| Foodborne disease outbreaks and microbial spoilage threaten public health and cause major financial loss to the food industry and the society. Proper detection of concerned microorganisms in both raw materials and final food productes is a key to control the problems associated with microbial contamination. Several platforms were used to develop accurate, rapid, quantitative, specific and sensitive detection methods for targeted, viable cells, including RNA-based amplification platforms, such as nucleic acid sequence based amplification (NASBA) and reverse transcriptase PCR (RT-PCR), as well as DNA amplification coupled with sample treatment with DNA-intercalating dye propidium monoazide (PMA).;In this study, a NASBA-molecular beacon assay targeting 18S ribosomal RNA has been established to investigate its potential to detect viable spoilage yeasts in juice products. Using the developed platform, less than 100 yeast cells per reaction was detected rapidly and specifically. In addition, significant decrease of amplification signals after lethal heat treatments indicated that this platform has the potential for rapid detection of viable spoilage yeasts if combined with quantitative analysis.;Listeria monocytogenes contamination is a serious public health issue. The second part of my project compared the suitability of using 16S rRNA, inlA mRNA and rplD mRNA as indictors to detect viable L. monocytogenes cells via Taqman real-time RT-PCR assay. Under the conditions examined, the amplification signals by all three transcripts were reduced in dead cells, while the inlA and rplD mRNA signals decreased more dramatically than 16S rRNA. However, residue signals were still detected from dead cells even after extreme heat treatments.;The ideal cell viability indictor should disappear rapidly and completely after cell inactivation. In the third part of my study, cDNA microarray analysis was conducted to select unstable mRNA targets. Using unstable transcript ornithine decarboxylase (ODC) mRNA screened by cDNA microarray assay, a Taqman real-time RT-PCR platform was established to detect viable and heat or disinfectant Pro-sanRTM inactivated spoilage Pseudomonas. Under the experimental condition, ODC-specific RT-PCR signals were almost undectable after Pseudomonas cells were exposed to mild heat treatments.;DNA-intercalating dye propidium monoazide (PMA) only can penetrate damaged cell membrane and form crosslinkage with DNA molecules, resulting in inhibition of amplification. PMA coupled Taqman real-time PCR was developed to examine viable Pseudomonas spp. PMA treatment successfully minimized false positive amplification signals by dead cells after heat, acid or disinfectant Pro-sanRTM inactivation.;Results from this study provided critical information regarding nucleic acid amplification-based methods for viable foodborne microbial detection, and the new knowledge regarding overall RNA stability will have significant impact on data interpretation for transcriptome related studies. The rapid detection platforms developed have direct applications in both food industry and basic scientific research. |
