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Solid Phase Rapid Detection of Viable Micro-Organisms using Nucleic Acid Amplification and Biosensing Techniques

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This project attempts to utilise both novel molecular biological methods and enhanced endpoint detection systems to distinguish between living and dead bacterial cells isolated from food matrices, using the presence of mRNA as the measure of cellular viability.

Culture-based methods are the most commonly used for the detection of living (viable) microorganisms, but these methods are labour intensive, time consuming and insensitive. In addition, microorganisms capable of causing disease may be present in foodstuffs at very low levels, and additional procedures are required to increase the number of microorganisms present to detectable levels. Another disadvantage of this type of method is that bacterial cells damaged during food processing may be incapable of growing, but remain viable and capable of producing toxic substances that can cause food poisoning. Also, starvation can trigger some bacteria to enter a state where they are viable, but can not be cultured. Numerous non-culture based methods have been developed with the aim of reducing the detection time, however none of these methods are capable of the very rapid detection (within a single working day) and identification of very low numbers of microorganisms. Nucleic acid-based methods are capable of much more rapid and sensitive detection. Two types of nucleic acid exist in cells, deoxyribonucleic acid (DNA) is present in both live and dead cells, and is therefore not suitable as a viability indicator, whereas a type of ribonucleic acid, messenger RNA (mRNA) is only found in live cells. Once isolated, the number of mRNA molecules can be increased by amplification to levels detectable by a specific sensing device known as a biosensor. In addition, the sensitivity of this approach can be further enhanced by first coupling it to a chemical or biological cascade system.

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Conclusions from data collected and observations made: Methodologies for the isolation of high quality, intact mRNA and its amplification to detectable limits were established for three different organisms which are important in human food poisoning namely, Escherichia co/i, Salmonella enteritidis and Listeria monocyto genes. Two nucleic acid-based amplification methods were used, the Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and Nucleic Acid Sequence Based Amplification NASBA), of which the latter was found to be superior in several key respects.

These amplification methods were then employed to ascertain if there was a correlation between the presence of mRNA and bacterial viability. Messenger RNA could still be detected when cells of E. co/i. S. enteritidis and L. monocytogenes were killed by a range of treatments including heat. chemical and exposure to ultraviolet light. Heat-killed cells of these three organisms were also used to artificially contaminate foodstuffs and, upon their subsequent isolation from the foods mRNA was detectable for more than twelve hours. On the basis of these results. the presence of mRNA does not appear to have a clear correlation with the viability of bacterial cells, however more fundamental research into this area is necessary before this can be established unequivocally.

A number of different nucleic acid detection systems were developed and evaluated during the course of this project. The aim was to develop a system which was rapid, sensitive, specific. capable of high throughput and suitable for automation. The use of multiwell plates enabled the simultaneous analysis of multiple samples. Two broad types of assay were developed, the first was an enhanced technique based on the formation of a coloured product (amplified colorimetric assay). and the second on the utilisation of the mammalian system for the clotting of blood (coagulation or clotting assay). Neither could be successfully applied to the detection of mRNA. due to its degradation during the course of the assays. The coagulation-based assays were found to be approximately one thousand times more sensitive than the amplified colorimetric assays. Three main variations of the coagulation-based assays were developed, of which a format utilising coloured beads for the visualisation of the clot was considered to be the most useful. This format could be developed for the detection of a wide range of targets other than DNA, such as bacterial cells, toxins, hormones and pesticides.

A biosensor was successfully applied to the detection of DNA, however significant modification of the instrument would be necessary in order for the procedure to be fully automated. This was however, the most rapid detection method developed, with a detection time of minutes rather than hours.

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Natural Toxins