Diseases caused by food- and water-borne pathogens have become a major public health problem. The Phase II objectives are as follows: <OL> <LI> To develop an improved, rapid, and sensitive sample preparation chip for real-time PCR-based detection of Escherichia coli O157:H7 and Salmonella in surface and irrigation water. The new chip will be able to isolate and concentrate a very small number of the pathogenic bacteria from a large volume of water and simultaneously remove PCR inhibitors. This will allow a considerable improvement in the sensitivity and specificity of PCR-based detection.<LI>To develop a quantitative real-time PCR assay for the detection of Salmonella. To insure that the real-time PCR results will be reliable, an internal DNA control will be genetically constructed for quality control of the sample preparation and PCR amplification. The real-time PCR assays for the detection of E. coli O157:H7 have been previously developed in Phase I. The sample preparation performance of the new chip for both E. coli O157:H7 and Salmonella will be evaluated and optimized in Phase II. The proposed sample preparation chip, in conjunction with real-time PCR assays, is expected to increase the detection sensitivity and reduce detection time.
Non-Technical Summary: Advances of molecular biology have greatly extended the detection sensitivity of food- and water-borne pathogens. Polymerase chain reaction (PCR) is one of most powerful technology in molecular biology that enables detection of the genomic DNA at single copy level. However, the contamination level of pathogenic bacteria in water samples is usually very low. In addition, a variety of substances in water sample are potential inhibitors to PCR amplification. More efficient sample preparation methods are needed to improve the PCR-based detections, especially for water-borne pathogens. This SBIR phase II project is to develop a rapid, sensitive PCR template preparation chip to remove amplification inhibitors and concentrate E. coli O157:H7 and Salmonella cells from water samples. The outcome of the phase II will be a sample preparation chip and PCR reagents that can help us to detect and precisely identify an outbreak source and to assess public water safety. <P> Approach: The proposed approach for Phase II study consists of two basic steps: immuno-capture of target bacteria on the sample preparation chip, followed by subsequent real-time PCR quantification and confirmation. The antibody specific to E. coli O157:H7 or Salmonella is immobilized on the surfaces of the sample preparation chip. When the water sample flows through the chip, the target bacteria are specifically captured on the immuno-affinity surfaces, and contaminants are washed away. This flow-through sample preparation method can be implemented in two assay formats: (1) enrichment and flow at the same time for a pre-selected period of time, and (2) continuous flow-through for unlimited sample size. The bacteria captured on the chip can be used in a variety of ways: (i) directly lysed using DNA extraction reagent for real-time PCR assays, (ii) cultured inside the sample preparation chip before lysis, or (iii) archived. Two real-time PCR assays, quantitative and multiplex real-time PCR assays, are developed in this study. The bacterial cell numbers from the sample preparation chip will be determined by quantitative real-time PCR assay. The strain identity and toxicity will be further confirmed by the multiplex real-time PCR assay. By examining the genetic information of the captured bacteria, we will evaluate the capability, specificity and recovery efficiency of the sample preparation chips.