丝菌酵母相对人急性单核细胞白血病细胞(THP-1)NF-κB信号通路激活和肿瘤坏死因子α(TNF-α)分泌的影响。方法:实时荧光定量PCR分析申克孢子丝菌酵母相刺激THP-1细胞TNF-αmRNA的表达,酶联免疫吸附法检测TNF-α分泌量,免疫印迹法分析申克孢子丝菌酵母相体外作用于THP-1细胞后IκBα和磷酸化IκBα的水平,免疫荧光法观察NF-κB-p65核转位。同时设置地塞米松(NF-κB抑制剂)抑制组,并检测100 n M地塞米松预处理THP-1细胞后TNF-αmRNA水平的变化。结果:申克孢子丝菌酵母相刺激THP-1细胞后6 hTNF-αmRNA水平显著高于空白对照组(P0.001)。申克孢子丝菌酵母相刺激THP-1细胞后24 h,TNF-α蛋白水平为(4610.419±121.501)pg/mL,显著高于空白对照组(186.964±98.073)pg/m L,差异具有统计学意义(P0.001)。申克孢子丝菌酵母相作用THP-1细胞后30~60 min,磷酸化IκBα蛋白水平显著升高,为时间依赖性。申克孢子丝菌酵母相组细胞核内NF-κB-p65较空白对照组荧光强度增强。100 nM地塞米松预处理各组THP-1细胞后,TNF-αmRNA水平较前明显降低。结论:人THP-1细胞体外与申克孢子丝菌酵母相作用后激活NF-κB信号通路并上调TNF-α分泌。