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Title: Using PCR to enumerate pathogen indicators in composted organic waste
Author: Mohamed Sunar, Norshuhaila
ISNI:       0000 0004 2742 1171
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2011
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Since the earliest work on controlled composting in the middle of the last century, the effectiveness of sanitisation has been determined using pathogen indicator organisms, such as E. coli and conventional enumeration techniques. Recent developments in molecular biology suggested the possibilities of replacing these 'standard' techniques with some form of PCR (polymerase chain reaction). The main part of the study related to the use of PCR to enumerate pathogen indicators in composted organic waste and compared it with conventional cultivation techniques recommended in the compost guidelines (PAS 100). The inactivation of pathogen indicators focus sed on the viability of their DNA and the survival of pathogen indicators in composted material. Membrane filtration was the standard conventional technique used to compare with molecular techniques. The main microorganism used in the study was Salmonella spp. Whilst PCR produced qualitative results' for detecting pathogen indicators, a quantitative approach was 'developed using cPCR (competitive polymerase chain reaction). The deletion method of pathogen-specific competitor fragments production for use in cPCR proved to be effective and was used to develop a competitor fragment. In this study, the cPCR technique was utilised to enumerate Salmonella spp. in aqueous samples. Results indicated that primers that produce a 670bp fragment from the E. coli gad AB gene did not represent the constructive choice for the cPCR method, because it resulted in PCR amplification products with faint bands and non-specific amplification. On the other hand, the primers that produced a 284 bp fragment from the invA gene were shown to be effective for the detection of Salmonella spp. by PCR owing to its high specificity. The clear and single band imaged in 2% agarose gel electrophoresis produced a good match; thus, it was highly significant in the cPCR and PMA-cPCR methods developed. The PMA (propidium monoazide) pretreatment step as DNA-intercalating dye was used in the cPCR protocol to provide viable/dead cell differentiation known as PMA-cPCR. The PMA-cPCR method developed was shown to be effective in enumerating the live Salmonella spp. DNA. The standard cultivation method gave approximately 1-2 log10 cfu/ml less compared to the PMA-cPCR results. These results indicated the existence of the VBNC (viable but non-culturable) state, whereas the viable cells failed to be cultured by the standard cultivation methods. Moreover, it also represented the non-loss of Salmonella spp. DNA during cross-linked upon bright light exposure. The results were approximately of the same magnitude order at average 108 cfu/ml for before and after the photo-activation procedure. The quantification results from the PMA-cPCR method for viable and non-viable DNA were analysed and compared with conventional methods and cPCR. This type of cPCR and PMA-cPCR methodology for quantifying Salmonella spp. shows very positive results, especially when utilised with an aqueous sample. Having developed the PCR based techniques for enumeration of Salmonella spp. work was then carried out in laboratory composters to study the survival of Salmonella spp. However, the DNA-based quantification for compost material was deemed to be an inappropriate method for use with quality monitoring of composting, as it always shows non- specific and non-amplification results. A few drawbacks concerning the use of PCR and PMA-cPCR in compost material were discussed, which led to various suggestions for future research. During the study, it became apparent that several different factors were responsible for the inactivation of Salmonella spp. as well as exposing the organisms to elevated temperatures. Different samples preparations was utilised to differentiate the biological, physical and chemical effects in composted samples. These results confirmed the effects of inactivation mechanisms involved, such as; antagonism and competition from other organisms, effect of chemicals changes and transportation. The PCR is a useful molecular technique for studying pathogen indicator detection and DNA-based quantification in solid waste. When being used it is recommended that highly competent individuals are needed if the nucleic acids amplification protocol is to be used for routine quality monitoring in the composting field. It is recommended that, despite false-positive results that depending on the elements contained in the compost materials, the standard enumeration techniques are the most appropriate for use in composting.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available