实时荧光PCR结合融解曲线分析快速鉴定临床常见曲霉菌

摘要/Abstract

摘要：

目的采用实时荧光PCR结合融解曲线分析的方法快速将临床常见曲霉菌鉴定到种的水平。方法 1普通PCR扩增真菌ITS区后进行序列比对,准确鉴定菌种并设计种特异性引物和探针。2采用实时荧光PCR的方法及融解曲线分析,根据不同的解链温度将5种临床常见曲霉菌鉴定到种的水平。3特异性、敏感性、重复性试验。结果 1解链曲线分析显示,不同种曲霉菌的种特异性探针有特异的Tm值,根据Tm值的不同可以将5种曲霉菌区分开来:烟曲霉Tm=61.4℃,黄曲霉Tm=57.4℃ ,黑曲霉Tm=67.7℃ ,土曲霉Tm=55.2℃和64.5℃,构巢曲霉Tm= 65.8℃。其中小孢根霉和疣状瓶霉与烟曲霉探针发生交叉反应,阴性对照不出现特异性的解链曲线。2该方法对5种曲霉菌的检测下限分别为:烟曲霉56.8 fg,黄曲霉1 110 fg,黑曲霉 13.7 fg,土曲霉123 fg,构巢曲霉780 fg。3重复性试验结果显示,同一种曲霉菌的Tm值波动范围不超过0.5℃。结论采用实时荧光PCR结合融解曲线分析的方法可以快速准确地将临床常见曲霉菌鉴定到种的水平,具有良好的敏感性、特异性和可重复性,有助于临床侵袭性曲霉感染的诊断和指导抗真菌药物使用。

关键词: 实时荧光PCR, 曲霉菌, 融解曲线

Abstract:

Objective Real-time PCR combined with melting curve analysis were carried out for rapid identification of clinical important Aspergillus to the species level.Method (1) PCR amplification of fungal ITS region, and then sequence alignment were done to identificate Aspergillus isolates correctly and design species specfic primers and probes. (2) Five species of clinical important Aspergillus were identified to species level according to different melting temperatures (Tm) of species specific probes in a biprobe real-time PCR.Results (1) The Tm value of A.terrus specific probe were 64.5℃ and 55.2℃, and that of A.flavus,A.fumigatus,A.nidulans and A.niger specific probe was 57.4℃,61.4℃,65.8℃,and 67.7℃ respectively.Phialophora verrucosa and Rhizopus microspores had cross reactions with A.fumigatus specific probe.(2)The detection limit of A.fumigatus was 56.8 fg, and that of A.flavus 1.11×10³ fg,A.niger 13.7 fg,A.terrus 1.23×10² fg and A.nidulans 7.80×10² fg.(3)The Tm value fluctuation was within 0.5℃ in repeated reactions.Conclusion The real-time PCR is time-saving and with high sensitivity, specificity and repeatablity. Therefore, it is helpful for diagnosis of invasive aspergillosis and the use of antifungal drugs.

Key words: real-time PCR, Aspergillus, melting curve

中图分类号:

R 379.6

史俊艳, 郭莉娜, 徐英春, 王瑶, 王贺, 刘文静. 实时荧光PCR结合融解曲线分析快速鉴定临床常见曲霉菌[J]. 中国真菌学杂志, 2012, 7(6): 330-334,338.

SHI Jun-yan, GUO Li-na, XU Ying-chun, WANG Yao, WANG He, LIU Wen-jing. Rapid identification of clinical important Aspergillus using real-time PCR combined with melting curve analysis[J]. Chinese Journal of Mycology, 2012, 7(6): 330-334,338.

参考文献

[1] Marr KA, ,Carter RA, Boeckh M, et al. Invasive aspergillosis in allogeneic stem cell transplant recipients: changes in epidemiology and risk factors
[J]. Blood,2002, 100(13): 4358-4366.

[2] Denning DW.Invasive aspergillosis
[J].Clinical Infectious Diseases,1998, 26(4): 781-803.

[3] Lass-Florl C,Griff K, Mayr A, et al. Epidemiology and outcome of infections due to Aspergillus terreus:10-year single centre experience
[J].British Journal of Haematology,2005,131(2): 201-207.

[4] Bretagne S, Costa M, Marmorat-Khuong A, et al. Detection of Aspergillus species DNA in bronchoalveolar lavage samples by competitive PCR
[J].Journal of Clinical Microbiology, 1995,33(5): 1164-1168.

[5] Trama JP, Mordechai E, Adelson ME. Detection of Aspergillus fumigatus and a mutation that confers reduced susceptibility to itraconazole and posaconazole by real-time PCR and pyrosequencing
[J]. Journal of Clinical Microbiology, 2005, 43(2): 906-908.

[6] Mellado E, Garcia-Effron G, Alcazar-Fuoli L, et al. A new Aspergillus fumigatus resistance mechanism conferring in vitro cross-resistance to azole antifungals involves a combination of cyp51A alterations
[J]. Antimicrobial Agents and Chemotherapy, 2007,51(6): 1897-1904.

[7] Kontoyiannis DP, Lewis RE, May GS, et al.Aspergillus nidulans is frequently resistant to amphotericin B
[J]. Mycoses, 2002,45(9-10):406-407.

[8] Sutton DA, Sanche SE, Recankar SG, et al. In vitro amphotericin B resistance in clinical isolates of Aspergillus terreus, with a head-to-head comparison to voriconazole
[J]. Journal of Clinical Microbiology, 1999, 37(7): 2343-2345.

[9] Balajee SA, L Sigler, and ME Brandt. DNA and the classical way: identification of medically important molds in the 21st century
[J]. Medical Mycology, 2007, 45(6): 475-490.

[10] Hope WW, Walsh TJ, and Denning DW. Laboratory diagnosis of invasive aspergillosis
[J]. The Lancet infectious diseases, 2005, 5(10):609-722.

[11] Tuon FF. A systematic literature review on the diagnosis of invasive aspergillosis using polymerase chain reaction (PCR) from bronchoalveolar lavage clinical samples
[J]. Revista iberoamericana de micologia : organo de la Asociacion Espanola de Especialistas en Micologia, 2007, 24(2): 89-94.

[12] del Palacio A, Cuetara MS and Ponton J. Laboratory diagnosis of invasive aspergillosis
[J]. Revista iberoamericana de micologia : organo de la Asociacion Espanola de Especialistas en Micologia, 2003, 20(3): 90-98.

[13] Mennink-Kersten MA, Ruegebrink D,Wasei N, et al. In vitro release by Aspergillus fumigatus of galactofuranose antigens,1,3-beta-D-glucan, and DNA, surrogate markers used for diagnosis of invasive aspergillosis
[J]. Journal of Clinical Microbiology, 2006, 44(5):1711-1718.

[14] 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]. Journal of Clinical Microbiology, 2007,45(3): 906-914.

[15] Logan JM, Edwards KJ, Saunders NA, et al. Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR
[J]. Journal of Clinical Microbiology, 2001,39(6): 2227-2232.

[16] Schabereiter-Gurtner C, Hirschl AM, Dragosics B, et al. Novel real-time PCR assay for detection of Helicobacter pylori infection and simultaneous clarithromycin susceptibility testing of stool and biopsy specimens
[J]. Journal of Clinical Microbiology, 2004,42(10): 4512-4518.

[17] Abdelbaqi K,Buissonniere A, Prouzet-Mauleon V, et al.Development of a real-time fluorescence resonance energy transfer PCR to detect arcobacter species
[J]. Journal of Clinical Microbiology, 2007,45(9): 3015-3021.

[18] Diaz MR and JW Fell. High-throughput detection of pathogenic yeasts of the genus trichosporon
[J]. Journal of Clinical Microbiology, 2004,42(8): 3696-3706.

[19] Pryce TM, Palladino, Kay ID, et al. Rapid identification of fungi by sequencing the ITS1 and ITS2 regions using an automated capillary electrophoresis system
[J]. Medical Mycology, 2003, 41(5): 369-381.

[20] Iwen PC, Hinrichs SH and Rupp ME. Utilization of the internal transcribed spacer regions as molecular targets to detect and identify human fungal pathogens
[J]. Medical Mycology, 2002, 40(1): 87-109.

[21] 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]. Journal of Clinical Microbiology, 2005,43(5): 2092-2103.

[22] Spiess B, Buchheidt D, Baust C, et al. Development of a LightCycler PCR assay for detection and quantification of Aspergillus fumigatus DNA in clinical samples from neutropenic patients
[J]. Journal of Clinical Microbiology, 2003,41(5): 1811-1818.

[23] Pham AS, Tarrand JJ,May GS, et al. Diagnosis of invasive mold infection by real-time quantitative PCR
[J]. American Journal of Clinical Pathology, 2003,119(1): 38-44.