新生隐球菌的AFLP分析

Amplified fragment length polymorphism analysis of Cryptococcus neoformans

目的评估AFLP-DNA指纹技术在新生隐球菌分类中应用情况。方法新生隐球菌基因组DNA用双酶酶切,双链接头连于其酶切末端,用与接头和酶切位点互补的引物扩增DNA片段,其产物在高分辨的变性聚丙酰胺凝胶上电泳分离,然后进行银染。结果分析来自5种血清型和临床分离株的18株新生隐球菌,可见有30多条大小在30～500bp的DNA-AFLP指纹,相同的血清型有不同的指纹图谱,来自同一患者不同病期的两株分离株和来自同一患者患者的不同部位的两株分离株都显示出相同的带型。结论显示了AFLP的高分辨率,是适用于新生隐球菌流行病学调查的有力工具。

Objective To evaluate AFLP-DNA fingerprinting method in the discrimination of C.neoformans.Methods Genomic DNA from C.neoformans was digested simultaneously with two restriction enzymes.Double-stranded adapters were ligated to the ends of restricted fragments and DNA fragments were amplified by PCR using primers complementary to the adapters and the restriction site sequences.AFLP products were separated using high resolution denaturing polyacrylamide gel electrophoresis and visualized by silver staining.Results DNA fingerprint patterns of 18 C.neoformans isolates from five serotypes and clinical isolates were analyzed by AFLP.AFLP fingerprints comprised over 30 bands detected in size range 30-500 bp.Different AFLP patterns were detected in isolates of the same serotype and identical banding patterns were observed from in isolates from the same patient at different time interals and different sources from one patiant.Conclusions AFLP is highly discriminatory,and powerful epidemiological tool for improving our understanding of C.neoformans.