从新生隐球菌DNA文库中筛选特异性探针

Screening of Specific Probes from the DNA Library of Cryptococcus neoformans

利用双重杂交筛选法从已经构建的 A 型新生隐球菌 DNA 克隆库(pCN)中筛选特异性探针。依据克隆库载体 pUC18的核苷酸序列设计并合成一对引物,PCR 扩增克隆库的插入片段,制备杂交膜。同位素标记26种鉴别菌及正常人的总 DNA 与克隆库的 PCR 产物杂交,获得28个候选克隆。同位素标记候选克隆的插入片段(以低熔点胶回收)与上述27种鉴别 DNA 杂交,获得特异性探针。结果:从已经构建的 pCN 中筛选出了3个特异性探针:pCNⅡA6、pCNⅡB5、pCNⅢG1,其中 pCNⅡA6只与A型新生隐球菌的 DNA 杂交,而不与其它鉴别 DNA 杂交,为 A 型特异性克隆;pCNⅡB5只与新生隐球菌的 DNA 杂交,为种特异性克隆;pCNⅢG1只与 A、D 型新生隐球菌的 DNA 杂交,为新生变种特异性克隆。应用上述特异性探针可以对隐球菌感染进行早期快速诊断及区分新生变种和格特变种。

To screen specific DNA probes by double hybridization from the constructed DNA li- brary of serotype A Cryptococcus neoformans.On the basis of the nucleotide sequence of vector pUC18, a pair of primers was synthesized.The insert fragments were amplified from the library on a PCR pro- cessor.The PCR products were spotted onto hybond-N^+ membranes.All inserts amplified from the ge- nomic library by PCR were screened by dot blot with 26 ^(32)P-labelled DNA from infectious agents for dif- ferentiation and from healthy persons.Twenty-eight candidate clones were obtained.The twenty-eight clone inserts got from low melting point agar were further characterized by dot blot with above 27 kinds of DNA for differentiation.Three specific DNA probes from the library of serotype A Cryptococcus neo- formans were obtained.Colony pCNIIA6 was serotype A-specific,which gave signals only with sertype A strain and did not cross hybridize with other DNAs.Colony pCNIIB5 was species-specific which gave signals only with DNA from Cryptococcus neoformans.Colony pCNIIIG1 was specific for var.neofor- mans,which gave signals only with serotype A and D strains.We can make a rapid diagnosis of Crypto- coccus neoformans infection at an early stage and distinguish the variants of C.neoformans and C.gattii using above specific probes.