阴道非白念珠菌的PCR-RFLP鉴定

目的对分离自外阴阴道念珠菌病(VVC)患者阴道的常见致病性非白念珠菌进行聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法的鉴定。方法首先采用芽管试验、厚壁孢子试验、法国科玛嘉(CHROMagar)念珠菌显色培养基及API20CAUX酵母菌鉴定系统将分离自VVC患者阴道内的念珠菌菌株鉴定到种,然后采用真菌通用引物将4种常见非白念珠菌(包括光滑念珠菌、近平滑念珠菌、克柔念珠菌和热带念珠菌)进行PCR扩增,并选用MspⅠ和HaeⅢ两种内切酶对扩增产物进行酶切分析。结果在4种非白念珠菌中光滑念珠菌15株(7.50%),近平滑念珠菌7株(3.50%),克柔念珠菌5株(2.50%),热带念珠菌2株(1.00%);PCR扩增后均产生稳定、清晰的条带,扩增产物经MspⅠ,HaeⅢ酶切后分别产生4种和2种特异性带型。结论外阴阴道念珠菌病非白念珠菌中以光滑念珠菌为主;PCR-RFLP方法在鉴定常见致病性非白念珠菌中比较可靠、稳定、特异,但仍有方法繁琐等不足之处。     

常见致病性念珠菌的PCR-RFLP鉴定研究

目的 研究常见致病性念珠菌的分子鉴定方法,为深部念珠菌病的分子诊断奠定基础。方法 对常见9种致病性念珠菌的11株标准株和39株临床株的核糖体DNA内转录间隔区进行聚合酶链反应扩增,对扩增产物进行MspⅠ、HaeⅢ和DdeⅠ三种内切酶的酶切分析。结果 9种念珠菌聚合酶链反应扩增后产生5种不同分子量的条带,扩增产物经MspⅠ、DdeⅠ和HaeⅢ酶切后分别产生8种、5种和4种特异性带型。结论 聚合酶链反应-限制性内切酶片段长度多态性方法在鉴定常见的致病性念珠菌中稳定、可靠、特异,是传统方法的有益补充。     

高分辨熔解曲线技术检测阴道念珠菌分型的研究

目的探讨采用高分辨熔解曲线方法鉴定常见致病性念珠菌的可行性,为外阴阴道念珠菌病的分子流行病学及病原学研究奠定基础。方法用芽管试验、厚壁孢子试验、科玛嘉(CHRO Magar)显色培养基及API20CAUX酵母菌鉴定系统对临床致病性念珠菌进行鉴定,然后对5株标准株和80株临床分离株的基因组DNA内转录间隔区进行聚合酶链反应,对扩增产物进行高分辨熔解曲线分型。结果 5株念珠菌标准株和80株临床株聚合酶链反应扩增后,通过熔解曲线技术分析产生5种不同的峰图,即得到念珠菌5种分型。结论外阴阴道念珠菌的主要致病菌是白念珠菌(77.50%),高分辨熔解曲线方法鉴定常见的致病性念珠菌特异性强、稳定性好,是传统鉴别方法的有益补充。     

用内转录间隔区通用引物的聚合酶链反应快速测定念珠菌菌种

目的：应用内转录间隔区（ITS）通用引物建立一种较为快速、简便的检测和鉴定念珠菌的聚合酶链反应（PCR）方法。方法：采用两对ITS通用引物ITS1、ITS4和ITS86、ITS4对7种（8株）念珠菌菌县液进行PCR扩增。结果：两对引物3.5h内对7种念珠菌的菌县液扩增出种特异的DNA多带，而第2对引物其种特异性更强。结论：采用ITS通用引物结合快速PCR检测法，可快速、简便、特异、敏感地检测出念珠菌，并能同时鉴定到种，这将在今后念珠菌病的早期诊断和治疗上有潜在的应用价值。     

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反向线点杂交鉴定常见念珠菌的研究

目的 建立PCR反向线点杂交技术（PCR-RLB）快速检测和鉴定常见念珠菌的方法。方法 以念珠菌属间隔序列Ⅱ（ITS2）为靶基因设计通用引物，用生物素标记反义引物，PCR扩增白念珠菌、热带念珠菌、克柔念珠菌、光滑念球菌、近平滑念珠菌、都柏林念珠菌DNA，然后与通过氨基标记固定在尼龙膜上的各特异性寡核苷酸探针杂交，并进行临床标本和分离株的检测。结果 念珠菌标准菌株可扩增出302 ~ 441 bp DNA片段，6种特异性寡核苷酸探针可分别与相应念珠菌PCR产物杂交，其敏感性为10 cfu/mL。通过对100例分离株的检测，然后与培养法鉴定（Merieux Vitek）的结果比较，有97株与培养结果一致，另外3株通过DNA测序结果证实与PCR-RLB测定结果一致。通过对200例女性阴道拭子进行检测，PCR-RLB阳性率为49%，而涂片法和培养法阳性率分别为27%和39%，明显高于涂片法和培养法（P < 0.05）。结论 该方法可快速、敏感、准确鉴定临床上常见的念珠菌。     

实时荧光定量PCR快速鉴定五种常见念珠菌

[摘要] 目的 建立实时荧光定量PCR快速检测和鉴定五种常见念珠菌的方法并探讨其可行性。方法 应用真菌通用引物ITS4和ITS86,分别构建五种常见念珠菌重组质粒,进行实时荧光定量PCR检测,绘制融解温度曲线图,并对融解温度进行统计学分析。结果 五种常见念珠菌融解温度为：（87.33±0.13）℃（白念珠菌）、（84.39±0.20）℃（热带念珠菌）、（85.53±0.18）℃（近平滑）、（86.47±0.10）℃（光滑念珠菌）、（91.41±0.18）℃（克柔念珠菌）。结论：本研究显示实时荧光定量PCR方法可快速检测和鉴定五种常见念珠菌。     

阴道念珠菌随机扩增多态性DNA分型研究

目的 对分离自外阴阴道念珠菌病患者阴道的念珠菌进行基因分型,并对不同菌株间的亲缘关系作相似性分析。方法 用形态学及生化方法对分离到的致病性念珠菌进行鉴定,然后采用随机扩增多态性DNA技术(RAPD)对45株临床分离株基因组DNA进行扩增,并将DNA扩增带型进行聚类分析。结果 30株白念株菌可以分成7群共19个亚群,15株非白念珠菌(包括光滑念珠菌6株,克鲁斯念珠菌、近平滑念珠菌和热带念珠菌各3株)至少可鉴定到种水平。白念珠菌种内不同菌株间的相似性系数在90%以上,不同念珠菌种间的相似性系数介于80%~90%。结论 外阴阴道念珠菌病的主要致病菌是白念珠菌,用RAPD技术可将之分成不同的基因组型别。     

PCR方法快速检测一些常见医学真菌

目的 为选择一种快速、敏感和特异的方法来检测临床感染的真菌。方法 选取真菌１８ＳｒＤＮＡ序列中两个片段设计引物,对１２种菌株进行ＰＣＲ扩增,均扩增出长约３１０ｂｐ产物。对一些临床常见细菌扩增均无反应。对酶母相真菌白念珠菌、新生隐球菌进行敏感性实验检测。结果 发现二者ＰＣＲ最小检出量均为菌体数达每个反应体系１０＾３。本实验过程总耗时３６ｈ以内。结论 本实验方法可快速特异性检测一些常见医学真菌。     

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 study of Candida species in cutaneous candidiasis based on PCR using a primer mix specific for the DNA topoisomerase II gene

BACKGROUND: We have previously reported a PCR-based identification system for pathogenic fungi by targeting the DNA topoisomerase II gene, in which primer mixes specific for this gene were used for the PCR amplifications. OBJECTIVE: To test the potential of the PCR using primer mix that is specific for the DNA topoisomerase II gene and are designated as PsVIc, for rapid identification of Candida species involved in cutaneous candidiasis, and to define the relation between Candida species and the infection lesion. METHODS: Scales from 48 patients with cutaneous candidiasis were cultured on GYEP agar plates, and the genomic DNAs were purified from the colonies and used as DNA templates for PCR amplifications. Candida was identified as individual species based on the sizes of the PCR products generated in the PCR amplifications using PsVlc. RESULTS: Four Candida species (five genotypes; Candida albicans, Candida glabrata, Candida parapsilosis I, Candida parapsilosis II and Candida tropicalis II) were identified in the patients' scales. In 19 of the patients (39.6%), multiple PCR products (two or three bands) were amplified in a DNA sample, especially derived from scales at the groin of bed-ridden older patients using napkins. CONCLUSION: The PCR-based identification using the primer mix was useful for an epidemiological study of Candida species in cutaneous candidiasis.     

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 PCR)扩增,扩增后的阳性产物分别用限制性内切酶Hinc II和Hinf I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3 390 bp、dPsD2能扩增出2 380 bp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Hinc II和Hinf I酶切后表现为58~1 670 bp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。     

Rapid identification of Candida albicans and its related species Candida stellatoidea and Candida dubliniensis by a single PCR amplification using primers specific for the repetitive sequence (RPS) of Candida albicans

BACKGROUND: Candidiasis is caused by several Candida species, of which Candida stellatoidea and C. dubliniensis are phenotypically close to C. albicans. Although current molecular biology-based techniques can distinguish between C. albicans and C. dubliniensis, a convenient tool that can distinguish C. stellatoidea from C. albicans has not yet been developed. OBJECTIVE: To develop a system that can simply, rapidly and specifically distinguish C. albicans from the related Candida species C. stellatoidea and C. dubliniensis. MATERIALS: Genomic DNAs were purified from various yeast species and amplified by primers specific for the repetitive sequence (RPS) of C. albicans. The PCR products were purified and sequenced in order to test the specificity of the PCR amplification. RESULTS: The PCR primers only amplified several products from C. albicans, C. stellatoidea and C. dubliniensis. Sequence analysis of the products revealed that C. stellatoidea and C. dubliniensis both had RPSs including alt repeats, similar to C. albicans. After the PCR amplification, each of the three Candida species showed a unique amplification profile. Furthermore, RFLP analysis of the PCR products using EcoRI and ClaI produced species-specific restriction profiles. CONCLUSIONS: This PCR-based technique targeting the alt repeats in the RPS is useful as a tool for the rapid identification and distinction of C. albicans, C. stellatoidea and C. dubliniensis.     

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。     

SPECIES IDENTIFICATION OF CANDIDA ISOLATES OBTAINED FROM ORAL LESIONS OF HIV INFECTED PATIENTS

A total of 60 patients suspected to have AIDS with oral lesions suggestive of oral candidiasis were studied. Candida species were isolated from 50 patients. Candida albicans was the commonest isolate (70 %) followed Candida parapsilosis(15%), Candida glabrata (7.5%) and Candida tropicalis (5%) respectively. Candida dubliniensis was isolated from a single case only. Though the reports from developed countries show more prevalence of the novel species Candida dubliniensis, in our study it was isolated in a single case. All the patients were treated successfully with oral fluconazole for 7 days except for the patients from which Candida glabrata was isolated, who were treated with Amphotericin B.