关键词: 不对称荧光聚合酶链反应, 白念珠菌, ITS-2基因, 单链DNA

Abstract:

Objective To investigate the efficiency of Candida albicans internal transcribed spacer 2 （ITS-2）single-stranded DNA （ssDNA） between 5.8S rDNA and 28S rDNA amplified by asymmetric fluorescence （AF）-polymerase chain reaction （PCR）,with the different dilution ratios of restrictive primers. Methods Compared with standard PCR,AF-PCR was carried out to amplify ITS-2 ssDNA of Candida albicans with the forward primers （P1,20 μmol/L） to the reverse primers （P2',20 μmol/L） of the different ratios and levels,and the PCR products were analyzed by electrophoresis and DNA sequencing. The amplification efficiency was evaluated. Results With the decreasing level of the forward primers,double-stranded DNA （dsDNA） bands of ITS-2 became weak,meanwhile,the ssDNA bands were brighten. When the forward primer dilution ratio reached 1:90 to 1:100,dsDNA bands were blurred,while the ssDNA bands became clear. It suggested that more ssDNA copies could be generated by AF-PCR in this amplification condition. Sequencing of PCR products also indicated that the bands locating above the dsDNA bands were ITS-2 ssDNA. Conclusions By optimizing the level of restrictive primer,AF-PCR could be used to successfully amplify ITS-2 ssDNA of Candida albicans effectively.

Key words: Asymmetric fluorescence-polymerase chain reaction, Candida albicans, Internal transcribed spacer 2, Single-stranded DNA