实时荧光PCR检测巴西孢子丝菌的实验方法的建立  

Establishment of a real time PCR method for detecting Sporothrix brasiliensis

作　　者：张明瑞[1] 周盈[1] 李福秋[1] 姚春丽[1] 杨鑫 龚杰 赵飞[3] ZHANG Ming-rui;ZHOU Ying;LIFu-qiu;YAO Chun-li;YANG Xin;GONG Jie;ZHAO Fei(Department of Dermatology,the Second Hospital of Jilin University,Changchun 130022,China;Changchun Central Hospital,Changchun 130051,China;National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]吉林大学第二医院皮肤科,长春130022 [2]长春市中心医院,长春130051 [3]中国疾病预防控制中心传染病预防控制所,北京102206

出　　处：《中国真菌学杂志》2019年第5期297-302,共6页Chinese Journal of Mycology

基　　金：吉林省孢子丝菌病流行病学调查(3D5177763429);病原细菌与突发急性真菌感染高通量快速检测与应急筛检技术研究(2018ZX10712-001)

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。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.

关 键 词：实时荧光PCR 巴西孢子丝菌 鉴定 感染动物模型 

分 类 号：R446.5[医药卫生—诊断学] R379.9[医药卫生—临床医学]