DNA from Listeria monocytogenes was used as the model system from this investigation, with PCR primers based on the listeriolysin O gene. Under standard polymerase chain reaction (PCR) conditions and with no prior treatment, amplification failed in the presence of more than 5% milk. Since inhibition of the PCR occurred at the same milk concentrations with full fat, half fat and fat-free milk, inhibition was not attributed to the fat content of the milk. Calcium ions were, however, identified as a major source of PCR inhibition. The results demonstrated that the inhibitory effects of calcium ions and milk could be partially reversed by increasing the magnesium concentration in the reaction to well above the standard levels normally required for PCR. This work has important implications for the use of the PCR in the direct detection of food pathogens.

The polymerase chain reaction is an immensely powerful technique for identification and detection purposes. Increasingly, competitive PCR is being used as the basis for quantification. However, sequence length, melting temperature and primary sequence have all been shown to influence the efficiency of amplification in PCR systems and may therefore compromise the required equivalent co-amplification of target and mimic in competitive PCR. The work discussed here not only illustrates the need to balance length and melting temperature when designing a competitive PCR assay, but also emphasises the importance of careful examination of sequences for GC-rich domains and other sequences giving rise to stable secondary structures which could reduce the efficiency of amplification by serving as pause or termination sites. We present data confirming that under particular circumstances such localised sequence, high melting temperature regions can act as permanent termination sites, and offer an explanation for the severity of this effect which results in prevention of amplification of a DNA mimic in competitive PCR. It is also demonstrated that when Taq DNA polymerase is used in the presence of betaine or a proof reading enzyme, the effect may be reduced or eliminated.

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HyBeacon probes are single-stranded oligonucleotides with one or more internal base(s) labeled with a fluorescent dye. When a probe forms a duplex with its target sequence, the level of fluorescence emission increases considerably. HyBeacons have been developed as new tools for rapid sequence detection and discrimination and have been employed in a wide variety of applications including infectious diagnostics and analysis of human polymorphisms. Single-labeled (FVG1) and dual-labeled (FVG11) probes were designed to analyze the factor V Leiden (R506Q) polymorphism which causes an increased risk of deep vein thrombosis and pulmonary embolism. Detection and identification of factor V alleles is performed by melting curve analysis and determination of probe melting temperature (T(m)). HyBeacon hybridization to the glutamine allele (Q) causes the formation of mismatched DNA duplexes that are detected through decreases in T(m). HyBeacon probes are included in homogeneous PCR assays to genotype samples with respect to the factor V polymorphism within 20 min, using purified DNAs and unpurified saliva/blood samples. This paper describes the preparation of homogeneous PCR assays, LightCycler target amplification, and subsequent melting curve analysis. This chapter also describes the use of homologous oligonucleotides and melting curve analysis as a method for probe evaluation.