Abstract
The quantitative real-time polymerase chain reaction (qPCR) is one of the most commonly molecular methods used today. It is central to numerous assays that have since been developed and described around its optimization. The Listeria monocytogenes prfA qPCR assay has been studied in great detail and due to its comprehensive knowledge, excellent performance (sensitivity of one single copy), and internal amplification control, it represents a suitable test platform for qPCR examinations. In this study, we compared ten different polymerases (or ready-to-use mastermixes) as possible (economic) alternatives to our gold standard Platinum Taq polymerase. We sought to determine the reproducibility of these assays under modified conditions, which are realistic because published assays are frequently used with substituted polymerases. Surprisingly, there was no amplification at all with some of the tested polymerases, even although the internal amplification control worked well. Since adaptation of the thermal profile and of MgCl2 concentration could restore amplification, simple replacement of the polymerase can destroy a well-established assay leading up to >106-fold less analytical sensitivity. Further, validation using Poisson and PCR-Stop analyses revealed limits to some assay-polymerase combinations and emphasize the importance of validation.
Highlights
Ever since the first description of the polymerase chain reaction (PCR) at the beginning of the 1980s by Kary Mullis, many researchers have improved upon this method to the extent that it is applicable to various fields of research and diagnostics
For initial comparison of the different polymerases, DNA from Listeria monocytogenes EGDe and ΔprfA were amplified at concentrations between 1.58 × 101 and 1.58 × 106 copies per reaction, which are normally used for calibration curves for quantification of L. monocytogenes
QPCR was performed with the thermal program optimized for the assay using Platinum Taq
Summary
Ever since the first description of the polymerase chain reaction (PCR) at the beginning of the 1980s by Kary Mullis, many researchers have improved upon this method to the extent that it is applicable to various fields of research and diagnostics. Development of the polymerase necessary for amplification in PCR moved from its origin in Escherichia coli towards heat stable polymerases that have the advantage of withstanding the high temperatures encountered with DNA denaturation [1]. For quantitative detection of DNA targets (quantitative PCR (qPCR)), fluorescent agents intercalating in double stranded DNA, such as SYBR or EVA green or fluorophores coupled to sequence-specific oligonucleotides can be detected by dedicated instruments [3]. Due to its high sensitivity, qPCR is able to detect down to one initial target molecule number (ITMN) under optimized conditions as demonstrated for the probe-based prfA assay using Poisson distribution in the boundary limit (< 10 ITMN) [4,5,6]
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