孢子丝菌病快速诊断方法的探究

Optimization of rapid detection method for sporotrichosis

目的研究申克孢子丝菌DNA提取方法;探索申克孢子丝茵的种特异性引物,运用聚合酶链反应方法鉴定申克孢子丝菌;从而为临床孢子丝茵病的分子诊断奠定基础。方法用Viscozyme L酶替代液氮研磨破壁提取孢子丝菌的基因组DNA;根据申克孢子丝茵钙调蛋白基因序列设计一对寡核苷酸引物,分别对52株申克孢子丝菌及3种6株普通真茵的基因组DNA进行PCR扩增。结果用Viscozyme L酶替代液氮研磨破壁成功提取出孢子丝菌的基因组DNA。特异性引物对52株申克孢子丝菌可扩增出一条约430 bp的片段,而对念珠菌、曲霉、黑霉基因组DNA扩增结果均为阴性。结论用Viscozyme L酶替代液氮研磨破壁提取孢子丝菌基因组DNA与传统的CTAB法相比不仅操作更简便,而且避免了液氮研磨过程中难于避免的污染。运用我们所设计的特异性引物,结合PCR方法对申克孢子丝菌进行分子鉴定,结果显示不仅该引物对申克孢子丝菌特异、敏感而且该方法简便、快捷;可用于申克孢子丝菌病的临床诊断。

Objectives A modified method for extracting DNA from Sporothrix.To study a rapid method to detect and identify sporothrix schenckii by using Species-specific oligonucleotide primer PCR techniques,and lay the foundation of molecular diagnosis for sporotrichosis.Methods Viscozyme L enzyme was used to replace of the traditional method.The species-specific oligonucleotide primer pair was used in this study.It was designed from nucleotide sequences of calmodulin gene in Sporothrix schenckii.52 strains of Sporothrix schenckii and 6 strains of common fungus were amplified by PCR.Results Genomic DNA of Sporothrix schenckii were extracted with the amended(CTAB)method.All strains of Sporothrix schenckii showed a specific fragment of about 430bp with the species-specific primer pair.Using the same primer pair,the other species had no specific amplification.Conclusions Compared with the traditional method,higher yield and purity of genomic DNA were obtained with less contamination.The result indicted that this was a simple and highly efficient method.This method was specific,sensitive,reliable,rapid and simple for identifying Sporothrix schenckii and could be used for clinical molecular diagnosis.