格特隐球菌交配型的多重PCR鉴定

Identification of mating types of Cryptococcus gattii by multiplex PCR

摘要:

【目的】建立一种基于格特隐球菌α交配型位点内SXI1α基因和a交配型位点内SXI2a基因的多重PCR分析，用于快速鉴定格特隐球菌的交配型。【方法】设计针对格特隐球菌α交配型位点内SXI1α基因部分片段和格特隐球菌a交配型位点内SXI2a基因部分片段的特异性引物，用于多重PCR鉴定格特隐球菌的交配型；并与交配试验以及已报道的扩增α交配型位点的引物MFα、STE12α，及扩增a交配型位点的引物STE20a、STE3a进行扩增效果的比较。【结果】基于SXI1α基因和SXI2a基因的多重PCR分析，准确鉴定所有受试格特隐球菌(包括VGI、VGII、VGIII和VGIV基因型)的交配型，引物STE12α、STE20a和STE3a在常规PCR鉴定中不能鉴定部分菌株的交配型；66.7%的受试菌株不能发生交配，交配试验无法鉴定其交配型。【结论】建立的多重PCR方法明显优于常规PCR或交配试验鉴定。

Abstract:

[Objective] To establish a multiplex PCR assay on the basis of the SXI1α gene located at the mating type α (MATα) locus and SXI2a gene located at the mating type a (MATa) locus for rapid identification of the mating types of Cryptococcus gattii. [Methods] The primers specific for the alleles of SXI1α gene fragment within the MATα locus of Cryptococcus gattii was designed in combination with the primers specific for the alleles of SXI2a gene fragment within the MATa locus for multiplex PCR analysis of mating types of Cryptococcus gattii. The results were compared with those obtained from regular PCR using primers MFα and STE12α specific for the MATα locus, and primers STE20a and STE3a specific for the MATa locus reported previously, as well as mating assays. [Results] The mating types of all strains including four major genotypes, VGI, VGII, VGIII and VGIV, were successfully identified by multiplex PCR analysis based on the SXI1α and SXI2a genes. However, the mating types of a part of strains could not be determined by regular PCR assays with primer STE12α, STE20a or STE3a; the mating types of 66.7% percent of strains involved here could not be determined due to their inability to mate with the tester strains. [Conclusion] The multiplex PCR analysis is superior to regular PCR or mating assay reported previously.