嗜酸氧化亚铁硫杆菌实时荧光定量PCR检测方法的建立  

Development of Fluorenscent Quantitative Real-time PCR Method for Detection of Acidithiobacillus ferrooxidans

作　　者：唐连飞[1] 肖家勇[1] 朱中武[1] 孟芳[1] 欧阳振宇[1] 陈盼[1] 白雪[1] 禹思宇[1] 

机构地区：[1]湖南出入境检验检疫局,长沙410004

出　　处：《中国国境卫生检疫杂志》2010年第5期339-342,共4页Chinese Journal of Frontier Health and Quarantine

摘　　要：〔目的〕建立一种检测进出境环保用微生物菌剂中嗜酸氧化亚铁硫杆菌(acidithiobacillus ferrooxidans,A.ferrooxidans)的实时荧光定量PCR方法。〔方法〕用9K培养基培养A.ferrooxidans标准菌株A.ferrooxidans ATCC23270,并提取基因组DNA作模板;根据GenBank中嗜酸氧化亚铁硫杆菌的16S基因序列设计合成引物和探针,用含有101 bp扩增目标产物的pGEM誖-T Easy载体质粒为阳性对照,构建标准曲线,建立荧光定量PCR检测方法,并进行方法学的评估。〔结果〕成功建立了嗜酸氧化亚铁硫杆菌的荧光定量PCR检测方法,该方法对嗜酸硫杆菌属中嗜酸氧化硫硫杆菌(Acidithiobacillus thiooxidans,A.thiooxidans)和嗜酸喜温硫杆菌(Acidithiobacillus caldus,A.caldus)无交叉反应;最少可检测到100个阳性质粒,说明有很好的敏感性;试验内变异系数为0.32%,具有很好的重复性。〔结论〕建立的嗜酸氧化亚铁硫杆菌的TaqMan探针荧光定量PCR检测方法特异性和灵敏度高、重复性好,可作为嗜酸氧化亚铁硫杆菌高通量的快速检测方法。Objective To develop a fluorescent quantitative real-time PCR for detection of A.ferrooxidans in import and export microbial manure.Methods Genomic DNA was extracted as template for PCR from A.ferrooxidans ATCC 23270 cultured in 9K culture medium.According to the A.ferrooxidans 16S gene sequence available in GenBank a pair of primes and a TaqMan probe were designed and synthesized in orde to develop a fluorescent quantitative real-time PCR for detection of A.ferrooxidans.To establish a standard curve,a plasmid containing the target 16S rRNA gene fragment（101 bp） was constructed using pGEM-T Easy vector（Promega） and severed as positive contro1.Results The fluorescent quantitative real-time PCR for detection of A.ferrooxidans was developed successfully.After methodological evaluation,the results showed that the developed method were specific enough to distinguish A.ferrooxidans from A.thiooxidans and A.caldus.A minimum of 100 positive plasmids could be detected,indicating a good sensitivity of the assay,as well as the coefficient of variance（CV%） was 0.32% for the intra-assay test,indicating a good repeatability.Conclusion This method is specific,sensitive and repetitive,and provides a high throughput and fast detection of A.ferrooxidans.

关 键 词：嗜酸氧化亚铁硫杆菌 实时荧光定量PCR 卫生检疫 检测方法 

分 类 号：R124[医药卫生—环境卫生学]