Real-time PCR is dependent upon a calibration function for quantification. While long-term storage of standards saves cost and time, solutions of DNA are prone to degradation. We present here the benchmark treatment for preservation of DNA standards, involving storage in 50% glycerol-double-distilled water, whereby a deviation of 0.2 threshold cycle (C T ) values resulted after 100 days of storage.
Establishment of molecular diagnostics offering quantitative technology is directly associated with real-time polymerase chain reaction (PCR). This rapid, accurate and sensitive method requires careful execution, including reliable calibration standards. The storage of such standards is crucial to prevent nucleic acid decay and to ensure stable results using real-time PCR. In this study, a broad investigation of possible causes of DNA degradation during storage was performed, including GC-content of the fragments, long-term storage, rapid freeze-and-thaw experiments, genomic DNA and short DNA fragments of different species, the influence of shear stress and the effect of nuclease remaining after DNA isolation. Several known chemical DNA degradation mechanisms have been matched with the experimental data through a process of elimination. Protocols for practical application, as well as a theoretical model describing the underlying mechanisms of deviation of real-time PCR results due to decay of standard DNA, have been developed. Primary amines in the buffer composition, which enhance depurination of the DNA helix, and shear stress due to ice crystal formation, could be identified as major sources of interaction. This results in degradation of the standard DNA, as well as in the probability of occurrence of mismatches affecting real-time PCR performance.
In this study, we present the concept of internal sample process controls (ISPCs) to monitor the efficiency of an analytical chain using sample preparation and quantitative PCR (qPCR). A recombinantListeria monocytogenesΔprfA(targeted deletion) strain containing a competitive artificial single-copy genomic target was applied to naturally contaminated samples to demonstrate its analytical suitability as an ISPC.
Aims: In the present study, chromogenic (red) bacteria were used to simulate actual target bacteria during set‐up and optimization of an isolation process of bacteria, designed for food samples. Isolation of bacteria from food in the context of molecular biological detection of food pathogens is a multistep process. Development of such a separation method requires continuous monitoring of the location of the presumable targets in the sample tubes. Therefore, red‐coloured pigmented bacteria were used as substitutes for the actual target bacteria, during the establishment of a new sample preparation technique.
Methods and Results: The chromogenic bacteria Micrococcus roseus and Serratia marcescens were confirmed to withstand the physical (e.g. centrifugal forces) and chemical (e.g. lysis buffer composition) conditions required during establishment of the new technique. Furthermore, the suitability of these model bacteria to substitute for the actual target pathogens (Salmonella enterica subsp. enterica serovar Typhimurium and Listeria monocytogenes) was assured by testing the physical properties of the model bacteria with respect to the proposed separation methods.
Conclusion: Visibility of the pigmented bacteria within the complex sample matrices served to allocate bacterial content during the various steps necessary for finalization of the method protocol. The presumptive bacterial targets can be allocated simply by visualization of their bright red colour silhouetted against the background sample matrix.
Significance and Impact of Study: The use of pigmented bacteria as substitutes for actual colourless target bacteria during design and development of a bacterial isolation method is a simple and inexpensive application. It saves a huge amount of time and resources, as the proof of principle of new methods is possible in rapid succession.
Conventional internal amplification controls (IAC) are DNA-based controls which monitor the amplification reaction of real-time PCR in food pathogen detection. Food pathogen detection using real-time PCR, however, includes necessarily sample preparation and DNA isolation/purification. This modular structure leads to an analytical chain. To cover the whole analytical chain, the concept of the IAC has to be extended to internal sample process controls (ISPCs) which include supporting pre-analytical steps. One concept for such ISPCs is the use of recombinant bacterial cells comprising a deleted target and an artificial competitive target instead, which are derived from the actual target strain. In this work, we present an ISPC for the molecular detection of Listeria monocytogenes. A Δ-prfA L. monocytogenes EGDe strain was cloned with a pPL2 phage insertion vector to include a single copy artificial DNA target, resulting in a fluorescence signal not interfering with the respective signal of the L. monocytogenes EGDe wild-type strain during realtime PCR. The recombinant strain was confirmed and characterized with conventional and real-time PCR including sequencing. Microbiological examination revealed a distinct phenotype pattern on selective plate media which enables discrimination of Δ-prfA L. monocytogenes EGDe from wild-type L. monocytogenes EGDe and Listeria innocua. The ISPC was applied in an examination of artificially contaminated ultra high temperature-treated milk to demonstrate its analytical suitability. The resulting corrected recovery values of the ISPC as obtained by the whole molecular quantification procedure correspond to the respective values determined for the actual target strain (P≤ 0.05).
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