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The objective of this SBIR project is to develop a detection method for water-borne pathogens. This method combines a proprietary sample preparation chip with multiplex real-time PCR to provide rapid, sensitive, specific and quantitative results. The method is applicable to a wide variety of water-borne pathogens. We will demonstrate that this method can eliminate PCR inhibition, minimize sample splitting, complete the test including pre-concentration in about three hours, determine specificity by six genes associated with E. coli O157:H7, and obtain sensitivity that meets EPA standards. Phase I will perform the proof-of-principle using E. coli O157:H7 in drinking water. Phase II will further improve the sample preparation method and detect E. coli O157:H7 in surface water.
Enterohemorrhagic Escherichia coli (E. coli O157:H7) is a causative agent of food- and water-borne diseases. Newly developed rapid immunoassay- and PCR-based techniques have been used for detection of E. coli O157:H7 in water and food samples. However, they have proven inadequate, because of the large sample size, an insufficient number of pathogens in the sample, sample splitting, antibody cross reaction, and PCR inhibition caused by sample contaminants. We propose a rapid, sensitive, specific and quantitative test for water-borne pathogens. The method can be employed wherever PCR is used to determine the presence, concentration and/or genetic characteristics of bacterial or viral pathogens. Greatest benefit will be seen in the analysis of samples that are large in relation to the typical 2-5 microliter template volume for PCR, particularly for water quality, food safety, and environment tests. We will develop the method initially to detect E. coli O157:H7 in drinking water in Phase I and source and recreational waters in Phase II. The public will benefit greatly from safer drinking water, rivers and beaches.
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The approach we are taking for the detection of water-borne pathogen is to utilize micro-fluidic chip to isolate and concentrate the bacteria from water sample, followed by cell-lysis and mutiplex real-time for strain identification. The details of the proposed approach is as below: Micro-fluidic chip. Immuno-concentration/purification chip to incorporate sonic turbulent mixing will be developed. The water sample will be circulated through a micro-fluidic chip. Sonic mixing will be employed to accelerate the capture process. The walls of the chip will be coated with specific monoclonal antibodies against the pathogen. This will selectively capture the target pathogen on the chip surface and simultaneously wash out the contaminants. The captured pathogens can be recovered in a small volume of lysis buffer for further genetic assays. Preparation of PCR template. The bacterial cells captured are directly lysed and centrifuged. The genomic DNA of E. coli O157:H7 in the supernatant is used as the template for quantitative and multiplex real-time PCRs. Real-time PCRs. Two types of real-time PCR assay will be applied in this study. (1) A quantitative real-time PCR will be used to determine genome copy numbers of E. coli O157:H7. The lacZ gene will be amplified as genetic marker and a standard curve of the lacZ gene will be constructed. The quantitative real-time PCR assay will be used to evaluate the capture efficiency of the micro-fluidic chip. (2) The multiplex real-time PCR will be used as the confirmation assays. Six genetic markers on the bacterial genome (16S rRNA, rfbEO157, flicH7, eaeA, stx1 and stx2) will be examined by multiplex real-time PCR for identifying the strain's classification and virulence. Specific primers and probes will be designed and synthesized. The protocols of multiplex real-time PCR will be developed and optimized in this Phase I study. The combination of proposed micro-fluidic chip and real-time PCRs will provides a rapid sample preparation, high sensitivity and specificity of the assays that will bring us a novel approach to detect water-borne pathogens.