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Novel methods of quantitative real-time PCR data analysis in a murine Helicobacter pylori vaccine model

机译：小鼠幽门螺杆菌疫苗模型中定量实时PCR数据分析的新方法

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Monitoring of Helicobacter pylori in the stomach is important to assess the efficacy of new vaccines against the pathogen. To realise the full potential of quantitative real-time PCR (q-PCR), this technology has to offer accurate and easy models of post-PCR data analysis. In this work, we used a variety of absolute and relative approaches of q-PCR data analysis to monitor the H. pylori infection in the stomach of immunized mice. Relative quantification was performed with Ct-based methods, with the DART program, and with two methods based on the mathematical analysis of raw fluorescence kinetics, the LinReg program and the Sigmoidal Curve Fitting Method. The different calculation methods were validated in mice immunized with cell lysates of Lactococcus lactis expressing the H. pylori urease subunit B in combination with cholera toxin. The H. pylori load was found to be reduced in immunized mice by a factor of 50-144, depending on the calculation method employed. We found that relative quantification using DART, LinReg and Sigmoidal Curve Fitting methods generated similar results (infection ratios of 54-58) with absolute quantification results (54-65). Results were very different to those using relative quantification Ct-based methods without a correction for PCR efficiency (ratio of 92-144) and with results based on conventional culture method (ratio of 34). Overall, this study demonstrates that q-PCR associated with a relative quantification analysis is a powerful tool for the monitoring of microorganisms in tissue. It could be used as an alternative to standard curve approach especially for the investigation of microbial load in vaccine models.
机译：胃中幽门螺杆菌的监测对于评估针对病原体的新疫苗的疗效很重要。为了充分发挥定量实时PCR（q-PCR）的潜力，该技术必须提供准确而简单的PCR后数据分析模型。在这项工作中，我们使用了各种绝对的和相对的q-PCR数据分析方法来监测免疫小鼠胃中的幽门螺杆菌感染。相对定量使用基于Ct的方法，DART程序以及基于原始荧光动力学的数学分析的两种方法LinReg程序和S曲线拟合法进行。在用表达幽门螺杆菌脲酶亚基B的乳酸乳球菌细胞裂解物结合霍乱毒素免疫的小鼠中验证了不同的计算方法。根据所采用的计算方法，发现在免疫小鼠中幽门螺杆菌的负荷降低了50-144倍。我们发现使用DART，LinReg和Sigmoidal曲线拟合方法进行的相对定量产生了相似的结果（感染比为54-58），并且具有绝对定量结果（54-65）。结果与使用相对定量基于Ct的方法的结果非常不同，没有校正PCR效率（比率为92-144），而基于常规培养方法的结果（比率为34）。总体而言，这项研究表明，与相对定量分析相关的q-PCR是监测组织中微生物的有力工具。它可以用作标准曲线方法的替代方法，特别是用于研究疫苗模型中的微生物负荷。

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