病原性丝状真菌快速诊断和鉴定的实验研究
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摘要
第一部分丝状真菌DNA提取方法的比较和优化
目的筛选适合提取丝状真菌模板DNA的方法。方法比较2个菌落培养时间段(3d内和7-14d)提取DNA质量的差异；运用氯化苄法、CTAB法、Biospin法和微波法分别提取黑曲霉基因组DNA,后用直接电泳、浓度测定、PCR扩增等方法比较所提DNA的浓度和质量；选出最优方法,用8种常见丝状真菌进行检验和验证。结果培养3天内的菌落提取的DNA纯度较高,无需纯化即可用于后续实验；4种方法制备的DNA均可用于PCR等后续实验,其中以CTAB法提取的DNA纯度好,产率最高。结论CTAB法是一种适用于丝状真菌DNA提取的简便方法。
第二部分PCR-RFLP技术快速鉴别8种致病丝状真菌病原菌的实验研究目的建立能应用于临床检测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MspⅠ5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一个工作日中完成；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深部感染丝状真菌的有效方法。
第三部分多重PCR检测深部曲霉感染病原菌的实验研究
目的建立能同时鉴别多种曲霉病原菌的多重PCR体系。方法筛选4种最常见深部曲霉感染病原菌(烟曲霉、黄曲霉、土曲霉和黑曲霉)的特异性引物建立多重PCR体系,用该体系检测4种曲霉单模板、双模板和三模板的扩增情况,用DNA模板连续稀释的方式检测该反应体系的敏感性。然后对22株临床株和2株环境分离株进行检测。结果该多重PCR体系有较好的特异性,4种曲霉分别扩增出250 bp,200bp,450 bp和150 bp的DNA片段,电泳图中能够很好区别；在合适的反应条件下,对单模板、双模板和三模板均能扩增出目的片段,未见明显非特异片段的干扰；多重PCR体系的敏感性为100pg,较单种特异性引物的敏感性(10pg)略差。结论多重PCR体系能够检测4种曲霉的单纯感染和混合感染,是一种快速、敏感、特异性强的诊断方法。
第四部分菌落PCR快速鉴别丝状真菌的实验研究
目的探讨菌落PCR在检测丝状真菌病原菌方面的运用。方法初步建立丝状真菌菌落PCR的检测技术,用19种丝状真菌标准株进行验证,选取部分菌种的菌落PCR产物和酶切结果与常规PCR产物和酶切结果进行比较；所有菌落PCR产物进行测序,检测其准确性。结果19株菌中有16株(占84.2%)菌落PCR扩增成功；其中有7株菌进行了菌落PCR与常规PCR产物及酶切图谱的比较,结果条带完全一致；16株扩增成功的菌株ITS区测序结果与传统形态学结果一致。结论菌落PCR可以用于丝状真菌的快速检测,不仅缩短了菌落形态学鉴定所需的较长时间,而且也节省了制备模板DNA的时间和成本,是一种快速、经济的PCR检测技术。
第五部分菌落PCR快速鉴定菌种的临床应用
目的探讨菌落PCR在临床标本检测中的应用。方法收集1例皮下真菌感染的临床标本,在真菌培养长出菌落伊始即进行菌落PCR检测,扩增ITS区进行测序,鉴定病原菌的菌种；收集浅部真菌感染的临床标本(指趾甲、皮屑)11例,选择真菌通用引物NS、皮肤癣菌特异性引物和酵母菌特异性引物组成的多重PCR体系,用菌落PCR的方法进行扩增后,根据扩增片段大小判断病原菌的种属。结果菌落PCR鉴定皮下真菌感染病例的病原菌在取材后的第4天完成,鉴定菌种与传统形态学方法鉴定结果一致；菌落PCR鉴定浅部真菌感染病原菌在3-5d内完成,能将病原菌初步鉴定至种属水平,较传统形态学鉴定节省10d左右的时间。结论菌落PCR技术检测临床标本快速简便,适合临床实验室常规使用。
Part I Comparison and optimization of DNA extraction method for filamentous fungi
Objective To find the best DNA extraction methods for filamentous fungi. Methods We compared the DNA quality of fungi cultured for 3d and 7-14d. As a sample, Aspergillus. niger DNA was extracted by four methods, including benzyl chloride method, CTAB method, Biospin kit and microwave method, and compared the purity and output of the products via agarose gel electrophoresis and PCR. The best method was used to extract 8 pathogenic mould. Results The extracted DNA from colony cultured for 3d was purer than that for 7-14d. Comparing the DNA agarose gel electrophoretogram made by the four methods, all the DNA was amplifiable using a standard PCR and the DNA extracted by CTAB showed the best result. Conclusions CTAB is suit to extract mould DNA and has the advantages of high extraction rate, good quality and simple operation.
Part II Experimental investigation on the identification for eight invasive pathogenic molds by PCR-RFLP
Objective To establish a diagnostic method for invasive mould infections using PCR-RFLP. Methods The ITS regions of Aspergillus. fumigatus, Aspergillus. flavus, Aspergillus. Terreus, Aspergillus. niger, Aspergillus. Versicolor, Aspergillus. Nidulans, Scedosporium apiospermum and Fusarium moniliforme were amplified with universal fungal primers, and RFLP analysis with HhaⅠ, HinfⅠ,HaeⅢ, TaqⅠand MspⅠ. Then 22 clinical strains and 2 environmental isolates were analyzed with PCR-RFLP. Results RFLP analysis of the PCR products with Hha I and Hinf I allowed discrimination of 8 invasive pathogenic molds. DNA extraction, PCR and restriction digestion could be carried out within one day.22 clinical strains and 2 environmental isolates were completely identical to those obtained by conventional morphological methods. Conclusions PCR-RFLP is a rapid method for the identification of deep mold infection.
PartⅢExperimental investigation on the detection for pathogens of invasive aspergillosis by multiplex PCR
Objective To develop a multiplex PCR method to identify simultaneously multiple aspergillus pathogens in a single reaction. Methods Four pairs of primers were selected in one multiplex PCR to identify the most frequent Aspergillus pathogens, A. fumigatus, A. flavus, A. terreus and A. niger. Mutiplex PCR with these 4 primer pairs was used to detect 1 template and 2 or 3 template mixtures. Sensitivity of multiplex PCR was tested with serial dilutions of PCR products. A total of 24 fungal isolates were tested with this method. Results The detection system demonstrated high specificity, that 4 aspergillus were identified through distinct amplicons of 250 bp,200 bp,450 bp and150 bp, respectively. Multiplex PCR could amplify the corresponding 1,2 or 3 DNA fragments, and the sensitivity of multiplex PCR was 100pg, which was less sensitive to single-primer-set PCR(10pg). Conclusions Multiplex PCR might be a rapid, sensitive and specific method for the diagnosis of invasive aspergillosis.
Part IV Experimental investigation on the detection of mould by colony PCR
Objective To detect the application of colony PCR in the field of identification of pathogenic mould. Methods 19 mould strains were examined using direct colony PCR to amplify the ITS region without DNA extraction. We compared parts of the strains by electrophoresis and PCR-RFLP using colony PCR and standard PCR. All of the strains were verified by analysis of fungal ITS sequences. Results 16 of the 19 strains(84.2%) yielded positive results on colony PCR. The cleavage maps of colony PCR-RFLP coincided with the standard PCR, and sequence analysis of the 16 strains were similar to those obtained by conventional morphological methods. Conclusions Colony PCR is a rapid method for the identification of mould, which not only reduces DNA template preparation time before PCR from mould colonies, but also reduces the cost of PCR.
Part V Clinical application of colony PCR in fast fungi identification
Objective To study the application of colony PCR in the detection of clinical specimens. Methods Clinical specimens from a boy with subcutaneous fungal infections were collected, and examined using direct colony PCR to amplify the ITS region. Skin and nail samples were taken from patients with superficial mycosis and detected by colony multiplex PCR based on 3 pairs of primers, universal fungus-specific primer, dermatophyte-specific primer and yeast-specific primer. Results Pathogens identification of subcutaneous fungal infections using colony PCR was similar to those obtained by conventional morphological methods, and completed in 5d. Use of colony PCR reduced the time for identification of superficial mycosis to 3-5d, which was considerably faster than morphology identification (another additional 10d). Conclusions Colony PCR is a rapid and convenient method for the detection of clinical specimens, and may be routinely used in clinical laboratory.
目的筛选适合提取丝状真菌模板DNA的方法。方法比较2个菌落培养时间段(3d内和7-14d)提取DNA质量的差异；运用氯化苄法、CTAB法、Biospin法和微波法分别提取黑曲霉基因组DNA,后用直接电泳、浓度测定、PCR扩增等方法比较所提DNA的浓度和质量；选出最优方法,用8种常见丝状真菌进行检验和验证。结果培养3天内的菌落提取的DNA纯度较高,无需纯化即可用于后续实验；4种方法制备的DNA均可用于PCR等后续实验,其中以CTAB法提取的DNA纯度好,产率最高。结论CTAB法是一种适用于丝状真菌DNA提取的简便方法。
第二部分PCR-RFLP技术快速鉴别8种致病丝状真菌病原菌的实验研究目的建立能应用于临床检测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MspⅠ5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一个工作日中完成；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深部感染丝状真菌的有效方法。
第三部分多重PCR检测深部曲霉感染病原菌的实验研究
目的建立能同时鉴别多种曲霉病原菌的多重PCR体系。方法筛选4种最常见深部曲霉感染病原菌(烟曲霉、黄曲霉、土曲霉和黑曲霉)的特异性引物建立多重PCR体系,用该体系检测4种曲霉单模板、双模板和三模板的扩增情况,用DNA模板连续稀释的方式检测该反应体系的敏感性。然后对22株临床株和2株环境分离株进行检测。结果该多重PCR体系有较好的特异性,4种曲霉分别扩增出250 bp,200bp,450 bp和150 bp的DNA片段,电泳图中能够很好区别；在合适的反应条件下,对单模板、双模板和三模板均能扩增出目的片段,未见明显非特异片段的干扰；多重PCR体系的敏感性为100pg,较单种特异性引物的敏感性(10pg)略差。结论多重PCR体系能够检测4种曲霉的单纯感染和混合感染,是一种快速、敏感、特异性强的诊断方法。
第四部分菌落PCR快速鉴别丝状真菌的实验研究
目的探讨菌落PCR在检测丝状真菌病原菌方面的运用。方法初步建立丝状真菌菌落PCR的检测技术,用19种丝状真菌标准株进行验证,选取部分菌种的菌落PCR产物和酶切结果与常规PCR产物和酶切结果进行比较；所有菌落PCR产物进行测序,检测其准确性。结果19株菌中有16株(占84.2%)菌落PCR扩增成功；其中有7株菌进行了菌落PCR与常规PCR产物及酶切图谱的比较,结果条带完全一致；16株扩增成功的菌株ITS区测序结果与传统形态学结果一致。结论菌落PCR可以用于丝状真菌的快速检测,不仅缩短了菌落形态学鉴定所需的较长时间,而且也节省了制备模板DNA的时间和成本,是一种快速、经济的PCR检测技术。
第五部分菌落PCR快速鉴定菌种的临床应用
目的探讨菌落PCR在临床标本检测中的应用。方法收集1例皮下真菌感染的临床标本,在真菌培养长出菌落伊始即进行菌落PCR检测,扩增ITS区进行测序,鉴定病原菌的菌种；收集浅部真菌感染的临床标本(指趾甲、皮屑)11例,选择真菌通用引物NS、皮肤癣菌特异性引物和酵母菌特异性引物组成的多重PCR体系,用菌落PCR的方法进行扩增后,根据扩增片段大小判断病原菌的种属。结果菌落PCR鉴定皮下真菌感染病例的病原菌在取材后的第4天完成,鉴定菌种与传统形态学方法鉴定结果一致；菌落PCR鉴定浅部真菌感染病原菌在3-5d内完成,能将病原菌初步鉴定至种属水平,较传统形态学鉴定节省10d左右的时间。结论菌落PCR技术检测临床标本快速简便,适合临床实验室常规使用。
Part I Comparison and optimization of DNA extraction method for filamentous fungi
Objective To find the best DNA extraction methods for filamentous fungi. Methods We compared the DNA quality of fungi cultured for 3d and 7-14d. As a sample, Aspergillus. niger DNA was extracted by four methods, including benzyl chloride method, CTAB method, Biospin kit and microwave method, and compared the purity and output of the products via agarose gel electrophoresis and PCR. The best method was used to extract 8 pathogenic mould. Results The extracted DNA from colony cultured for 3d was purer than that for 7-14d. Comparing the DNA agarose gel electrophoretogram made by the four methods, all the DNA was amplifiable using a standard PCR and the DNA extracted by CTAB showed the best result. Conclusions CTAB is suit to extract mould DNA and has the advantages of high extraction rate, good quality and simple operation.
Part II Experimental investigation on the identification for eight invasive pathogenic molds by PCR-RFLP
Objective To establish a diagnostic method for invasive mould infections using PCR-RFLP. Methods The ITS regions of Aspergillus. fumigatus, Aspergillus. flavus, Aspergillus. Terreus, Aspergillus. niger, Aspergillus. Versicolor, Aspergillus. Nidulans, Scedosporium apiospermum and Fusarium moniliforme were amplified with universal fungal primers, and RFLP analysis with HhaⅠ, HinfⅠ,HaeⅢ, TaqⅠand MspⅠ. Then 22 clinical strains and 2 environmental isolates were analyzed with PCR-RFLP. Results RFLP analysis of the PCR products with Hha I and Hinf I allowed discrimination of 8 invasive pathogenic molds. DNA extraction, PCR and restriction digestion could be carried out within one day.22 clinical strains and 2 environmental isolates were completely identical to those obtained by conventional morphological methods. Conclusions PCR-RFLP is a rapid method for the identification of deep mold infection.
PartⅢExperimental investigation on the detection for pathogens of invasive aspergillosis by multiplex PCR
Objective To develop a multiplex PCR method to identify simultaneously multiple aspergillus pathogens in a single reaction. Methods Four pairs of primers were selected in one multiplex PCR to identify the most frequent Aspergillus pathogens, A. fumigatus, A. flavus, A. terreus and A. niger. Mutiplex PCR with these 4 primer pairs was used to detect 1 template and 2 or 3 template mixtures. Sensitivity of multiplex PCR was tested with serial dilutions of PCR products. A total of 24 fungal isolates were tested with this method. Results The detection system demonstrated high specificity, that 4 aspergillus were identified through distinct amplicons of 250 bp,200 bp,450 bp and150 bp, respectively. Multiplex PCR could amplify the corresponding 1,2 or 3 DNA fragments, and the sensitivity of multiplex PCR was 100pg, which was less sensitive to single-primer-set PCR(10pg). Conclusions Multiplex PCR might be a rapid, sensitive and specific method for the diagnosis of invasive aspergillosis.
Part IV Experimental investigation on the detection of mould by colony PCR
Objective To detect the application of colony PCR in the field of identification of pathogenic mould. Methods 19 mould strains were examined using direct colony PCR to amplify the ITS region without DNA extraction. We compared parts of the strains by electrophoresis and PCR-RFLP using colony PCR and standard PCR. All of the strains were verified by analysis of fungal ITS sequences. Results 16 of the 19 strains(84.2%) yielded positive results on colony PCR. The cleavage maps of colony PCR-RFLP coincided with the standard PCR, and sequence analysis of the 16 strains were similar to those obtained by conventional morphological methods. Conclusions Colony PCR is a rapid method for the identification of mould, which not only reduces DNA template preparation time before PCR from mould colonies, but also reduces the cost of PCR.
Part V Clinical application of colony PCR in fast fungi identification
Objective To study the application of colony PCR in the detection of clinical specimens. Methods Clinical specimens from a boy with subcutaneous fungal infections were collected, and examined using direct colony PCR to amplify the ITS region. Skin and nail samples were taken from patients with superficial mycosis and detected by colony multiplex PCR based on 3 pairs of primers, universal fungus-specific primer, dermatophyte-specific primer and yeast-specific primer. Results Pathogens identification of subcutaneous fungal infections using colony PCR was similar to those obtained by conventional morphological methods, and completed in 5d. Use of colony PCR reduced the time for identification of superficial mycosis to 3-5d, which was considerably faster than morphology identification (another additional 10d). Conclusions Colony PCR is a rapid and convenient method for the detection of clinical specimens, and may be routinely used in clinical laboratory.
引文
[1]Arendrup MC. Invasive fungal infections:past achievements and challenges ahead[J]. Clin Microbiol Infect,2009,15(7):599-601.
[2]White PL, Perry MD, Barnes RA. An update on the molecular diagnosis of invasive fungal disease[J]. FEMS Microbiol Lett.2009,296(1):1-10.
[3]Bow EJ. Invasive fungal infection in haematopoietic stem cell transplant recipients: epidemiology from the transplant physician's viewpoint[J]. Mycopathologia,2009, 168(6):2832-2897.
[4]McCoy D, Depestel DD, Carver PL. Primary antifungal prophylaxis in adult hematopoietic stem cell transplant recipients:current therapeutic concepts[J]. Pharmacotherapy,2009,29(11):1306-1325.
[5]Baddley JW, Pappas PG, Smith AC, et al. Epidemiology of Aspergillus terreus at a university hospital[J]. J Clin Microbiol,2003,41(12):5525-5529.
[6]Nucci M, Marr KA. Emerging fungal diseases[J]. Clin Infect Dis,2005,41(4): 521-526.
[7]McCoy D, Depestel DD, Carver PL. Primary antifungal prophylaxis in adult hematopoietic stem cell transplant recipients:current therapeutic concepts[J]. Pharmacotherapy,2009,29(11):1306-1325.
[8]Diekema DJ, Messer SA, Hollis RJ, et al. Activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B against 448 recent clinical isolates of filamentous fungi[J]. J Clin Microbiol,2003,41(8):3623-3626.
[9]Husain S, Alexander BD, Munoz P, et al. Opportunistic mycelial fungal infections in organ transplant recipients:emerging importance of non-Aspergillus mycelial fungi[J]. Clin Infect Dis,2003,37(2):221-229.
[10]Caillot D, Couaillier JF, Bernard A, et al. Increasing volume and changing characteristics of invasive pulmonary aspergillosis on sequential thoracic computed tomography scans in patients with neutropenia[J]. J Clin Oncol,2001,19 (1) 253-259.
[11]Kelaher A. Two non-invasive diagnostic tools for invasive aspergilosis: (1-3)-beta-Dglucan and the galactomannan assay[J]. Clin Lab Sci,2006,19 (4) 222-224.
[12]Haugland RA, Brinkman N, Vesper SJ. Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using realtime PCR analysis[J]. J Microbiol Methods,2002,50(3):319-323.
[13]Maaroufi Y, Ahariz N, Husson M, et al. Comparison of different methods of isolation of DNA of commonly encountered Candida species and its quantitation by using a real-time PCR-based assay[J]. J Clin Microbiol,2004,42(7): 3159-3163.
[14]Griffin DW, Kellogg CA, Peak KK, et al. A rapid and efficient assay for extracting DNA from fungi[J]. Lett Appl Microbiol,2002,34(3):210-214.
[15]Hinrikson HP, Hurst SF, Lott TJ, et al. Assessment of ribosomal large-subunit D1-D2, internal transcribed spacer 1, and internal transcribed spacer 2 regions as targets for molecular identification of medically important Aspergillus species[J]. J Clin Microbiol,2005,43(5):2092-2103.
[16]Nilsson RH, Ryberg M, Kristiansson E, et al. Taxonomic reliability of DNA sequences in public sequence databases:a fungal perspective[J]. PLoS One,2006, 1:e59.
[17]Kamiya A, Kikuchi A, Tomita Y, et al. PCR and PCR-RFLP techniques targeting the DNA topoisomerase Ⅱ gene for rapid clinical diagnosis of the etiologic agent of dermatophytosis[J]. J Dermatol Sci,2004,34(1):35-48.
[18]Dean TR, Kohan M, Betancourt D, et al. A simple polymerase chain reaction/restriction fragment length polymorphism assay capable of identifying medically relevant filamentous fungi[J]. Mol Biotechnol,2005,31(1):21-28.
[19]Vastag M, Papp T, Kasza Z, et al. Intraspecific variation in two species of Rhizomucor assessed by random amplified polymorphic DNA analysis[J]. J Basic Microbiol,2000,40(4):269-277.
[20]Raclasky V, Trtkova J, Ruskova L, et al. Primer R108 performs best in the RAPD strain typing of three Aspergillus species frequently isolated from patients[J]. Folia Microbiol,2006,51(2):136-140.
[21]Healy M, Reece K, Walton D,et al. Identification to the species level and differentiation between strains of Aspergillus clinical isolates by automated repetitive-sequence-based PCR[J]. J Clin Microbiol,2004,42(9):4016-24.
[22]Kanbe T, Yamaki K, Kikuchi A. Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene[J]. Microbiol Immunol,2002,46(12):841-848.
[23]Haugland RA, Varma M, Wymer LJ, et al. Quantitative PCR analysis of selected Aspergillus, Penicillium and Paecilomyces species[J]. Syst Appl Microbiol,2004, 27(2):198-210.
[1]Chen SC, Halliday CL, Meyer W. A review of nucleic acid-based diagnostic tests for systemic mycoses with an emphasis on polymerase chain reaction-based assays[J]. Med Mycol,2002,40(4):333-357.
[2]Maaroufi Y, Ahariz N, Husson M, et al. Comparison of different methods of isolation of DNA of commonly encountered Candida species and its quantitation by using a real-time PCR-based assay[J]. J Clin Microbiol,2004,42(7):3159-3163.
[3]Haugland RA, Brinkman N, Vesper SJ. Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using realtime PCR analysis[J]. J Microbiol Methods,2002,50(3):319-323.
[4]Haugland RA, Heckman JL, Wymer LJ. Evaluation of different methods for the extraction of DNA from fungal conidia by quantitative competitive PCR analysis[J]. J Microbiol Methods,1999,37(2):165-176.
[5]Griffin DW, Kellogg CA, Peak KK, et al. A rapid and efficient assay for extracting DNA from fungi[J]. Lett Appl Microbiol,2002,34(3):210-214.
[6]Clarkson AI, Lefevre P, Titchener-Hooker NJ. A study of process interactions between cell disruption and debris clarification stages in the recovery of yeast intracellular products[J]. Biotechnol Prog,1993,9(5):462-467.
[7]Cenis JL. Rapid extraction of fungal DNA for PCR amplification[J]. Nucleic Acids Res,1992,20(9):2380.
[8]Specht CA, DiRusso CC, Novotny CP, et al. A method for extracting high-molecular-weight deoxyribonucleic acid from fungi[J]. Anal. Anal Biochem, 1982,119(1):158-63.
[9]Walsh TJ, Francesconi A, Kasai M, et al. PCR and single-strand conformational polymorphism for recognition of medically important opportunistic fungi[J]. J Clin Microbiol,1995,33(12):3216-3220.
[1]Martin GS, Mannino DM, Eaton S, et al. The epidemiology of sepsis in the United States from 1979 through 2000[J]. N Engl J Med,2003,348(16):1546-1554.
[2]Marr KA, Carter RA, Crippa F, et al. Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients[J]. Clin Infect Dis,2002,34(7): 909-917.
[3]Cornillet A, Camus C, Nimubona S, et al. Comparison of epidemiological, clinical, and biological features of invasive aspergillosis in neutropenic and nonneutropenic patients:a 6-year survey[J]. Clin Infect Dis,2006,43(5):577-584.
[4]Pfaller MA, Diekema DJ. Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus[J]. J Clin Microbiol,2004,42(10):4419-4431.
[5]Baddley JW, Pappas PG, Smith AC, et al. Epidemiology of Aspergillus terreus at a
university hospital[J]. J Clin Microbiol,2003,41(12):5525-5529.
[6]Nucci M, Marr KA, Queiroz-Telles F, et al. Fusarium infection in hematopoietic stem cell transplant recipients[J]. Clin Infect Dis,2004,38(9):1237-1242.
[7]Walsh TJ, Groll A, Hiemenz J, et al. Infections due to emerging and uncommon medically important fungal pathogens[J]. Clin Microbiol Infect,2004,10(Suppl 1):48-66.
[8]Diekema DJ, Messer SA, Hollis RJ, et al. Activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B against 448 recent clinical isolates of filamentous fungi[J]. J Clin Microbiol,2003,41(8):3623-3626.
[9]Kelaher A. Two non-invasive diagnostic tools for invasive aspergilosis: (1-3)-beta-Dglucan and the galactomannan assay[J]. Clin Lab Sci,2006,19 (4) 222-224.
[10]Mirhendi H, Makimura K, Khoramizadeh M, et al. A one-enzyme PCR-RFLP assay for identification of six medically important Candida species[J]. Nippon Ishinkin Gakkai Zassh,2006,47(3):225-229.
[1]Barnes RA, White PL, Bygrave C, et al. Clinical impact of enhanced diagnosis of invasive fungal disease in high-risk haematology and stem cell transplant patients[J]. J Clin Pathol,2009,62(1):64-69.
[2]Baddley JW, Pappas PG, Smith AC, et al. Epidemiology of Aspergillus terreus at a university hospital[J]. J Clin Microbiol,2003,41(12):5525-5529.
[3]刘军,刘维达.聚合酶链反应检测深部致病真菌的实验研究[J].中华皮肤科杂志,2005,38：503-505.
[4]Greene RE, Schlamm HT, Oestmann JW, et al. Imaging findings in acute invasive pulmonary aspergillosis:clinical significance of the halo sign[J]. Clin Infect Dis, 2007,44(3):373-379.
[5]Cooper EE, O'Reilly MA, Guest DI, et al. Influence of building construction work on Aspergillus infection in a hospital setting[J]. Infect Control Hosp Epidemiol,2003, 24(7):472-476.
[6]Bow EJ. Invasive fungal infection in haematopoietic stem cell transplant recipients: epidemiology from the transplant physician's viewpoint[J]. Mycopathologia,2009, 168(6):2832-2897.
[7]McCoy D, Depestel DD, Carver PL. Primary antifungal prophylaxis in adult hematopoietic stem cell transplant recipients:current therapeutic concepts[J]. Pharmacotherapy,2009,29(11):1306-1325.
[8]Meersseman W, Lagrou K, Maertens J, et al. Invasive aspergillosis in the intensive care unit[J]. Clin InfectDis,2007,45(2):205-216.
[9]Caillot D, Couaillier JF, Bernard A, et al. Increasing volume and changing characteristics of invasive pulmonary aspergillosis on sequential thoracic computed tomography scans in patients with neutropenia[J]. J Clin Oncol,2001,19 (1) 2532259.
[10]Kelaher A. Two non-invasive diagnostic tools for invasive aspergilosis: (1-3)-beta-Dglucan and the galactomannan assay[J]. Clin Lab Sci,2006,19 (4): 222-224.
[11]Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH. Multiplex PCR: critical parameters and step-by-step protocol[J]. Biotechniques,1997,23(3): 504-511.
[12]Orzechowski Xavier M, Pasqualotto AC, Uchoa Sales Mda P, et al. Invasive pulmonary aspergillosis due to a mixed infection caused by Aspergillus flavus and Aspergillus fumigatus[J]. Rev Iberoam Micol,2008,25(3):176-178.
[1]Maaroufi Y, Ahariz N, Husson M, et al. Comparison of different methods of isolation of DNA of commonly encountered Candida species and its quantitation by using a real-time PCR-based assay[J]. J Clin Microbiol,2004,42(7):3159-3163.
[2]Gussow D, Clackson T. Direct clone characterization from plaques and colonies by the polymerase chain reaction[J]. Nucleic Acids Res,1989,17(10):4000.
[3]Haugland RA, Brinkman N, Vesper SJ. Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using realtime PCR analysis[J]. J Microbiol Methods,2002,50(3):319-323.
[4]Fredricks DN, Smith C, Meier A. Comparison of Six DNA Extraction Methods for Recovery of Fungal DNA as Assessed by Quantitative PCR[J]. J Clin Microbiol, 2005,43(10):5122-5128.
[5]唐哗盛,李英.菌落PCR在大规模基因组测序中的应用[J].生物化学与生物物理进展,2002,29(2)：316-318.
[6]Dallas-Yang Q, Jiang G, Sladek FM. Avoiding false positives in colony PCR[J]. Biotechniques,1998,24(4):580-582.
[7]Suzuki S, Taketani H, Kusumoto K, et al. High-throughput genotyping of filamentous fungus Aspergillus oryzae based on colony direct polymerase chain reaction[J]. J Biosci Bioeng,2006,102(6):572-574.
[8]Alshahni MM, Makimura K, Yamada T, et al. Direct colony PCR of several medically important fungi using Ampdirect plus[J]. Jpn J Infect Dis,2009,62(2):164-167.
[9]Griffin DW, Kellogg CA, Peak KK, et al. A rapid and efficient assay for extracting DNA from fungi[J]. Lett Appl Microbiol,2002,34(3):210-214.
[10]van den Brink HJ, van Zeijl CM, Brons JF, et al. Cloning and characterization of the NADPH cytochrome P450 oxidoreductase gene from the filamentous fungus Aspergillus niger[J].DNA Cell Biol,1995,14(8):719-729.
[11]H Mirhendi, K Diba, A Rezaei, et al. Colony-PCR is a rapid and sensitive method for DNA amplification in yeasts[J]. J Public Health,2007,36(1):40-44.
[12]Agrawal V, Roy N. Contaminating insert degradation by preincubation colony PCR: a method for avoiding false positives in transformant[J]. Anal Biochem,2008, 375(1):159-161.
[1]Gussow D, Clackson T. Direct clone characterization from plaques and colonies by the polymerase chain reaction[J]. Nucleic Acids Res,1989,17(10):4000.
[2]Kelaher A. Two non-invasive diagnostic tools for invasive aspergilosis: (1-3)-beta-Dglucan and the galactomannan assay[J]. Clin Lab Sci,2006,19 (4): 222-224.
[3]吴绍熙.现代医学真菌学检验手册[M].北京：中国协和医科大学出版社,2005：137.
[4]Bretagne S, Costa JM. Towards a molecular diagnosis of invasive aspergillosis and disseminated candidosis[J]. FEMS Immunol Med Microbiol,2005,45(3):361-318.
[5]Gerald Pierard. Onychomycosis and Other Superficial Fungal Infections of the Foot in the Elderly:A Pan-European Survey[J]. Dermatology,2001,202(3):220-224.
[6]李筱芳,吕桂霞,陈辉,等.多重PCR诊断甲真菌病的实验研究[J].中国皮肤性病学杂志,2007,21(1)：10-13.
[7]Lau A, Sorrell TC, Lee O, et al. Colony multiplex-tandem PCR for rapid, accurate identification of fungal cultures[J]. J Clin Microbiol,2008,46(12):4058-4060.
[1]Arendrup MC. Invasive fungal infections:past achievements and challenges ahead[J]. Clin Microbiol Infect,2009,15(7):599-601.
[2]Meersseman W, Lagrou K, Maertens J, et al. Invasive aspergillosis in the intensive care unit[J]. Clin InfectDis,2007,45(2):205-216.
[3]Greene RE, Schlamm HT, Oestmann JW, et al. Imaging findings in acute invasive pulmonary aspergillosis:clinical significance of the halo sign[J]. Clin Infect Dis, 2007,44(3):373-379.
[4]Bow EJ. Invasive fungal infection in haematopoietic stem cell transplant recipients: epidemiology from the transplant physician's viewpoint[J]. Mycopathologia,2009, 168(6):2832-2897.
[5]McCoy D, Depestel DD, Carver PL. Primary antifungal prophylaxis in adult hematopoietic stem cell transplant recipients:current therapeutic concepts[J]. Pharmacotherapy,2009,29(11):1306-1325.
[6]Baddley JW, Pappas PG, Smith AC, et al. Epidemiology of Aspergillus terreus at a university hospital[J]. J Clin Microbiol,2003,41(12):5525-5529.
[7]Hagerty JA, Ortiz J, Reich D, et al. Fungal infections in solid organ transplant patients[J]. Surg Infect (Larchmont),2003,4(3):263-271.
[8]Barnes RA, White PL, Bygrave C, et al. Clinical impact of enhanced diagnosis of invasive fungal disease in high-risk haematology and stem cell transplant patients[J]. J Clin Pathol,2009,62(1):64-69.
[9]Nucci M, Marr KA. Emerging fungal diseases[J]. Clin Infect Dis,2005,41(4): 521-526.
[10]Diekema DJ, Messer SA, Hollis RJ, et al. Activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B against 448 recent clinical isolates of filamentous fungi[J]. J Clin Microbiol,2003,41(8):3623-3626.
[11]Husain S, Alexander BD, Munoz P, et al. Opportunistic mycelial fungal infections in organ transplant recipients:emerging importance of non-Aspergillus mycelial fungi[J]. Clin Infect Dis,2003,37(2):221-229.
[12]Maertens J. Evaluating prophylaxis of invasive fungal infections in patients with haematologic malignancies[J]. Eur J Haematol,2007,78(4):275-82.
[13]Nucci M, Anaissie E. Fusarium infections in immunocompromised patients[J]. Clin Microbiol Rev,2007,20(4):695-704.
[14]Pfaller MA, Diekema DJ. Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus[J]. J Clin Microbiol,2004,42(10):4419-4431.
[15]Pagano L, Caira M, Candoni A, et al. The epidemiology of fungal infections in patients with hematologic malignancies:the SEIFEM-2004 study[J]. Haematologica,2006,91(8):1068-1075.
[16]Walsh TJ, Groll A, Hiemenz J, et al. Infections due to emerging and uncommon medically important fungal pathogens[J]. Clin Microbiol Infect,2004,10(1): 48-66.
[17]Husain S, Munoz P, Forrest G, et al. Infections due to Scedosporium apiospermum and Scedosporium prolificans in transplant recipients:clinical characteristics and impact of antifungal agent therapy on outcome[J]. Clin Infect Dis,2005,40(1): 89-99.
[18]Araujo R, Costa-de-Oliveira S, Coutinho I, et al. Evaluating the resistance to posaconazole by E-test and CLSI broth microdilution methodologies of Candida spp. and pathogenic moulds[J]. Eur J Clin Microbiol Infect Dis,2009,28(9): 1137-1140.
[19]Chamilos G, Lewis RE, Kontoyiannis DP. Delaying amphotericin B-based frontline therapy significantly increases mortality among patients with hematologic malignancy who have zygomycosis[J]. Clin Infect Dis,2008,47(4):503-509.
[20]Roden MM, Zaoutis T, Buchanan WL, et al. Epidemiology and outcome of zygomycosis:a review of 929 reported cases[J]. Clin Infect Dis,2005,41(5): 634-653.
[21]Chayakulkeeree M, Ghannoum MA, Perfect JR. Zygomycosis:the re-emerging fungal infection[J]. Eur J Clin Microbiol Infect Dis,2006,25(4):215-229.
[22]Schabereiter-Gurtner C, Selitsch B, Rotter ML, et al. Development of novel real-time PCR assays for detection and differentiation of eleven medically important Aspergillus and Candida species in clinical specimens[J]. J Clin Microbiol,2007,45(3):906-914.
[23]Hayette MP, Vaira D, Susin F, et al. Detection of Aspergillus species DNA by PCR in bronchoalveolar lavage fluid[J]. J Clin Microbiol,2001,39(6):2338-2340.
[24]Kami M, Fukui T, Ogawa S, et al. Use of real-time PCR on blood samples for diagnosis of invasive aspergillosis[J]. Clin Infect Dis,2001,33(9):1504-1512.
[25]Halliday C, Hoile R, Sorrell T, et al. Role of prospective screening of blood for invasive aspergillosis by polymerase chain reaction in febrile neutropenic recipients of haematopoietic stem cell transplants and patients with acute leukaemia[J]. Brit J Haematol,2006,132(4):478-486.
[26]Hall L, Wohlfiel S, Roberts GD. Experience with the MicroSeq D2 large-subunit ribosomal DNA sequencing kit for identification of filamentous fungi encountered in the clinical laboratory[J]. J Clin Microbiol,2004,42(2):622-626.
[27]Schwarz P, Bretagne S, Gantier JC, et al. Molecular identification of zygomycetes from culture and experimentally infected tissues[J]. J Clin Microbiol.2006, 44(2):340-349.
[28]Nilsson RH, Ryberg M, Kristiansson E, et al. Taxonomic reliability of DNA sequences in public sequence databases:a fungal perspective[J]. PLoS One,2006, I:e59.
[29]Dean TR, Kohan M, Betancourt D, et al. A simple polymerase chain reaction/restriction fragment length polymorphism assay capable of identifying medically relevant filamentous fungi[J], Mol Biotechnol,2005,31(1):21-28.
[30]Kamiya A, Kikuchi A, Tomita Y, et al. PCR and PCR-RFLP techniques targeting the DNA topoisomerase II gene for rapid clinical diagnosis of the etiologic agent of dermatophytosis[J]. J Dermatol Sci,2004,34(1):35-48.
[31]Vastag M, Papp T, Kasza Z, et al. Intraspecific variation in two species of Rhizomucor assessed by random amplified polymorphic DNA analysis[J]. J Basic Microbiol,2000,40(4):269-277.
[32]Raclasky V, Trtkova J, Ruskova L, et al. Primer R108 performs best in the RAPD strain typing of three Aspergillus species frequently isolated from patients[J]. Folia Microbiol,2006,51(2):136-140.
[33]Healy M, Reece K, Walton D,et al. Identification to the species level and differentiation between strains of Aspergillus clinical isolates by automated repetitive-sequence-based PCR[J]. J Clin Microbiol,2004,42(9):4016-24.
[34]Dewey Hansen, Mimi Healy, Kristy Reece, et al. Repetitive-Sequence-Based PCR Using the DiversiLab System for Identification of Aspergillus Species[J]. J Clin Microbiol,2008,46(5):1835-1839.
[35]Luo G, Mitchell TG. Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR[J]. J Clin Microbiol,2002,40(8):2860-2865.
[36]Araujo R, Pina-Vaz C, Rodrigues AG, et al. Simple and highly discriminatory microsatellite-based multiplex PCR for Aspergillus fumigatus strain typing[J]. Clin Microbiol Infect,2009,15(3):260-266.
[37]Kanbe T, Yamaki K, Kikuchi A. Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene[J]. Microbiol Immunol,2002,46(12):841-848.
[38]Sugita C, Makimura K, Uchida K, et al. PCR identification system for the genus Aspergillus and three major pathogenic species:Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger[J]. Med Mycol,2004,42(5):433-437.
[39]Wu Z, Tsumura Y, Blomquist G, et al.18S rRNA gene variation among common airborne fungi, and development of specific oligonucleotide probes for the detection of fungal isolates[J]. Appl Environ Microbiol,2003,69(9):5389-5397.
[40]Hsiao CR, Huang L, Bouchara JP, et al. Identification of medically important molds by an oligonucleotide array[J]. J Clin Microbiol,2005,43(8):3760-3768.
[41]Huang A, Li JW, Shen ZQ, et al. High-throughput identification of clinical pathogenic fungi by hybridization to an oligonucleotide microarray[J]. J Clin Microbiol,2006,44(9):3299-3305.
[42]Haugland RA, Varma M, Wymer LJ, et al. Quantitative PCR analysis of selected Aspergillus, Penicillium and Paecilomyces species[J]. Syst Appl Microbiol,2004, 27(2):198-210.
[43]Cuenca-Estrella M, Meije Y, Diaz-Pedroche C, et al. Value of serial quantification of fungal DNA by a real-time PCR-based technique for early diagnosis of invasive Aspergillosis in patients with febrile neutropenia[J]. J Clin Microbiol,2009,47(2): 379-84.
[44]Challier S, Boyer S, Abachin E, et al. Development of a serum-based Taqman real-time PCR assay for diagnosis of invasive aspergillosis[J]. J Clin Microbiol,
2004,42(2)：844-846.
[2]White PL, Perry MD, Barnes RA. An update on the molecular diagnosis of invasive fungal disease[J]. FEMS Microbiol Lett.2009,296(1):1-10.
[3]Bow EJ. Invasive fungal infection in haematopoietic stem cell transplant recipients: epidemiology from the transplant physician's viewpoint[J]. Mycopathologia,2009, 168(6):2832-2897.
[4]McCoy D, Depestel DD, Carver PL. Primary antifungal prophylaxis in adult hematopoietic stem cell transplant recipients:current therapeutic concepts[J]. Pharmacotherapy,2009,29(11):1306-1325.
[5]Baddley JW, Pappas PG, Smith AC, et al. Epidemiology of Aspergillus terreus at a university hospital[J]. J Clin Microbiol,2003,41(12):5525-5529.
[6]Nucci M, Marr KA. Emerging fungal diseases[J]. Clin Infect Dis,2005,41(4): 521-526.
[7]McCoy D, Depestel DD, Carver PL. Primary antifungal prophylaxis in adult hematopoietic stem cell transplant recipients:current therapeutic concepts[J]. Pharmacotherapy,2009,29(11):1306-1325.
[8]Diekema DJ, Messer SA, Hollis RJ, et al. Activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B against 448 recent clinical isolates of filamentous fungi[J]. J Clin Microbiol,2003,41(8):3623-3626.
[9]Husain S, Alexander BD, Munoz P, et al. Opportunistic mycelial fungal infections in organ transplant recipients:emerging importance of non-Aspergillus mycelial fungi[J]. Clin Infect Dis,2003,37(2):221-229.
[10]Caillot D, Couaillier JF, Bernard A, et al. Increasing volume and changing characteristics of invasive pulmonary aspergillosis on sequential thoracic computed tomography scans in patients with neutropenia[J]. J Clin Oncol,2001,19 (1) 253-259.
[11]Kelaher A. Two non-invasive diagnostic tools for invasive aspergilosis: (1-3)-beta-Dglucan and the galactomannan assay[J]. Clin Lab Sci,2006,19 (4) 222-224.
[12]Haugland RA, Brinkman N, Vesper SJ. Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using realtime PCR analysis[J]. J Microbiol Methods,2002,50(3):319-323.
[13]Maaroufi Y, Ahariz N, Husson M, et al. Comparison of different methods of isolation of DNA of commonly encountered Candida species and its quantitation by using a real-time PCR-based assay[J]. J Clin Microbiol,2004,42(7): 3159-3163.
[14]Griffin DW, Kellogg CA, Peak KK, et al. A rapid and efficient assay for extracting DNA from fungi[J]. Lett Appl Microbiol,2002,34(3):210-214.
[15]Hinrikson HP, Hurst SF, Lott TJ, et al. Assessment of ribosomal large-subunit D1-D2, internal transcribed spacer 1, and internal transcribed spacer 2 regions as targets for molecular identification of medically important Aspergillus species[J]. J Clin Microbiol,2005,43(5):2092-2103.
[16]Nilsson RH, Ryberg M, Kristiansson E, et al. Taxonomic reliability of DNA sequences in public sequence databases:a fungal perspective[J]. PLoS One,2006, 1:e59.
[17]Kamiya A, Kikuchi A, Tomita Y, et al. PCR and PCR-RFLP techniques targeting the DNA topoisomerase Ⅱ gene for rapid clinical diagnosis of the etiologic agent of dermatophytosis[J]. J Dermatol Sci,2004,34(1):35-48.
[18]Dean TR, Kohan M, Betancourt D, et al. A simple polymerase chain reaction/restriction fragment length polymorphism assay capable of identifying medically relevant filamentous fungi[J]. Mol Biotechnol,2005,31(1):21-28.
[19]Vastag M, Papp T, Kasza Z, et al. Intraspecific variation in two species of Rhizomucor assessed by random amplified polymorphic DNA analysis[J]. J Basic Microbiol,2000,40(4):269-277.
[20]Raclasky V, Trtkova J, Ruskova L, et al. Primer R108 performs best in the RAPD strain typing of three Aspergillus species frequently isolated from patients[J]. Folia Microbiol,2006,51(2):136-140.
[21]Healy M, Reece K, Walton D,et al. Identification to the species level and differentiation between strains of Aspergillus clinical isolates by automated repetitive-sequence-based PCR[J]. J Clin Microbiol,2004,42(9):4016-24.
[22]Kanbe T, Yamaki K, Kikuchi A. Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene[J]. Microbiol Immunol,2002,46(12):841-848.
[23]Haugland RA, Varma M, Wymer LJ, et al. Quantitative PCR analysis of selected Aspergillus, Penicillium and Paecilomyces species[J]. Syst Appl Microbiol,2004, 27(2):198-210.
[1]Chen SC, Halliday CL, Meyer W. A review of nucleic acid-based diagnostic tests for systemic mycoses with an emphasis on polymerase chain reaction-based assays[J]. Med Mycol,2002,40(4):333-357.
[2]Maaroufi Y, Ahariz N, Husson M, et al. Comparison of different methods of isolation of DNA of commonly encountered Candida species and its quantitation by using a real-time PCR-based assay[J]. J Clin Microbiol,2004,42(7):3159-3163.
[3]Haugland RA, Brinkman N, Vesper SJ. Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using realtime PCR analysis[J]. J Microbiol Methods,2002,50(3):319-323.
[4]Haugland RA, Heckman JL, Wymer LJ. Evaluation of different methods for the extraction of DNA from fungal conidia by quantitative competitive PCR analysis[J]. J Microbiol Methods,1999,37(2):165-176.
[5]Griffin DW, Kellogg CA, Peak KK, et al. A rapid and efficient assay for extracting DNA from fungi[J]. Lett Appl Microbiol,2002,34(3):210-214.
[6]Clarkson AI, Lefevre P, Titchener-Hooker NJ. A study of process interactions between cell disruption and debris clarification stages in the recovery of yeast intracellular products[J]. Biotechnol Prog,1993,9(5):462-467.
[7]Cenis JL. Rapid extraction of fungal DNA for PCR amplification[J]. Nucleic Acids Res,1992,20(9):2380.
[8]Specht CA, DiRusso CC, Novotny CP, et al. A method for extracting high-molecular-weight deoxyribonucleic acid from fungi[J]. Anal. Anal Biochem, 1982,119(1):158-63.
[9]Walsh TJ, Francesconi A, Kasai M, et al. PCR and single-strand conformational polymorphism for recognition of medically important opportunistic fungi[J]. J Clin Microbiol,1995,33(12):3216-3220.
[1]Martin GS, Mannino DM, Eaton S, et al. The epidemiology of sepsis in the United States from 1979 through 2000[J]. N Engl J Med,2003,348(16):1546-1554.
[2]Marr KA, Carter RA, Crippa F, et al. Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients[J]. Clin Infect Dis,2002,34(7): 909-917.
[3]Cornillet A, Camus C, Nimubona S, et al. Comparison of epidemiological, clinical, and biological features of invasive aspergillosis in neutropenic and nonneutropenic patients:a 6-year survey[J]. Clin Infect Dis,2006,43(5):577-584.
[4]Pfaller MA, Diekema DJ. Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus[J]. J Clin Microbiol,2004,42(10):4419-4431.
[5]Baddley JW, Pappas PG, Smith AC, et al. Epidemiology of Aspergillus terreus at a
university hospital[J]. J Clin Microbiol,2003,41(12):5525-5529.
[6]Nucci M, Marr KA, Queiroz-Telles F, et al. Fusarium infection in hematopoietic stem cell transplant recipients[J]. Clin Infect Dis,2004,38(9):1237-1242.
[7]Walsh TJ, Groll A, Hiemenz J, et al. Infections due to emerging and uncommon medically important fungal pathogens[J]. Clin Microbiol Infect,2004,10(Suppl 1):48-66.
[8]Diekema DJ, Messer SA, Hollis RJ, et al. Activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B against 448 recent clinical isolates of filamentous fungi[J]. J Clin Microbiol,2003,41(8):3623-3626.
[9]Kelaher A. Two non-invasive diagnostic tools for invasive aspergilosis: (1-3)-beta-Dglucan and the galactomannan assay[J]. Clin Lab Sci,2006,19 (4) 222-224.
[10]Mirhendi H, Makimura K, Khoramizadeh M, et al. A one-enzyme PCR-RFLP assay for identification of six medically important Candida species[J]. Nippon Ishinkin Gakkai Zassh,2006,47(3):225-229.
[1]Barnes RA, White PL, Bygrave C, et al. Clinical impact of enhanced diagnosis of invasive fungal disease in high-risk haematology and stem cell transplant patients[J]. J Clin Pathol,2009,62(1):64-69.
[2]Baddley JW, Pappas PG, Smith AC, et al. Epidemiology of Aspergillus terreus at a university hospital[J]. J Clin Microbiol,2003,41(12):5525-5529.
[3]刘军,刘维达.聚合酶链反应检测深部致病真菌的实验研究[J].中华皮肤科杂志,2005,38：503-505.
[4]Greene RE, Schlamm HT, Oestmann JW, et al. Imaging findings in acute invasive pulmonary aspergillosis:clinical significance of the halo sign[J]. Clin Infect Dis, 2007,44(3):373-379.
[5]Cooper EE, O'Reilly MA, Guest DI, et al. Influence of building construction work on Aspergillus infection in a hospital setting[J]. Infect Control Hosp Epidemiol,2003, 24(7):472-476.
[6]Bow EJ. Invasive fungal infection in haematopoietic stem cell transplant recipients: epidemiology from the transplant physician's viewpoint[J]. Mycopathologia,2009, 168(6):2832-2897.
[7]McCoy D, Depestel DD, Carver PL. Primary antifungal prophylaxis in adult hematopoietic stem cell transplant recipients:current therapeutic concepts[J]. Pharmacotherapy,2009,29(11):1306-1325.
[8]Meersseman W, Lagrou K, Maertens J, et al. Invasive aspergillosis in the intensive care unit[J]. Clin InfectDis,2007,45(2):205-216.
[9]Caillot D, Couaillier JF, Bernard A, et al. Increasing volume and changing characteristics of invasive pulmonary aspergillosis on sequential thoracic computed tomography scans in patients with neutropenia[J]. J Clin Oncol,2001,19 (1) 2532259.
[10]Kelaher A. Two non-invasive diagnostic tools for invasive aspergilosis: (1-3)-beta-Dglucan and the galactomannan assay[J]. Clin Lab Sci,2006,19 (4): 222-224.
[11]Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH. Multiplex PCR: critical parameters and step-by-step protocol[J]. Biotechniques,1997,23(3): 504-511.
[12]Orzechowski Xavier M, Pasqualotto AC, Uchoa Sales Mda P, et al. Invasive pulmonary aspergillosis due to a mixed infection caused by Aspergillus flavus and Aspergillus fumigatus[J]. Rev Iberoam Micol,2008,25(3):176-178.
[1]Maaroufi Y, Ahariz N, Husson M, et al. Comparison of different methods of isolation of DNA of commonly encountered Candida species and its quantitation by using a real-time PCR-based assay[J]. J Clin Microbiol,2004,42(7):3159-3163.
[2]Gussow D, Clackson T. Direct clone characterization from plaques and colonies by the polymerase chain reaction[J]. Nucleic Acids Res,1989,17(10):4000.
[3]Haugland RA, Brinkman N, Vesper SJ. Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using realtime PCR analysis[J]. J Microbiol Methods,2002,50(3):319-323.
[4]Fredricks DN, Smith C, Meier A. Comparison of Six DNA Extraction Methods for Recovery of Fungal DNA as Assessed by Quantitative PCR[J]. J Clin Microbiol, 2005,43(10):5122-5128.
[5]唐哗盛,李英.菌落PCR在大规模基因组测序中的应用[J].生物化学与生物物理进展,2002,29(2)：316-318.
[6]Dallas-Yang Q, Jiang G, Sladek FM. Avoiding false positives in colony PCR[J]. Biotechniques,1998,24(4):580-582.
[7]Suzuki S, Taketani H, Kusumoto K, et al. High-throughput genotyping of filamentous fungus Aspergillus oryzae based on colony direct polymerase chain reaction[J]. J Biosci Bioeng,2006,102(6):572-574.
[8]Alshahni MM, Makimura K, Yamada T, et al. Direct colony PCR of several medically important fungi using Ampdirect plus[J]. Jpn J Infect Dis,2009,62(2):164-167.
[9]Griffin DW, Kellogg CA, Peak KK, et al. A rapid and efficient assay for extracting DNA from fungi[J]. Lett Appl Microbiol,2002,34(3):210-214.
[10]van den Brink HJ, van Zeijl CM, Brons JF, et al. Cloning and characterization of the NADPH cytochrome P450 oxidoreductase gene from the filamentous fungus Aspergillus niger[J].DNA Cell Biol,1995,14(8):719-729.
[11]H Mirhendi, K Diba, A Rezaei, et al. Colony-PCR is a rapid and sensitive method for DNA amplification in yeasts[J]. J Public Health,2007,36(1):40-44.
[12]Agrawal V, Roy N. Contaminating insert degradation by preincubation colony PCR: a method for avoiding false positives in transformant[J]. Anal Biochem,2008, 375(1):159-161.
[1]Gussow D, Clackson T. Direct clone characterization from plaques and colonies by the polymerase chain reaction[J]. Nucleic Acids Res,1989,17(10):4000.
[2]Kelaher A. Two non-invasive diagnostic tools for invasive aspergilosis: (1-3)-beta-Dglucan and the galactomannan assay[J]. Clin Lab Sci,2006,19 (4): 222-224.
[3]吴绍熙.现代医学真菌学检验手册[M].北京：中国协和医科大学出版社,2005：137.
[4]Bretagne S, Costa JM. Towards a molecular diagnosis of invasive aspergillosis and disseminated candidosis[J]. FEMS Immunol Med Microbiol,2005,45(3):361-318.
[5]Gerald Pierard. Onychomycosis and Other Superficial Fungal Infections of the Foot in the Elderly:A Pan-European Survey[J]. Dermatology,2001,202(3):220-224.
[6]李筱芳,吕桂霞,陈辉,等.多重PCR诊断甲真菌病的实验研究[J].中国皮肤性病学杂志,2007,21(1)：10-13.
[7]Lau A, Sorrell TC, Lee O, et al. Colony multiplex-tandem PCR for rapid, accurate identification of fungal cultures[J]. J Clin Microbiol,2008,46(12):4058-4060.
[1]Arendrup MC. Invasive fungal infections:past achievements and challenges ahead[J]. Clin Microbiol Infect,2009,15(7):599-601.
[2]Meersseman W, Lagrou K, Maertens J, et al. Invasive aspergillosis in the intensive care unit[J]. Clin InfectDis,2007,45(2):205-216.
[3]Greene RE, Schlamm HT, Oestmann JW, et al. Imaging findings in acute invasive pulmonary aspergillosis:clinical significance of the halo sign[J]. Clin Infect Dis, 2007,44(3):373-379.
[4]Bow EJ. Invasive fungal infection in haematopoietic stem cell transplant recipients: epidemiology from the transplant physician's viewpoint[J]. Mycopathologia,2009, 168(6):2832-2897.
[5]McCoy D, Depestel DD, Carver PL. Primary antifungal prophylaxis in adult hematopoietic stem cell transplant recipients:current therapeutic concepts[J]. Pharmacotherapy,2009,29(11):1306-1325.
[6]Baddley JW, Pappas PG, Smith AC, et al. Epidemiology of Aspergillus terreus at a university hospital[J]. J Clin Microbiol,2003,41(12):5525-5529.
[7]Hagerty JA, Ortiz J, Reich D, et al. Fungal infections in solid organ transplant patients[J]. Surg Infect (Larchmont),2003,4(3):263-271.
[8]Barnes RA, White PL, Bygrave C, et al. Clinical impact of enhanced diagnosis of invasive fungal disease in high-risk haematology and stem cell transplant patients[J]. J Clin Pathol,2009,62(1):64-69.
[9]Nucci M, Marr KA. Emerging fungal diseases[J]. Clin Infect Dis,2005,41(4): 521-526.
[10]Diekema DJ, Messer SA, Hollis RJ, et al. Activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B against 448 recent clinical isolates of filamentous fungi[J]. J Clin Microbiol,2003,41(8):3623-3626.
[11]Husain S, Alexander BD, Munoz P, et al. Opportunistic mycelial fungal infections in organ transplant recipients:emerging importance of non-Aspergillus mycelial fungi[J]. Clin Infect Dis,2003,37(2):221-229.
[12]Maertens J. Evaluating prophylaxis of invasive fungal infections in patients with haematologic malignancies[J]. Eur J Haematol,2007,78(4):275-82.
[13]Nucci M, Anaissie E. Fusarium infections in immunocompromised patients[J]. Clin Microbiol Rev,2007,20(4):695-704.
[14]Pfaller MA, Diekema DJ. Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus[J]. J Clin Microbiol,2004,42(10):4419-4431.
[15]Pagano L, Caira M, Candoni A, et al. The epidemiology of fungal infections in patients with hematologic malignancies:the SEIFEM-2004 study[J]. Haematologica,2006,91(8):1068-1075.
[16]Walsh TJ, Groll A, Hiemenz J, et al. Infections due to emerging and uncommon medically important fungal pathogens[J]. Clin Microbiol Infect,2004,10(1): 48-66.
[17]Husain S, Munoz P, Forrest G, et al. Infections due to Scedosporium apiospermum and Scedosporium prolificans in transplant recipients:clinical characteristics and impact of antifungal agent therapy on outcome[J]. Clin Infect Dis,2005,40(1): 89-99.
[18]Araujo R, Costa-de-Oliveira S, Coutinho I, et al. Evaluating the resistance to posaconazole by E-test and CLSI broth microdilution methodologies of Candida spp. and pathogenic moulds[J]. Eur J Clin Microbiol Infect Dis,2009,28(9): 1137-1140.
[19]Chamilos G, Lewis RE, Kontoyiannis DP. Delaying amphotericin B-based frontline therapy significantly increases mortality among patients with hematologic malignancy who have zygomycosis[J]. Clin Infect Dis,2008,47(4):503-509.
[20]Roden MM, Zaoutis T, Buchanan WL, et al. Epidemiology and outcome of zygomycosis:a review of 929 reported cases[J]. Clin Infect Dis,2005,41(5): 634-653.
[21]Chayakulkeeree M, Ghannoum MA, Perfect JR. Zygomycosis:the re-emerging fungal infection[J]. Eur J Clin Microbiol Infect Dis,2006,25(4):215-229.
[22]Schabereiter-Gurtner C, Selitsch B, Rotter ML, et al. Development of novel real-time PCR assays for detection and differentiation of eleven medically important Aspergillus and Candida species in clinical specimens[J]. J Clin Microbiol,2007,45(3):906-914.
[23]Hayette MP, Vaira D, Susin F, et al. Detection of Aspergillus species DNA by PCR in bronchoalveolar lavage fluid[J]. J Clin Microbiol,2001,39(6):2338-2340.
[24]Kami M, Fukui T, Ogawa S, et al. Use of real-time PCR on blood samples for diagnosis of invasive aspergillosis[J]. Clin Infect Dis,2001,33(9):1504-1512.
[25]Halliday C, Hoile R, Sorrell T, et al. Role of prospective screening of blood for invasive aspergillosis by polymerase chain reaction in febrile neutropenic recipients of haematopoietic stem cell transplants and patients with acute leukaemia[J]. Brit J Haematol,2006,132(4):478-486.
[26]Hall L, Wohlfiel S, Roberts GD. Experience with the MicroSeq D2 large-subunit ribosomal DNA sequencing kit for identification of filamentous fungi encountered in the clinical laboratory[J]. J Clin Microbiol,2004,42(2):622-626.
[27]Schwarz P, Bretagne S, Gantier JC, et al. Molecular identification of zygomycetes from culture and experimentally infected tissues[J]. J Clin Microbiol.2006, 44(2):340-349.
[28]Nilsson RH, Ryberg M, Kristiansson E, et al. Taxonomic reliability of DNA sequences in public sequence databases:a fungal perspective[J]. PLoS One,2006, I:e59.
[29]Dean TR, Kohan M, Betancourt D, et al. A simple polymerase chain reaction/restriction fragment length polymorphism assay capable of identifying medically relevant filamentous fungi[J], Mol Biotechnol,2005,31(1):21-28.
[30]Kamiya A, Kikuchi A, Tomita Y, et al. PCR and PCR-RFLP techniques targeting the DNA topoisomerase II gene for rapid clinical diagnosis of the etiologic agent of dermatophytosis[J]. J Dermatol Sci,2004,34(1):35-48.
[31]Vastag M, Papp T, Kasza Z, et al. Intraspecific variation in two species of Rhizomucor assessed by random amplified polymorphic DNA analysis[J]. J Basic Microbiol,2000,40(4):269-277.
[32]Raclasky V, Trtkova J, Ruskova L, et al. Primer R108 performs best in the RAPD strain typing of three Aspergillus species frequently isolated from patients[J]. Folia Microbiol,2006,51(2):136-140.
[33]Healy M, Reece K, Walton D,et al. Identification to the species level and differentiation between strains of Aspergillus clinical isolates by automated repetitive-sequence-based PCR[J]. J Clin Microbiol,2004,42(9):4016-24.
[34]Dewey Hansen, Mimi Healy, Kristy Reece, et al. Repetitive-Sequence-Based PCR Using the DiversiLab System for Identification of Aspergillus Species[J]. J Clin Microbiol,2008,46(5):1835-1839.
[35]Luo G, Mitchell TG. Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR[J]. J Clin Microbiol,2002,40(8):2860-2865.
[36]Araujo R, Pina-Vaz C, Rodrigues AG, et al. Simple and highly discriminatory microsatellite-based multiplex PCR for Aspergillus fumigatus strain typing[J]. Clin Microbiol Infect,2009,15(3):260-266.
[37]Kanbe T, Yamaki K, Kikuchi A. Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene[J]. Microbiol Immunol,2002,46(12):841-848.
[38]Sugita C, Makimura K, Uchida K, et al. PCR identification system for the genus Aspergillus and three major pathogenic species:Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger[J]. Med Mycol,2004,42(5):433-437.
[39]Wu Z, Tsumura Y, Blomquist G, et al.18S rRNA gene variation among common airborne fungi, and development of specific oligonucleotide probes for the detection of fungal isolates[J]. Appl Environ Microbiol,2003,69(9):5389-5397.
[40]Hsiao CR, Huang L, Bouchara JP, et al. Identification of medically important molds by an oligonucleotide array[J]. J Clin Microbiol,2005,43(8):3760-3768.
[41]Huang A, Li JW, Shen ZQ, et al. High-throughput identification of clinical pathogenic fungi by hybridization to an oligonucleotide microarray[J]. J Clin Microbiol,2006,44(9):3299-3305.
[42]Haugland RA, Varma M, Wymer LJ, et al. Quantitative PCR analysis of selected Aspergillus, Penicillium and Paecilomyces species[J]. Syst Appl Microbiol,2004, 27(2):198-210.
[43]Cuenca-Estrella M, Meije Y, Diaz-Pedroche C, et al. Value of serial quantification of fungal DNA by a real-time PCR-based technique for early diagnosis of invasive Aspergillosis in patients with febrile neutropenia[J]. J Clin Microbiol,2009,47(2): 379-84.
[44]Challier S, Boyer S, Abachin E, et al. Development of a serum-based Taqman real-time PCR assay for diagnosis of invasive aspergillosis[J]. J Clin Microbiol,
2004,42(2)：844-846.