应用锁定核酸水解探针实时荧光定量PCR检测烟曲霉  

Performance of locked nucleic acid probe real-time polymerase chain reaction in the detection of AspergiUus fumigatus

作　　者：王利君[1] 朱敏[1] 曹晶晶[1] 顾海彤[1] 鲁辛辛[1] 

机构地区：[1]首都医科大学附属北京同仁医院检验科,北京100730

出　　处：《中华医学杂志》2011年第18期1268-1271,共4页National Medical Journal of China

基　　金：国家自然科学基金（30772066）

摘　　要：目的建立检测烟曲霉的锁定核酸（LNA）水解探针实时荧光定量PCR（qPCR）方法。方法实验菌株来自北京同仁医院临床分离曲霉菌株，均通过形态学和DNA测序方法鉴定到种。实验组：烟曲霉48株；对照组：黄曲霉55株、杂色曲霉16株、构巢曲霉10株、聚多曲霉5株、寄生曲霉1株；临床标本组：已经纯培养出烟曲霉的冻存临床标本，鼻窦炎鼻窦组织标本20份、肺泡灌洗液1份。提取标本的DNA，LNA水解探针qPCR检测烟曲霉特异性p-微管蛋白基因，评价此方法的分析特异性、扩增效率、线性动态范围和检测限。结果烟曲霉特异性p-微管蛋白基因LNA水解探针qPCR检测烟曲霉方法的分析特异性为100％，扩增效率为98．2％，线性动态范围6个数量级，检测限2．5Pg。结论烟曲霉特异性β-微管蛋白基因LNA水解探针qPCR法能快速、准确地检测烟曲霉，而且此方法易操作、经济实惠．应用前景广阔。Objective To evaluate the performance of locked nucleic acid （LNA） probe Real-time polymerase chain reaction （PCR） in the detection of Aspergillus fumigatus （ A. fumigatus）. Methods All clinically cultured isolates of Aspergillus at our hospital were identified by morphology and DNA sequencing assay. The experimental group consists of A. fumigatus （ n = 48 ） while the control group was made up of A. flavus （ n =55）, a. versicolor （n = 16）, A. nidulans （n = 10）, A. sydowii （ n =5） and a. parasiticus （ n = 1 ）. The clinical samples consisted of A. fumigatus sinusitis tissue （ n = 20） and bronchoalveolar lavage fluid （ n = 1 ）. DNA was extracted from all samples. A. fumigatus β-tubulin gene was targeted with LNA probe Real-time PCR assay. LNA probe Real-time PCR was evaluated with regards to specificity, efficiency, linear dynamic range in PCR amplification and limits of detection. Results All clinical samples were positively amplified. The specificity was 100% and the PCR efficiency 98. 2%. Linear dynamic range was at least six orders of magnitude and the limit of detection 2. 5 pg. Condttsion LNA probe Real-time PCR is a promisingly accurate assay of rapidly detecting A. fumigatus practically and cost-efficiently.

关 键 词：曲霉 烟 核酸探针 聚合酶链反应 

分 类 号：R440[医药卫生—诊断学]