临床皮炎外瓶霉荧光定量PCR检测方法的建立

目的 建立基于TaqMan探针技术的皮炎外瓶霉荧光定量PCR检测方法.方法 通过对皮炎外瓶霉ITS区域基因组序列（GenBank：JN675373.1）进行分析,设计合成特异性引物和荧光标记探针,优化荧光定量PCR反应条件.以临床标本中分离的皮炎外瓶霉为阳性菌株,及其他种类真菌和细菌作为阴性对照菌株,从特异性、敏感性及重复性方面对该方法检测效果进行评价.结果 该研究设计的引物和探针能扩增皮炎外瓶霉特异性序列.临床分离得到的皮炎外瓶霉在反应中有明显扩增曲线,而甄氏外瓶霉、棘状外瓶霉、烟曲霉、白色念珠菌、新生隐球菌、马内菲青霉等20株菌在CT值≤38范围内均未有扩增;利用基因重组构建的标准品完成了标准曲线的绘制,在1.0×103～1.0×107拷贝数（Cp）内具有良好的线性关系（R2=1.000）,最低可检出量为10 Cp/μL.结论 成功建立了荧光定量PCR检测皮炎外瓶霉方法,该法特异度强、敏感度高、重复性好,将有助于临床皮炎外瓶霉感染的早期诊断和针对性治疗.

Establishment of a real-time fluorescence quantitative PCR method for the detection of clinical Exophiala dermatitidis

Objective To establish a novel fluorescent quantitative PCR （qPCR） method for the detection of Exophiala dermatitidis based on TaqMan probe technology.Methods Specific primers and fluorescent labeling probes were designed,according to the sequence of internal transcribed spacer （ITS） regions of E.dermatitis genomic sequence （GenBank：JN675373.1）.And the condition of qPCR was optimized.E.dermatitis isolated from clinical specimens were tested as positive control,and other species of fungi and bacteria were used as negative control,to evaluate the detective effect of specificity,sensitivity and repeatability.Results In this study,primers and probes were designed to amplify Exophiala dermatitidis specific sequence,and Exophiala dermatitidis isolated from clinical specimens obtained obvious amplification curve in the reaction,whereas the 20 negative control strains,such as Exophiala spinous,Aspergillus fumigat us,Candida albicans,Cryptococcus neoformans,Penicillium marneffei,et al were not amplified in the CT value≤38 range.The qPCR exhibited high sensitivity （10 Cp/μL） and a good linearity from 103 to 107 copies （R2 =1.000）.Conclusion We successfully established a quantitative PCR method with high specificity,sensitivity,good repeatability for detection of E.dermatitis.The method could contribute to the early diagnosis of E.dermatitis infection and targeted therapy.