H.C. Parkes

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.

With a continued increase in the range of transgenes, and plant species for which genetic modification is possible, this review attempts to bring together some of the factors that will influence the eventual fate of transgenes in the environment, and the effects that such a dispersal may have. The review is developed from papers presented at the SEB Swansea meeting (April, 1994). Using experiments with GM (genetically modified) plants, and markers in non-GM plants, as well as observations on natural and crop populations, it is possible to predict isolation distances required for limiting the unintentional release from GM crops, and the probable fate of both GM pollen and seed if it is released beyond the GM plot. Knowledge of wild relatives of crop plants, and ecological mechanisms can also give insights into the possible effects of different transgenes on native plants, and other agricultural crops. A large number of limited scale releases of GM plants have now taken place from which we can gain information on the performance of GM crops in an agricultural environment, and the stability of the GM phenotype. All this information, can help to form a sound basis for regulations on the release of GM plants, an assessment of the need for, and scope of monitoring, and the best way in which to use GM crops.