多重PCR技术快速鉴定血培养阳性标本中酵母菌菌种的应用研究

目的：探讨多重PCR技术在快速鉴定引起真菌血症的重要医学酵母菌中的应用。方法：扩增酵母菌位于18S rRNA和5．8S rRNA基因之间的内转录间区（ITS）1的片段，以及白念珠菌位于ITS2区的特殊DNA片段，建立多重PCR方法，并鉴定血培养液中的酵母菌。结果：通过扩增片段长度分析显示光滑念珠菌482bp，季也蒙念珠菌248bp，近平滑念珠菌229bp，白念珠菌218bp和110bp，热带念珠菌219bp，新生隐球菌201bp，克柔念珠菌182bp。采用多重PCR方法共检测29例患者47份血培养阳性标本共50个分离株，其中白念珠菌27株，热带念珠菌8株，光滑念珠菌4株，近平滑念珠菌4株，新生隐球菌2株，克柔念珠菌1株，季也蒙念珠菌1株，均与培养鉴定结果一致，实验敏感性96％（47／50），特异性100％。多重PCR实验检测全过程共需8h。结论：多重PCR方法快速、敏感、特异，有助于播散眭念珠菌感染的早期诊断，具有较好的应用前景。

Rapid identification of yeasts in positive blood cultures by a multiplex PCR method

Objective:To establish a method for detecting and identifying clinically important yeasts that cause fungemia by multiplex PCR.Methods:The internal transcribed spacer 1(ITS1) region between the 18srRNA and 5.8srRNA genes,and a specific DNA fragment within the ITS2 region of Candida albicans were amplified to develop a multiplex PCR method.The method was used to identify the yeasts in the clinical blood specimens.Results:With this method,C.albicans produced two amplicons,whereas other species produced only one.Through sequence analysis,the precise lengths of the PCR products were found as follows:C.glabrata (482 bp ),C.guilliermondii (248 bp),C.parapsilosis (229 bp),C.albicans (218 bp and 110 bp),C.tropicalis (219 bp),Cryptococcus neoformans (201 bp) and C.krusei (182 bp).The method was used to test 47 positive blood cultures (50 isolates),from which the following strains were isolated:C.albicans (27),C.tropicalis(8),C.glabrata (4),C.parapsilosis (4),C.neoforman (2),C.krusei (1),C.guilliermondii (1),and other rare Candidas(3).The test sensitivity of the method was 96%(47 of 50 isolates ),and specificity 100%.The multiplex PCR could be completed within 8 hours.Conclusion:The present method is proved to be a rapid,sensitive and specific technique,and is helpful in the early diagnosis of disseminated candidiasis.It might be used widely in the future.