聚合酶链反应结合反向线点杂交技术在检测及鉴定真菌性鼻窦炎常见致病曲霉菌中的应用

Application of PCR combined with reverse line blot assay in detection and identification of common pathogenic AspergiUus in fungal sinusitis

目的探讨聚合酶链反应结合反向线点杂交（PCR／RLB）技术在检测和鉴定真菌性鼻窦炎（FS）常见致病曲霉菌中应用的可行性。方法收集首都医科大学附属北京同仁医院2009年5月至2010年2月住院手术的FS患者活检石蜡包埋组织26例和活检新鲜组织8例，全部病例进行了病理学真菌检查、真菌培养及真菌ITS2区测序；提取各标本中真菌的DNA，用真菌特异性引物行PCR扩增，针对真菌的ITS2区设计曲霉菌种特异性探针，用5根曲霉菌种特异性探针与PCR产物进行反向线点杂交；将PCR／RLB与ITS2区测序、真菌培养及病理学检查的结果进行比较。阳性对照为5株曲霉菌株，阴性对照为真菌培养为非曲霉的石蜡包埋组织。结果活检标本病理学检查均可见菌丝；真菌培养14例阳性（41．2％）；测序16例（47．1％）得到结果；PCR／RLB鉴定34例均得出鉴定结果：黄曲霉14例，烟曲霉10例，黑曲霉4例，构巢曲霉1例，黄曲霉和烟曲霉同时阳性3例，烟曲霉与黑曲霉同时阳性2例。结论PCR／RLB技术可以对FS常见致病曲霉菌进行检测及鉴定，与病理组织学检查、真菌培养及DNA测序方法相比较，具有经济省时、敏感性高、特异性强、高通量等优势。

Objective To evaluate the feasibility of PCR/reverse line blot hybridization （RLB） assay in the detection and identification of clinical pathogens in fungal sinusitis （FS）. Methods Twentysix formalin-fixed and paraffin-embedded tissues and 8 fresh tissues of FS were collected from Beijing Tongren Hospital, Capital Medical University from May 2009 to February 2010. Pathological examination, fungal culture and ITS2 region sequencing were carried out. The DNA of all samples was extracted by standard procedure and fungal universal primers ITS3 and ITS4 were used for PCR amplification of all tissues. Then the amplified products were used for RLB with five fungal species-specific probes. The resuhs of PCR/RLB were compared with ITS region sequencing, fungal culture and pathological examination. Results For the biopsy tissues, fungal cultures were positive in 14 cases （41.2%）; pathologic examination demonstrated fungal hyphae in all cases; ITS2 region sequencing was successful in 16 cases （47. 1% ） ; PCR/RLB showed A. flavus in 14 cases, A. fumigatus in 10 cases, A. niger in four cases, A. nidulans in one case, A. flavus and A. fumigatus in three cases, and A. fumigatus and A. niger in two cases. Conclusions The PCR/RLB assay is suitable for rapid and accurate detection and identification of the pathogenic fungus of FS. Compared with the conventional fungal culture and microscopy, pathologic examination and DNA sequencing, the PCR/RLB has the advantages of more economy, time saving, and higher sensitivity, specificity and throughput.