【摘要】 目的建立一种快速、特异、灵敏的荧光PCR方法检测巴西孢子丝菌。方法比对NCBI数据库中所有巴西孢子丝菌内转录间隔区(internal transcribed spacer, ITS)序列,在保守区域设计并合成特异性引物和探针,建立并优化荧光PCR检测方法。对优化后的方法使用标准浓度核酸进行扩增效率、灵敏度及特异度评价。通过巴西孢子丝菌小鼠感染模型,与组织培养比较,对本研究中的方法进行评价。结果建立的实时荧光PCR方法对巴西孢子丝菌的检测限为100fg。该方法对申克孢子丝菌、球形孢子丝菌、其他常见致病真菌28种、常见细菌3种以及人类基因组和小鼠基因组扩增结果均为阴性,特异度为100%。对巴西孢子丝菌感染小鼠脑、肝、肺、脾、肾及淋巴结检测与培养结果相一致。结论本研究建立的荧光PCR方法可快速、灵敏、特异地鉴定巴西孢子丝菌,并能够有效的对感染小鼠模型标本进行检测,有助于孢子丝菌病的早期特异性病原学诊断。更多还原

【Abstract】 Objective To establish a rapid, specific and sensitive real-time PCR method for the detection of Sporothrix brasiliensis. Methods The internal transcribed spacer(ITS) sequence of S. brasiliensis were downloaded from the NCBI database. The conserved regions were obtained by software comparison. Specific primers and probes were designed and synthesized to establish and optimize the real time PCR method. The optimized method was used to evaluate amplification efficiency, sensitivity, and specificity using standard concentration nucleic acids. The method in this study was evaluated by mouse infection model of S. brasiliensis and compared with culture. Results The detection limit of the real-time PCR method established in this study for S. brasiliensis was 100 fg. The method was negative for the amplification of S. shenckii, S. globosa, 28 other common pathogenic fungi, 3 common bacteria, human genome and mouse genome, and the specificity was 100%. The detection results of the method and culture of brain, liver, lung, spleen, kidney and lymph nodes in mice infected with S. brasiliensis were consistent. Conclusion The real time PCR method established in this paper is a rapid, sensitive and specific method for the identification of S. brasiliensis. It can also detect specimens of infected mouse models, which is helpful for early diagnosis of sporotrichosis caused by S. brasiliensis.更多还原