多重PCR快速鉴定19种致病真菌

摘要:目的：建立快速鉴定致病真菌的多重PCR体系。 方法：选取19种致病真菌的rDNA内部转录间隔区（ITS）序列设计引物,建立两个多重PCR体系,扩增临床与环境分离真菌菌株、人类细胞及9种常见细菌DNA,检测体系的特异性;以浓度梯度的DNA模板检测体系的灵敏度。 结果：对氟康唑天然耐药的克柔念珠菌、光滑念珠菌以及对两性霉素B耐药的土曲霉、镰刀菌、尖端赛多孢、根霉在该体系中可扩增出特异性条带,其他真菌可同时进行种属鉴定。74株被测真菌菌株的鉴定结果与常规鉴定的符合率为95.9%。两个多重PCR体系对人类基因组及9种常见细菌DNA的扩增结果均为阴性;扩增阳性的最低模板浓度均为10 fg/μL。 结论：建立的两个多重PCR体系可对致病真菌进行检测和种属鉴定。     

荧光定量聚合酶链反应检测常见致病真菌方法的建立

目的建立快速的检测临床致病真菌的荧光定量聚合酶链反应(PCR)方法。方法使用真菌的通用引物ITS86和ITS4,检测临床常见的致病性真菌,并观察检测方法的敏感性和特异性。结果建立的荧光定量PCR可以准确地检测念珠菌属、曲霉菌属和新型隐球菌等常见的致病真菌,白色念珠菌的敏感性为0.87fg,光滑念珠菌的敏感性为4.2fg,与细菌和人类基因组DNA以及病毒DNA均无交叉反应,根据融解曲线的Tm值可以进行菌种的初步鉴定。结论荧光定量PCR方法检测真菌的敏感性和特异性好,可以应用于临床进行快速检测。     

临床常见念珠菌的PCR-RFLP鉴定方法研究

目的 建立临床常见念珠菌的聚合酶链反应-限制性片段长度多态性(PCR-RFLP)检测技术.方法 用真菌通用引物ITS1-ITS4 分别扩增白色念珠菌、热带念珠菌、光滑念珠菌、克柔念珠菌和近平滑念珠菌5 种菌株的ITS1-5.8 S rDNA-ITS2 区域,然后对扩增产物进行MspⅠ酶切分析.结果 5种念珠菌标准菌株经PCR-RFLP 分析后产生了5 种不同的特异性条带,成功地将临床上常见的5 种念珠菌区分开来.应用此方法鉴定了60 株临床分离的念珠菌,其PCR-RFLP 结果与标准菌株一致,测序结果进一步证实了此方法的可靠性.结论 PCR-RFLP 检测技术具有快速、稳定、特异、准确性高的特点,在常见致病念珠菌鉴定方面具有良好的临床应用前景.     

PCR结合反向膜杂交快速鉴定临床常见念珠菌方法学的建立

目的建立PCR结合反向膜杂交技术快速检测临床常见致病念珠菌的方法。方法根据真菌rDNA高度可变区设计白色念珠菌、热带念珠菌,近平滑念珠菌,都柏林念珠菌,光滑念珠菌、克柔念珠菌的种特异性探针,加尾后固定在尼龙膜上,生物素标记的通用引物扩增转录间隔区ITS1,利用反向膜杂交技术快速检测念珠菌种群。结果以上六种念珠菌均能扩增出大小为218 bp的条带,每张膜上只有与靶序列对应的探针点有阳性结果,其他探针点均为阴性。正常人类基因组DNA及临床常见的几种细菌DNA扩增结果均为阴性。结论 PCR结合反向膜杂交技术方法学的建立,可以快速检测临床常见致病的念珠菌。该方法特异性高,耗费时间短。可以快速、准确的为临床抗真菌药物的使用提供指导依据。     

基因芯片技术检测临床常见致病性念珠菌和新型隐球菌及ERG11基因点突变的实验分析

目的建立基因芯片技术检测临床常见致病性念珠菌和新型隐球菌及氟康唑耐药白念珠菌相关ERG11基因点突变的试验方法。方法针对临床常见的5种致病性念珠菌和新型隐球菌5.8S r DNA与28S r DNA间的内转录间区2(ITS-2)基因设计、合成一系列寡核苷酸探针,制备寡核苷酸芯片,以鉴定5种致病性念珠菌和新型隐球菌;设计、合成能够特异性扩增ERG11基因点突变的引物,并采用不对称荧光聚合酶链反应(PCR)扩增ERG11基因,将PCR产物与芯片进行杂交。结果采用特异性引物和不对称荧光PCR从12株临床分离耐药株中成功扩增出ERG11基因4个突变点;采用基因芯片杂交技术成功鉴定5种临床常见致病性念珠菌和新型隐球菌。结论制备的寡核苷酸基因芯片,可以用于鉴定临床常见的致病性念珠菌和新型隐球菌。     

新生和格特隐球菌种、变种、基因型和交配型的分子鉴定

目的 评价限制性片段长度多态性(Restriction fragment length polymorphism,RFLP)方法检测α和a交配型位点内GEF1α/a基因片段在鉴定新生和格特隐球菌种、变种、基因型和交配型中的作用.方法 筛选交配型位点内的GEF1α/a基因进行PCR-RFLP分析.根据各基因型和交配型参考株的GEF1α/a基因保守序列设计引物,扩增受试新生和格特隐球菌GEF1α/a基因部分片段.利用DNAMAN和Vector NTI软件进行序列比对、限制性图谱分析、限制性内切酶的筛选和模拟电泳.选择EcoTl4 Ⅰ和Hap Ⅱ限制性内切酶分别对125株新生和格特隐球菌的GEF1α/a基因扩增片段进行RFLP分型.结果 所有受试的82株新生隐球菌和43株格特隐球菌均扩增出1 300 bp大小片段,而罗伦隐球菌、白念珠菌、热带念珠菌、近平滑念珠菌、克柔念珠菌、光滑念珠菌、阿萨希毛孢子菌、烟曲霉和黄曲霉参考株均扩增阴性.RFLP分型准确鉴定所有125株新生和格特隐球菌的种、变种、基因型和交配型.结论 针对GEF1α/a基因片段的PCR-RFLP方法特异性高、稳定性好,适用于新生和格特隐球菌种、变种、基因型和交配型的同步和快速鉴定以及进一步的分子流行病学分析.     

一种快速提取新生隐球菌及其他酵母样真菌DNA方法

目的寻找一种快速提取酵母样真菌DNA的方法。方法使用酶溶解法提取新生隐球菌及其他酵母样真菌DNA,并对提取的DNA通过特异性引物进行PCR,所得产物进行酶切验证。结果用此方法提取的新生隐球菌基因组DNA浓度为280～395ng/μL,OD260/OD280比值为1.6～1.8,所提取的DNA可用于PCR的扩增检测特异性酵母样真菌。结论该方法可以用于提取新生隐球菌及其他酵母样真菌。     

聚合酶链反应检测真菌感染的临床评价

用聚合酶链反应 (PCR)检测真菌 ,已有较多报道[1 2 ] ,我们也曾报道过初步研究[3 ] 。为给临床提供快速、灵敏、非创伤的真菌感染诊断方法 ,本文就采用真菌通用引物PCR检测真菌DNA的灵敏度、特异性、可检测真菌范围及临床适用性作进一步评价。1　材料和方法1 1　引物　真菌 18S亚基rRNA多拷贝基因的保守序列通用引物序列为 5′ ATTGGAGGGCAAGTCTGGTG 3′和 5′ CCGATCCCTAGTCGGCATAG 3′ ,扩增产物大小因真菌病原体而异 ,为 4 82～ 5 0 3bp[4] ,白色念珠菌为 4 90bp。由北京赛百盛生物工程公司合成。1 2　临床无菌性…     

探讨实时荧光定量PCR法检测血液新生隐球菌的临床意义

目的探讨实时荧光定量PCR(real-time fluorescent quantitative polymerase chain reaction,RT-FQ-PCR)法检测新生隐球菌感染的临床应用价值。方法选择2014年2月-2014年7月就诊的47例疑似新生隐球菌感染患者的血液标本,采用常规培养基培养方法以及RT-FQ-PCR法进行检测,计算检测新生隐球菌的阳性率,并进行统计学分析。采用RT-FQ-PCR法对新生隐球菌、光滑念珠菌、克柔念珠菌、烟曲霉、黑曲霉、大肠杆菌、肺炎球菌、金黄色葡萄球菌和链球菌这些常见真菌和细菌的DNA进行检测,评价RT-FQ-PCR法的特异性。采用RT-FQ-PCR和常规培养基培养法对稀释至5个/m L的新生隐球菌菌液进行检测,评价RT-FQ-PCR法的灵敏度。结果采用RT-FQ-PCR法,以新生隐球菌特异性引物为探针对新生隐球菌和其他常见真菌及细菌进行检测,只有新生隐球菌出现荧光信号。RTFQ-PCR在5个/m L浓度的新生隐球菌菌液中检测出新生隐球菌,而常规培养基培养法未检测出。在47例疑似新生隐球菌感染患者中,常规培养基培养方法检测出11例,阳性率为23.40%,而RT-FQPCR法检测出14例,阳性率为29.79%,二者经比较差异无统计学意义(P0.05)。结论 RT-FQ-PCR法与常规培养基培养法对新生隐球菌的检测具有很好的一致性。且RT-FQ-PCR法能够检测出较低浓度的新生隐球菌,较常规培养基培养法快速、灵敏度高、特异性强,可缩短报告时间,为临床早期诊断及治疗新生隐球菌感染提供可靠依据。     

聚合酶链反应反向斑点杂交快速检测及鉴定念珠菌

目晨培养为基础的快速检测与鉴定念珠菌的方法。方法 将白色念珠菌、热带念珠菌、光滑念珠菌种特异探针加尾后固定在硝酸纤维素膜上;用氯化苄法提取念珠菌ＤＮＡ,采用真菌特异通用引物ＰＣＲ扩增ＤＮＡ,在扩增过程中用Ｂｉｏ－１１－ｄＵＴＰ标记扩增产物,然后分别与白色念珠菌、热带念珠菌、光滑念珠菌特异探针杂交。结果 对１４种念珠菌扩增均为阳性,对３种常见细菌扩增均为阴性。３种念珠菌只与其对应探针杂交。结论 该方     

常见病原真菌鉴定基因芯片方法的建立

目的 建立临床常见的病原真菌的基因芯片检测体系.方法 选取8种临床常见真菌,包括白色假丝酵母菌、光滑假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌、土曲霉、黄曲霉、米根霉和烟曲霉.根据真菌ITS区设计引物和探针,进行扩增和芯片制备.通过将待检真菌的PCR产物变性后与点布探针的芯片杂交,观察并分析荧光信号强度,进行对真菌的快速检测.并选取临床真菌阳性标本和细菌阳性标本共25份进行验证.结果 25份临床阳性标本的芯片检测结果显示,10份细菌阳性的标本,经真菌基因芯片检测,全部为阴性,无荧光信号.其余15份真菌标本中,12份临床真菌阳性标本均鉴定出芯片中的某一种真菌的结果.另外采用克柔假丝酵母菌人为制备的3份待检样本,经检测,芯片中8种真菌位点无荧光信号,为阴性,仅有真菌通用探针得到荧光信号,表明该标本中有真菌存在,但无法确认是哪一种.结论 本研究建立的基因芯片可对常见病原真菌进行快速准确的鉴定,为基因芯片应用于临床奠定了基础.     

多重荧光PCR-熔解曲线法同时检测常见病原真菌方法学的建立及诊断价值

目的建立多重荧光PCR-熔解曲线法，并评价其同时检测假丝酵母菌、曲霉菌及新型隐球菌的诊断价值。 方法建立多重荧光PCR-熔解曲线法并对其检测体系进行优化。收集临床高度怀疑为侵袭性真菌感染（IFI）的各类临床样本179例，其中血液65例、深部痰液35例、尿液30例、脑脊液18例、胸腹水12例、肺泡灌洗液10例、新鲜肺组织9例。通过敏感性、特异性、重复性实验验证多重荧光PCR-熔解曲线法的检测性能，并应用受试者工作特征（ROC）曲线评价该方法的诊断效能。 结果建立的多重荧光PCR-熔解曲线法可同时检测8种常见病原真菌，包括4种假丝酵母菌（白假丝酵母菌、热带假丝酵母菌、光滑假丝酵母菌、克柔假丝酵母菌）、3种曲霉菌（烟曲霉、黄曲霉、黑曲霉）和新型隐球菌。其最低检测限为1 × 10⁴ cfu/mL，且常见细菌无扩增反应，重复性实验解链温度波动小于0.5 ℃。179例临床样本中，以培养法、镜检法、病理诊断等"金标准"方法诊断IFI阳性为112例，多重荧光PCR-熔解曲线法检测阳性为96例。多重荧光PCR-熔解曲线法同时检测假丝酵母菌、曲霉菌及新型隐球菌的敏感度为0.857，特异度为0.970，阳性预测值为0.980，阴性预测值为0.802，ROC曲线下面积为0.914，95%置信区间为0.868 ~ 0.959，P < 0.001。 结论本研究建立的多重荧光PCR-熔解曲线法可快速、准确、高通量同时检测假丝酵母菌、曲霉菌及新型隐球菌，对于IFI的早期诊断具有重要意义。     

In Vitro Antifungal Activity of Nikkomycin Z in Combination with Fluconazole or Itraconazole

Nikkomycins are nucleoside-peptide antibiotics produced by Streptomyces species with antifungal activities through the inhibition of chitin synthesis. We investigated the antifungal activities of nikkomycin Z alone and in combination with fluconazole and itraconazole. Checkerboard synergy studies were carried out by a macrobroth dilution procedure with RPMI 1640 medium at pH 6.0. At least 10 strains of the following fungi were tested: Candida albicans, other Candida spp., Cryptococcus neoformans, Coccidioides immitis, Aspergillus spp., and dematiacious fungi (including Exophiala jeanselmei, Exophiala spinifera, Bipolaris spicifera, Wangiella dermatitidis, Ochroconis humicola, Phaeoannellomyces werneckii, and Cladophialophora bantiana), and 2 strains each of Fusarium, Scedosporium, Paecilomyces, Penicillium, and Trichoderma spp. A total of 110 isolates were examined. Inocula of fungal elements were standardized by hemacytometer counting or spectrophotometrically. MICs and minimum lethal concentrations (MLCs) were determined visually by comparison of growth in drug-treated tubes with growth in drug-free control tubes. Additive and synergistic interactions between nikkomycin and either fluconazole or itraconazole were observed against C. albicans, Candida parapsilosis, Cryptococcus neoformans, and Coccidioides immitis. Marked synergism was also observed between nikkomycin and itraconazole against Aspergillus fumigatus and Aspergillus flavus. No antagonistic interaction between the drugs was observed with any of the strains tested.     

Rapid identification of fungal pathogens in BacT/ALERT, BACTEC, and BBL MGIT media using polymerase chain reaction and DNA sequencing of the internal transcribed spacer regions

We report a direct polymerase chain reaction/sequence (d-PCRS)-based method for the rapid identification of clinically significant fungi from 5 different types of commercial broth enrichment media inoculated with clinical specimens. Media including BacT/ALERT FA (BioMérieux, Marcy l'Etoile, France) (n = 87), BACTEC Plus Aerobic/F (Becton Dickinson, Microbiology Systems, Sparks, MD) (n = 16), BACTEC Peds Plus/F (Becton Dickinson) (n = 15), BACTEC Lytic/10 Anaerobic/F (Becton Dickinson) (n = 11) bottles, and BBL MGIT (Becton Dickinson) (n = 11) were inoculated with specimens from 138 patients. A universal DNA extraction method was used combining a novel pretreatment step to remove PCR inhibitors with a column-based DNA extraction kit. Target sequences in the noncoding internal transcribed spacer regions of the rRNA gene were amplified by PCR and sequenced using a rapid (24 h) automated capillary electrophoresis system. Using sequence alignment software, fungi were identified by sequence similarity with sequences derived from isolates identified by upper-level reference laboratories or isolates defined as ex-type strains. We identified Candida albicans (n = 14), Candida parapsilosis (n = 8), Candida glabrata (n = 7), Candida krusei (n = 2), Scedosporium prolificans (n = 4), and 1 each of Candida orthopsilosis, Candida dubliniensis, Candida kefyr, Candida tropicalis, Candida guilliermondii, Saccharomyces cerevisiae, Cryptococcus neoformans, Aspergillus fumigatus, Histoplasma capsulatum, and Malassezia pachydermatis by d-PCRS analysis. All d-PCRS identifications from positive broths were in agreement with the final species identification of the isolates grown from subculture. Earlier identification of fungi using d-PCRS may facilitate prompt and more appropriate antifungal therapy.     

The influence of 5-fluorocytosine on nucleic acid synthesis in Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus.

5-Fluorocytosine (5-FC) has a rapid inhibitory effect on the synthesis of RNA and DNA in the yeast and hyphal form of Candida albicans. 5-FC has a less marked effect on the RNA and DNA content of sensitive strains of Cryptococcus neoformans and has no effect on the nucleic acid content of Aspergillus fumigatus nor of resistant strains of C. albicans and C. neoformans. 5-FC has a slower inhibitory effect on yeast cell number increase and no effect on hyphal length of hyphal phase C. albicans over a 7-h incubation period. Rapidly growing yeasts of C. albicans and C. neoformans decrease in volume and in dry weight per cell. 5-FC prevents this decrease in sensitive strains. These results have been discussed with respect to the known metabolic pathway of 5-FC, the fungistatic and fungicidal action of 5-FC and the development of resistance to 5-FC.     

In vitro activities of two new antifungal azoles

The antifungal activities of equimolar quantities of three azole compounds, Bay n 7133 [1-(4-chlorophenoxy)-3,3-dimethyl-2-(1,2,4-triazole-1-yl)methylbutan-2-O1], Bay 1 19139 [1-(4-chlorophenoxy)-1-(1-imidazolyl)-3,3-dimethyl-2-butanol hydrochloride], and ketoconazole, were compared by testing the susceptibility in vitro of 10 clinical isolates each of Candida albicans, Candida parapsilosis, Torulopsis glabrata, Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Rhizopus spp., Mucor spp., and Coccidioides immitis. Molecule for molecule, ketoconazole was consistently the most active drug. All three azoles were primarily fungistatic, although they were fungicidal at clinically relevant concentrations against some strains of A. niger.     

真菌基因组DNA的提取和通用PCR检测方法的建立

目的改良真菌基因组DNA的提取方法,建立真菌通用聚合酶链反应（PCR）,为目前临床真菌感染的早期诊断、预防和治疗提供有效工具。方法参考前人报道的多种真菌基因组DNA的提取方法,作改良以确立本研究中所采纳的手段,并应用真菌核糖体DNA（rDNA）通用引物,以实验室保存的标准菌株及临床分离菌株来建立临床真菌感染检测用通用PCR。结果白念珠菌和烟曲霉在75℃温度下分别作用60和80min可以完全被灭活。经目前多种破壁方法的探讨,发现对于白念珠菌（单细胞真菌）选用酶消化法,破壁效率高达98．29％,而烟曲霉（多细胞真菌）则需要酶消化法与打击器振荡法联合应用,其破壁率也可达66．68％,进一步用酚氯仿法抽提其基因组DNA,能够获得相对纯度高且有一定得量。当选择真菌rDNAITS2区间的一对通用引物,通过PCR反应体系的优化,使得本研究中建立的通用PCR,对于白念珠菌和烟曲霉的检测下限分别为5个和9．7个,其PCR产物测序结果与数据库比对完全一致,同时选择临床分离的或实验室保存的其他真菌、细菌和病毒株进行验证,该方法仅针对真菌群,结合测序分析可以实现种属水平的鉴定。结论改良真菌基因组DNA提取后所建立的针对真菌rDNA的通用PCR敏感且特异,适宜实验室操作。     

Antifungal activity of C-27 steroidal saponins

As part of our search for new antifungal agents from natural resources, 22 C-27 steroidal saponins and 6 steroidal sapogenins isolated from several monocotyledonous plants were tested for their antifungal activity against the opportunistic pathogens Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, and Aspergillus fumigatus. The results showed that the antifungal activity of the steroidal saponins was associated with their aglycone moieties and the number and structure of monosaccharide units in their sugar chains. Within the 10 active saponins, four tigogenin saponins (compounds 1 to 4) with a sugar moiety of four or five monosaccharide units exhibited significant activity against C. neoformans and A. fumigatus, comparable to the positive control amphotericin B. The antifungal potency of these compounds was not associated with cytotoxicity to mammalian cells. This suggests that the C-27 steroidal saponins may be considered potential antifungal leads for further preclinical study.     

In Vitro and In Vivo Activities of CS-758 (R-120758), a New Triazole Antifungal Agent

The activity of CS-758 (R-120758), a new triazole antifungal agent, was evaluated and compared with those of fluconazole, itraconazole, and amphotericin B in vitro and with those of fluconazole and itraconazole in vivo. CS-758 exhibited potent in vitro activity against clinically important fungi. The activity of CS-758 against Candida spp. was superior to that of fluconazole and comparable or superior to those of itraconazole and amphotericin B. CS-758 retained potent activity against Candida albicans strains with low levels of susceptibility to fluconazole (fluconazole MIC, 4 to 32 microg/ml). Against Aspergillus spp. and Cryptococcus neoformans, the activity of CS-758 was at least fourfold superior to those of the other drugs tested. CS-758 also exhibited potent in vivo activity against murine systemic infections caused by C. albicans, C. neoformans, Aspergillus fumigatus, and Aspergillus flavus. The 50% effective doses against these infections were 0.41 to 5.0 mg/kg of body weight. These results suggest that CS-758 may be useful in the treatment of candidiasis, cryptococcosis, and aspergillosis.