一种基于环介导等温扩增技术原理的组织胞浆菌分子诊断技术

中国真菌学杂志　 2016, Vol. 11 　Issue (2): 79-84.

一种基于环介导等温扩增技术原理的组织胞浆菌分子诊断技术

李娟, 陈敏, 潘搏, 雷文知, 方文捷, 刘加, 洪南, 李颖芳, 廖万清, 潘炜华

上海市医学真菌分子生物学重点实验室上海长征医院皮肤病与真菌病研究所全军皮肤病重点实验室第二军医大学附属长征医院皮肤科, 上海 200003

收稿日期:2015-12-29 出版日期:2016-04-28 发布日期:2016-04-28
通讯作者: 潘炜华,E-mail:panweihua@medmail.com.cn E-mail:panweihua@medmail.com.cn
作者简介:李娟,女(汉族),硕士研究生在读.E-mail:lijuanluck@gmail.com
基金资助:
重大传染病防治科技重大专项(2013ZX10004612);国家973项目(2013CB531601,2013CB531606);国家自然科学基金(81201269);上海市科委专项(14DZ2272900)

A loop-mediated isothermal amplification method for molecular detection of Histoplasma capsulatum

LI Juan, CHEN Min, PAN Bo, LEI Wen-zhi, FANG Wen-jie, LIU Jia, HONG Nan, LI Ying-fang, LIAO Wan-qing, PAN Wei-hua

Shanghai Key Laboratory of Molecular Medical Mycology, Institute of dermatology and fungal diseases, PLA Key laboratory of the fungal diseases, Department of dermatology, Changzheng hospital, Shanghai 200003

Received:2015-12-29 Online:2016-04-28 Published:2016-04-28

摘要/Abstract

摘要：

目的研发一种基于环介导等温扩增(1oop-mediated isothermal amplification,LAMP)技术原理的组织胞浆菌感染的分子诊断技术。方法以组织胞浆菌为研究对象,针对其种特异性的M抗原基因序列,设计并筛选数套基于LAMP技术的特异性引物,同时优化其反应条件,建立一种针对组织胞浆菌感染的快速分子鉴定技术。结果该LAMP技术在65℃、90 min反应条件下,对组织胞浆菌基因组DNA的检测敏感度达到5.3 pg/反应(即5.3×10^-12 g,约160个基因组DNA拷贝),且与其相近种属真菌无交叉反应。结论我们研发的这种基于LAMP技术原理的组织胞浆菌分子诊断技术具有操作简便、特异性及敏感性高等优点,具有临床推广应用的潜力。

关键词: 组织胞浆菌, 环介导等温扩增技术, 分子诊断, 快速检测

Abstract:

Objective To develop a molecular diagnostic approach for the diagnosis of Histoplasma capsulatum based on the LAMP technology.Methods To establish and optimize a specific primer set of the LAMP technology targeting the sequence of M Antigen for rapid and molecular detection of Histoplasma capsulatum at species level.Results In our study, no cross-reaction was observed after 90 min of LAMP reaction, including DNA of the phylogenetically related species such as Coccidioides immitis, Sporothrix schenckii, Penicillium marneffei, etc.The limit-of-detection of the LAMP for genomic Histoplasma capsulatum DNA was approximately 5.3×10^-12 g per run (approximately 160 genomic copies).Conclusion Our proposed LAMP assay could represent a potential molecular detection tool for rapid diagnosis of Histoplasma capsulatum as a point of care test to be used in clinics in the future.

Key words: histoplasmosis, loop-mediated isothermal amplification, molecular diagnostics, rapid detection

中图分类号:

R379.9

李娟, 陈敏, 潘搏, 雷文知, 方文捷, 刘加, 洪南, 李颖芳, 廖万清, 潘炜华. 一种基于环介导等温扩增技术原理的组织胞浆菌分子诊断技术[J]. 中国真菌学杂志, 2016, 11(2): 79-84.

LI Juan, CHEN Min, PAN Bo, LEI Wen-zhi, FANG Wen-jie, LIU Jia, HONG Nan, LI Ying-fang, LIAO Wan-qing, PAN Wei-hua. A loop-mediated isothermal amplification method for molecular detection of Histoplasma capsulatum[J]. Chinese Journal of Mycology, 2016, 11(2): 79-84.

参考文献

[1] Singgh N.Fungal infections in the recipients of solid organ transplantaion[J].Infect Dis Clin North Am,2003,17(1):113-134.
[2] Kauffman CA.Histoplasmosis:a clinical and laboratory update[J].Clin Microbiol Rev,2007,20(1):115-132.
[3] Craig SC.The tropical world of Samuel Taylor Darling:parasites,pathology,and philanthropy[J].J Hist Med Allied Sci,2009,64(3):375-377.
[4] 赵蓓蕾,施毅,印洁,等.我国部分地区组织胞浆菌感染的流行病学调查[J].医学研究生学报,2003,16(3):199-202.
[5] 廖万清,潘搏,潘炜华.警惕在我国长江流域的组织胞浆菌病[J].皮肤性病诊疗学杂志,2014,21(3):171-172.
[6] Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63.
[7] Tomita N,Mori Y,Kanda H,et al.Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products[J].Nat Protoc,2008,3(5):877-882.
[8] 朱衡,瞿峰,朱立煌.利用氯化苄提取适于分子生物学分析的真菌DNA[J].真菌学报,1994,13(1):34-40.
[9] Ramos-e-Silva M,Lima CM,Schechtman RC,et al.Systemic mycoses in immunodepressed patients (AIDS)[J].Clin Dermatol,2012,30(6):616-627.
[10] Baddley JW,Sankara IR,Rodriquez JM,et al.Histoplasmosis in HIV-infected patients in a southern regional medical center:poor prognosis in the era of highly active antiretroviral therapy[J].Diagn Microbiol Infect Dis,2008,62(2):151-156.
[11] Florence H,Matthieu N,Christine A,et al.AIDS-related Histoplasma capsulatum var.capsulatum infection:25 years experience of French Guiana[J].Aids,2008,22(9):1047-1053.
[12] Pontes LB,Leitão Tdo M,Lima GG,et al.Clinical and evolutionary characteristics of 134 patients with disseminated histoplasmosis associated with AIDS in the State of Ceara[J].Rev Soc Bras Med Trop,2010,43(1):27-31.
[13] 桂希恩,管立人.内脏利什曼病、播散型组织胞浆菌病及马内菲青霉菌病的诊断和鉴别[J].中国寄生虫学与寄生虫病杂志,2007,25(1):69-72.
[14] Supparatpinyo K,Khamwan C,Baosoung V,et al.Disseminated Penicillium marneffei infection in southeast Asia[J].The Lancet,1994,344(8915):110-113.
[15] Guimarães AJ,Pizzini CV,De Matos Guedes HL,et al.ELISA for early diagnosis of histoplasmosis[J].J Med Microbiol,2004,53(6):509-514.
[16] Hung MN,Sun HY,Hsueh PR,et al.Meningitis due to Histoplasma capsulatum and Mycobacterium tuberculosis in a returned traveler with acquired immunodeficiency syndrome[J].J Formos Med Assoc,2005,104(11):860-863.
[17] Garringer TO,Wheat LJ,Brizendine EJ.Comparison of an established antibody sandwich method with an inhibition method of Histoplasma capsulatum antigen detection[J].J Clin Microbiol,2000,38(8):2909-2913.
[18] Scheel CM,Zhou Y,Theodoro RC,et al.Development of a loop-mediated isothermal amplification method for detection of Histoplasma capsulatum DNA in clinical samples[J].J Clin Microbiol,2014,52(2):483-488.
[19] Gago S,Esteban C,Valero C,et al.A multiplex real-time PCR assay for identification of Pneumocystis jirovecii,Histoplasma capsulatum,and Cryptococcus neoformans/Cryptococcus gattii in samples from AIDS patients with opportunistic pneumonia[J].J Clin Microbiol,2014,52(4):1168-1176.
[20] Hall GS,Pratt-Rippin K,Washington JA.Evaluation of a chemiluminescent probe assay for identification of Histoplasma capsulatum isolates[J].J Clin Microbiol,1992,30(11):3003-3004.
[21] Chemaly RF,Tomford JW,Hall GS,et al.Rapid diagnosis of Histoplasma capsulatum endocarditis using the AccuProbe on an excised valve[J].J Clin Microbiol,2001,39(7):2640-2641.