嗜酸氧化亚铁硫杆菌实时荧光定量PCR检测方法的建立

    唐连飞 肖家勇 朱中武 孟 芳 欧阳振宇 陈 盼 白 雪 禹思宇

    （湖南出入境检验检疫局，长沙 410004）

    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101bp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（Acidithiobacillusthiooxidans，A. thiooxidans）和嗜酸喜温硫杆菌（Acidithiobacilluscaldus，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。

    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

    〔中图分类号〕　R124 〔文献标识码〕　B

    Development of Fluorenscent QuantitativeReal-time PCR Method for Detection of Acidithiobacillusferrooxidans

    Tang Lianfei, Xiao Jiayong, Zhu Zhongwu, MengFang, Ouyang Zhenyu, Chen Pan, Bai Xue, Yu Siyu. (Hunan Entry-ExitInspection and Quarantine Bureau, Changsha 410004, China)

    [Abstract]   Objective  To  develop a fluorescent quantitative real-time PCR fordetection of A.ferrooxidans in import and export microbialmanure.  Methods   Genomic DNA was extracted astemplate for PCR from A.ferrooxidans ATCC 23270 cultured in 9Kculture medium. According to the A.ferrooxidans 16S gene sequenceavailable in GenBank a pair of primes and a TaqMan probe weredesigned and synthesized in orde to develop a fluorescentquantitative real-time PCR for detection of A.ferrooxidans. Toestablish  a standard curve, a plasmid containing the target16S rRNA gene fragment （101 bp） was constructed using pGEM?誖-T Easyvector (Promega) and severed as positive contro1.Results    The fluorescent quantitative real-timePCR for detection of A.ferrooxidans  was developedsuccessfully. After methodological evaluation, the results showedthat the developed method were specific enough to distinguishA.ferrooxidans from A. thiooxidans and A.caldus. A minimum of 100positive plasmids could be detected, indicating a good sensitivityof the assay, as well as the coefficient of variance (CV%) was0.32% for the intra-assay test, indicating a goodrepeatability.  Conclusion  This method is specific,sensitive and repetitive, and provides a high throughput and fastdetection of A.ferrooxidans.

    [Key words]   Acidithiobacillusferrooxidans； Fluorescent quantitative real-time PCR； Healthyquarantine；Detection method