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;l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检测炯曲霉特异性β-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β-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.

abstractsObjective 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.Conclusion LNA probe Real-time PCR is a promisingly accurate assay of rapidly detecting A.fumigatus practically and cost-efficiently.