CAJ | 학술논문

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.
목적 탐토취합매련반응결합반향선점잡교(PCR/RLB)기술재검측화감정진균성비두염(FS)상견치병곡매균중응용적가행성.방법 수집수도의과대학부속북경동인의원2009년5월지2010년2월주원수술적FS환자활검석사포매조직26례화활검신선조직8례,전부병례진행료병이학진균검사、진균배양급진균ITS2구측서；제취각표본중진균적DNA,용진균특이성인물행PCR확증,침대진균적ITS2구설계곡매균충특이성탐침,용5근곡매균충특이성탐침여PCR산물진행반향선점잡교；장PCR/RLB여ITS2구측서、진균배양급병이학검사적결과진행비교.양성대조위5주곡매균주,음성대조위진균배양위비곡매적석사포매조직.결과 활검표본병이학검사균가견균사；진균배양14례양성(41.2％)；측서16례(47.1％)득도결과；PCR/RLB감정34례균득출감정결과:황곡매14례,연곡매10례,흑곡매4례,구소곡매1례,황곡매화연곡매동시양성3례,연곡매여흑곡매동시양성2례.결론 PCR/RLB기술가이대FS상견치병곡매균진행검측급감정,여병리조직학검사、진균배양급DNA측서방법상비교,구유경제성시、민감성고、특이성강、고통량등우세.
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 Twenty- six 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 results 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.