环介导等温扩增快速检测临床常见曲霉菌方法的建立和应用

Establishment and application of loop-mediated isothermal amplification for detecting clinical isolates of Aspergillus spp

目的建立一种环介导等温扩增（LAMP）反应快速检测临床常见曲霉菌方法。方法根据GenBank上提交的烟曲霉（登录号AY660917）、土曲霉（登录号AF454183）、黄曲霉（登录号AF454158）以及黑曲霉（登录号AF454169）菌的特异28SrRNA基因序列比对后的相对保守区设计特异LAMP引物，分别提取标准曲霉、临床对照菌株以及临床标本DNA，然后以LAMP技术扩增靶基因，通过反应体系中的荧光染料显色作用，直接肉眼观察结果。并将每种曲霉菌约10。孢子加入到正常对照血清中模拟临床标本，用LAMP方法进行检测验证。结果15种非曲霉菌属以及人全血基因组的LAMP和PCR检测结果均为阴性、30份临床非曲霉菌血液感染标本以及10份正常对照血清标本的LAMP检测结果也均为阴性，无非特异反应发生。4种上述标准曲霉菌属的LAMP和PCR检测结果均为阳性；每种曲霉菌的双份共8份模拟临床标本，约含10。孢子，经LAMP检测也均为阳性。另对系列每反应管分别含有500、50、5、0．5、0．05Pg模板进行灵敏度分析，LAMP最低检测限可达到0．05～0．5pg每反应管。结论LAMP是一种简便、快速、特异和敏感的检测方法，可以用来检测临床和环境标本中的常见曲霉菌属。（中华检验医学杂五占，2014，37：140—143）

Objective To establish a method for detecting Aspergillus spp. by Loop-mediated isothermal amplification. Methods Aspergillus spp. specific primers were designed from the relative conservation region of the published sequence of 28S rRNA genes of A.fumigatus （ GenBank accession number AY660917 ）, A. terreus （ GenBank accession number AF454183 ）, A.flavus （ GenBank accession number AF454158 ）, and A. niger （GenBank accession number AF454169 ）. Genomic DNA were extracted from Aspergillus standard strains, clinical control strains and clinical samples, and amplified by LAMP. The amplified results could be read with the naked eye by the coloring effect of fluorescent nucleotide dye without the DNA electrophoresis. Approximately 103 each AspergiUus conidia suspension was added to the serum from healthy volunteers, and detected these simulative clinical samples by LAMP assay. Results The LAMP and PCR assays obtained positive results for all four Aspergillus species and 8 simulative clinical samples （ double samples for each AspergiUus species） including 103 conidia,but negative in the remaining 15 non-Aspergillus species,human total blood genomic DNA, 30 clinical serum samples infected with non-Aspergillus and 10 healthy volunteers. The LAMP assay had a minimum detection of 0. 05-0. 5pg, by means of detect different levels of 500,50,5, O. 5,0.05 pg template in each reaction tube. Conclusion The results confirm that LAMP is a simple, rapid, sensitive and specific method, and can be used for detection of Aspergillus strains in clinical and environmental specimens. （Chin J Lab Med,2014,37：140-143）