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马尔尼菲青霉菌是一种温度依赖性双相型条件致病真菌,可感染免疫缺陷人群,区域流行于东南亚地和我国南方,但其双相转换和致病性分子机制尚不清楚。实时荧光定量PCR是马尔尼菲青霉菌双相转换和致病性分子机制研究的重要手段,但仍缺乏用于马尔尼菲青霉菌实时荧光定量PCR标准化分析理想的内参基因。本研究根据转录组测序数据筛选了四个表达最稳定的Pfp、Rp123、FacpA、DigA基因与传统的内参基因18Sr RNA、β-actin(Act1)、β-tubulin(Btu)作为候选内参基因,用Best Keeper和ge Norm软件进行基因表达稳定性分析。结果表明FacpA和DigA基因在马尔尼菲青霉菌双相转换过程中表达最为稳定,可作为用于马尔尼菲青霉菌实时荧光定量PCR标准化分析的内参基因。
Penicillium marneffei is a temperature-dependent bi-phasic pathogenic fungus that can infect immunodeficient people. The region is endemic to Southeast Asia and southern China. However, the mechanism of biphasic transformation and pathogenicity is not yet clear. Real-time PCR is an important method for the study of biphasic transformation and pathogenic molecular mechanism of Penicillium marneffei, but it still lacks the ideal reference gene for the standardized analysis of Penicillium marneffei real-time quantitative PCR. In this study, the four most stable Pfp, Rp123, FacpA, DigA genes and 18Sr RNA, β-actin (Act1) and β-tubulin (Btu) were selected as candidate reference genes according to the transcriptome sequencing data. Best Keeper and ge Norm software for gene expression stability analysis. The results showed that FacpA and DigA genes were most stable during the biphasic transformation of Penicillium marneffei and could be used as internal reference genes for the standardized analysis of Penicillium marneffei real-time fluorescence quantitative PCR.