中华皮肤科杂志 ›› 1996, Vol. 29 ›› Issue (5): 313-315.

• 论著 • 上一篇    下一篇

从新生隐球菌DNA文库中筛选特异性探针

吴建华1, 廖万清1, 柴建华2, 陈江汉1   

  1. 1. 第二军医大学长征医院皮肤科真菌病研究室, 上海200003;
    2. 复旦大学遗传学研究所
  • 收稿日期:1996-03-26 修回日期:1996-06-13 出版日期:1996-10-15 发布日期:1996-10-15
  • 基金资助:
    国家自然科学基金资助

Screening of Specific Probes from the DNA Library of Cryptococcus neoformans

Wu Jianhua1, Liao Wanqing1, Chai Jianhua2   

  1. Department of Dermatology, Changzheng Hospital, Shanghai 200003
  • Received:1996-03-26 Revised:1996-06-13 Online:1996-10-15 Published:1996-10-15

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摘要： 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。

关键词: 新生隐球菌, DNA文库, 探针

Abstract: To screen specific DNA probes by double hybridization from the constructed DNA library 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 processor.The PCR products were spotted onto hybond-N+ membranes.All inserts amplified from the genomic library by PCR were screened by dot blot with 26 32P-labelled DNA from infectious agents for differentiation 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 neoformans 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.neoformans,which gave signals only with serotype A and D strains.We can make a rapid diagnosis of Cryptococcus neoformans infection at an early stage and distinguish the variants of C.neoformans and C.gattii using above specific probes.

Key words: Cryptoeoccus neoformans, Genomic library, DNA probe