PCR-based detection of microorganisms in complex biological samples

University dissertation from Applied Microbiology, PO Box 124, SE-221 00 Lund, Sweden

Abstract: The detection of microorganisms by PCR can be divided into four steps: (1) sample collection, (2) sample preparation, (3) DNA amplification and (4) detection of PCR products. The major problem in developing PCR-based detection methods is the sample preparation step. This step is necessary to overcome problems caused by PCR inhibitors and it determines, to a large extent, the sensitivity of the PCR-based method. A sample preparation method based on aqueous two-phase systems, composed of water soluble polymers, have been developed. An aqueous two-phase system, composed of polyethylene glycol 4000 and dextran 40, was found to separate PCR-inhibitory compounds in soft cheese from Listeria monocytogenes. The PCR detection level was lowered by a factor of more than 1000 using the sample preparation method developed. An identical aqueous two-phase system have been used to extract PCR-inhibitory faecal samples. The majority of the PCR-inhibitory substances, including bile salts, was found to partition to the polyethylene glycol-rich top phase whereas the bacteria were detected by PCR in the dextran-rich bottom phase. Three sample preparation methods — an aqueous two-phase system, density gradient centrifugation and DNA extraction — have been compared with regard to their efficiency in removing PCR inhibitors from raw minced pork and enrichment media when detecting Yersinia enterocolitica. Individual components of the samples were examined regarding PCR inhibition. A PCR assay has been developed for the detection of yeast in industrial sugar solutions. A concentration step, based on filtration, was employed prior to PCR. A PCR detection level of naturally contaminated sugar solutions of 0.1 CFU per ml was obtained. The developed detection method is rapid (<5 hours) and easy to perform. The sample preparation method, prior to PCR, must be carefully chosen. The type of PCR inhibitors, the structure of sample and the required detection level are some of the factors that must be considered when selecting a sample preparation method.

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