Water and Health Research Centre, Faculty of Health Sciences, University of Johannesburg, Johannesburg, South Africa.
PLoS One. 2021 Nov 29;16(11):e0260082. doi: 10.1371/journal.pone.0260082. eCollection 2021.
Quantifying pathogenic genes with q-PCR in complex samples to determine the pathogen loads is influenced by a wide range of factors, including choice of extraction method, standard curve, and the decision to use relative versus absolute quantification of the genes. The aim was to investigate the standardisation of q-PCR methods to determine enumerated E. coli gene ratios grown with the IDEXX Colilert® Quanti-Trays® using enteropathogenic E. coli as the model pathogen. q-PCR targeting the eaeA and gadAB genes was used to calculate the eaeA: gadAB ratios for clinical strains collected between [2005-2006 (n = 55)] and [2008-2009 (n = 19)] using the LinRegPCR software and Corbett Research Thermal cycler software. Both programs grouped the isolates into two distinct groups based on the gene ratios although the Corbett Research Thermal cycler software gave results one log higher than the LinRegPCR program. Although the eaeA: gadAB ratio range was determined using extracted E. coli DNA, the impact of free DNA and other bacteria present in the sample needed to be understood. Standard curve variations using serially diluted extracted E. coli DNA, serially diluted pure E. coli culture followed by DNA extraction from each dilution with or without other bacteria was tested using the eaeA q-PCR to quantify the genes. Comparison of the standard curves showed no significant difference between standard curves prepared with diluted DNA or with cells diluted before the DNA is extracted (P = 0.435). Significant differences were observed when background DNA was included in the diluent or Coliform cells added to the diluent to dilute cells before the DNA is extracted (P < 0.001). The "carrier" DNA and Coliform cells enhanced the DNA extraction results resulting in better PCR efficiency. This will have an influence on the quantification of gene ratios and pathogen load in samples containing lower numbers of E. coli.
在复杂样本中使用 q-PCR 对病原体进行定量分析以确定病原体负荷会受到多种因素的影响，包括提取方法、标准曲线的选择，以及使用基因相对定量或绝对定量的决定。本研究旨在调查使用 IDEXX Colilert® Quanti-Trays® 培养肠致病性大肠杆菌作为模型病原体时，q-PCR 方法标准化的情况。使用 LinRegPCR 软件和 Corbett Research 热循环仪软件，针对 eaeA 和 gadAB 基因进行 q-PCR 检测，以计算 2005-2006 年（n=55）和 2008-2009 年（n=19）期间收集的临床分离株的 eaeA:gadAB 比值。虽然 Corbett Research 热循环仪软件给出的结果比 LinRegPCR 程序高出一个对数级，但这两个程序都根据基因比值将分离株分为两个不同的组。虽然使用提取的大肠杆菌 DNA 确定了 eaeA:gadAB 比值范围，但需要了解样品中游离 DNA 和其他细菌的影响。使用连续稀释提取的大肠杆菌 DNA、连续稀释的纯大肠杆菌培养物以及在有无其他细菌的情况下从每个稀释度提取 DNA 进行标准曲线变化测试，使用 eaeA q-PCR 定量基因。比较标准曲线表明，用稀释 DNA 或用细胞稀释后再提取 DNA 制备的标准曲线之间没有显著差异（P=0.435）。当在稀释液中包含背景 DNA 或将大肠菌群细胞添加到稀释液中以在提取 DNA 之前稀释细胞时，观察到显著差异（P<0.001）。“载体”DNA 和大肠菌群细胞增强了 DNA 提取结果，从而提高了 PCR 效率。这将对含有较少大肠杆菌数量的样品中基因比值和病原体负荷的定量产生影响。
