摘要
目的:拟建立多重RT-PCR诊断甲真菌病的方法。方法:采用Trizol提取真菌的RNA;选择3对引物(真菌通用引物,皮肤癣菌特异性引物和酵母特异性引物)建立3重RT-PCR体系,采用红色毛癣菌、白念珠菌和短帚霉摸索反应条件和验证体系的适用性;并用红色毛癣菌来测定反应的灵敏度。结果:(1)所采用的真菌通用引物28srDNA、皮肤癣菌特异性引物ACT和酵母特异性引物ACT1有较好的通用性和特异性;(2)在适宜的反应条件下,无论对单模板,还是多模板,该反应体系均能扩增出目的片段,未见明显非特异片段的干扰;(3)该反应体系对模拟临床标本的检测灵敏度为102cfu/mL。结论:多重RT-PCR技术可在较短时间内检测出皮肤癣菌、酵母和霉菌等,并可判断菌的活力,将为临床快速诊断和及时、合理地治疗甲真菌病和其他真菌性疾病提供参考。
Objective: To develop a multiplex reverse transcription- PCR method to detect pathogenic fungi of onychomycosis. Methods: RNAs of tested fungi were extracted with Trizol. The specificity of RT- PCR was determined with universal fungus- specific primer, dermatophyte - specific primer and yeast - specific primer palm respectively. Multiplex RT- PCR with these 3 primer palm was used to detect 1 template ( T. rubrum, C. albieans, S. brevicaulis ) and 2 or 3 template mixtures. Sensitivity of multiplex RT- PCR was measured with simulated clinical specimen. Results: The detection system was of high specificity. Multiplex RT - PCR amplified the corresponding 1, 2 or 3 cDNA fragments, depending on the number of template DNA added, and the sensitivity of multiplex RT - PCR was 102 cfu/ml. Conclusion: This method might be a potential tool for rapidly assessing fungal viability in the diagnosis of onyehomyeosis in light of the simultaneous presence of two or three different fungal species.
出处
《中国麻风皮肤病杂志》
2007年第11期956-959,共4页
China Journal of Leprosy and Skin Diseases
关键词
聚合酶链反应
甲真菌病
诊断
polymerase chain reaction
onyehomyeosis
diagnosis