FungiQuant: a broad-coverage fungal quantitative real-time PCR assay.
FungiQuant: a broad-coverage fungal quantitative real-time PCR assay.
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DOI：
10.1186/1471-2180-12-255
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发表时间：
2012-11-08
期刊：
影响因子：
4.2
通讯作者：
Price LB
中科院分区：
文献类型：
--
作者：
Liu CM;Kachur S;Dwan MG;Abraham AG;Aziz M;Hsueh PR;Huang YT;Busch JD;Lamit LJ;Gehring CA;Keim P;Price LB
真菌负荷定量是真菌群落分析的关键组成部分。目前用于量化人类微生物组中真菌组分的方法的局限性表明需要新的广泛覆盖技术。我们分析了来自SILVA数据库的2，085个18 S rRNA基因序列，用于试验设计。我们使用基于qPCR的方法生成并量化了质粒标准品。我们评估了4，968个序列的检测覆盖率，并根据定量实时PCR实验（MIQE）指南的最低信息发布进行了检测验证。我们设计了FungiQuant，这是一种靶向真菌18 S rRNA基因中351 bp区域的TaqMan® qPCR测定法。我们的计算机模拟分析表明，FungiQuant与所分析的2，617种真菌物种中的90.0%完全匹配。我们表明，FungiQuant的灵敏度为100%，其扩增效率范围为76.3%至114.5%，针对测试的69种真菌物种的r2值>0.99。此外，FungiQuant运行间和运行内的方差系数范围分别为<10%和<20%。我们进一步表明，FungiQuant的定量限为25个拷贝，检测限为5个拷贝。最后，通过比较仅人类背景DNA与低水平真菌DNA的结果，我们表明，一式三份进行的两个或三个FungiQuant的扩增对于真阳性真菌检测具有统计学意义。FungiQuant对各种真菌具有全面的覆盖范围，是一种强大的定量和检测工具，用于区分真正的真菌检测和非靶人类DNA。
Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for new broad-coverage techniques. We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. We designed FungiQuant, a TaqMan® qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuant’s is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r2-values of >0.99 against the 69 fungal species tested. Additionally, FungiQuant inter- and intra-run coefficients of variance ranged from <10% and <20%, respectively. We further showed that FungiQuant has a limit of quantification 25 copies and a limit of detection at 5 copies. Lastly, by comparing results from human-only background DNA with low-level fungal DNA, we showed that amplification in two or three of a FungiQuant performed in triplicate is statistically significant for true positive fungal detection. FungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA.