Quantification of M13 and T7 bacteriophages by TaqMan and SYBR green qPCR.
Quantification of M13 and T7 bacteriophages by TaqMan and SYBR green qPCR.
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DOI：
10.1016/j.jviromet.2017.11.012
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发表时间：
2018-03
影响因子：
3.1
通讯作者：
Ghosh D
中科院分区：
文献类型：
--
作者：
Peng X;Nguyen A;Ghosh D
TaqMan和SYBR Green定量聚合酶链式反应(QPCR)方法被发展为基于DNA的方法，可分别和同时对噬菌体展示筛选实验中的M13和T7噬菌体进行重复计数。以已知浓度的M13和T7 DNA标准曲线为参照，用TaqMan或SYBR Green定量分析M13和T7噬菌体的基因组拷贝。TaqMan qPCR能同时定量M13和T7噬菌体DNA，检测范围分别为2.75×10 1~2.75×10 8个基因组拷贝(GC)/μL和2.6 6×10 1~2.66×10 8基因组拷贝(GC)/μL。TaqMan qPCR对M13和T7噬菌体DNA的有效扩增效率(ES)分别为0.97和0.90。SYBR Green的灵敏度是TaqMan的10倍，能定量检测M13和T7噬菌体的2.75~2.75×107GC/μL和2.66×101~2.66×107GC/μL，扩增效率分别为1.06和0.78。由于其优越的灵敏度，SYBR Green qPCR被用来计数针对细胞系选择的M13和T7噬菌体展示克隆，其定量滴度的准确性与传统的双层空斑试验相当。与酶联免疫吸附试验相比，两种定量聚合酶链式反应方法的检测灵敏度和重复性都有所提高。这些定量聚合酶链式反应方法重复性好、灵敏度高、省时，可单独或同时定量大量噬菌体样品，从而避免了耗时的双层空斑分析。这些发现突显了qPCR用于噬菌体计数的吸引力，应用范围从选择到下一代测序(NGS)。
TaqMan and SYBR Green quantitative PCR (qPCR) methods were developed as DNA-based approaches to reproducibly enumerate M13 and T7 phages from phage display selection experiments individually and simultaneously. The genome copies of M13 and T7 phages were quantified by TaqMan or SYBR Green qPCR referenced against M13 and T7 DNA standard curves of known concentrations. TaqMan qPCR was capable of quantifying M13 and T7 phage DNA simultaneously with a detection range of 2.75*101 – 2.75*108 genome copies(gc)/μL and 2.66*101 – 2.66*108 genome copies(gc)/μL respectively. TaqMan qPCR demonstrated an efficient amplification efficiency (Es) of 0.97 and 0.90 for M13 and T7 phage DNA, respectively. SYBR Green qPCR was ten-fold more sensitive than TaqMan qPCR, able to quantify 2.75 – 2.75*107 gc/μL and 2.66*101 – 2.66*107 gc/μL of M13 and T7 phage DNA, with an amplification efficiency Es of 1.06 and 0.78, respectively. Due to its superior sensitivity, SYBR Green qPCR was used to enumerate M13 and T7 phage display clones selected against a cell line, and quantified titers demonstrated accuracy comparable to titers from traditional double-layer plaque assay. Compared to enzyme linked immunosorbent assay, both qPCR methods exhibited increased detection sensitivity and reproducibility. These qPCR methods are reproducible, sensitive, and time-saving to determine their titers and to quantify a large number of phage samples individually or simultaneously, thus avoiding the need for time-intensive double-layer plaque assay. These findings highlight the attractiveness of qPCR for phage enumeration for applications ranging from selection to next-generation sequencing (NGS).
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影响因子：
64.8
作者：
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通讯作者：
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通讯作者：
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