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Modification of Sample Preparation to Differentiate Live, Injured and Dead Bacteria in Polymerase Chain Reaction Assay

Award No.: 
2010-ST-061-FD0001
Status: 
Complete
Principal Investigator: 
Teshome Yehualaeshet
PI Organization: 
Tuskegee University
Abstract: 
Accurate determination of live, damaged or injured, and dead bacteria is important in microbiology detection to avoid false alarm. Dead bacteria present after processes such as pasteurization or disinfection might present no hazard but still can be detected by Polymerase chain reaction (PCR). Injured cells are virulent but may or may not be detected by standard procedures. PCR offers a more rapid, sensitive method than culture-based techniques but the major limitation is lack of differentiating the DNA from live, injured or dead bacteria. In food matrices, DNA can be very stable and persist for extended periods of time. Therefore, further studies are imperative to develop a DNA-based assay to identify only viable organisms. Pretreatment of the cells with DNA-intercalating molecules, that can enter cells with compromised membranes, can be of value with end-point PCR by suppressing amplification of DNA from non-viable cells. The objectives of this proposal are: 1) to select and validate DNA-intercalating chemicals which are highly selective in penetrating only into dead bacterial cells with compromised membrane integrity, but not into viable including injured cells, 2) to determine the efficiency of the chemicals to bind the nuclear DNA and block the amplification of the targeted gene, and 3) to examine if the chemical works similar for samples from both culture and food samples. The major biothreat pathogens included in the experiment will be surrogate of Yersinia pestis (Yersinia pestis A122, Yersinia pestis KIM10+and Y. pseudotuberculosis) and Francisella tularensis (Francisella novicida). The pathogen will be isolated from culture and inoculated food matrix (milk). The isolated bacteria will be grouped to viable, injured (incremental heating) and dead cells (heat killed at 100 degrees C for 2 min). The bacteria will be pre-treated with the selected DNA intercalating chemicals. DNA will be extracted, quantified and subjected to real time PCR . A number of viability stains or dyes have been previously reported to identify viable cells. Bromide derivative dyes (like propidium monoazide and ethidium monoazide bromide) have been successfully used to differentiate viable and non-viable bacteria in conjunction with PCR. The irreversible binding of the dyes will prevent the DNA from being amplified in the PCR assay. The method presented here will overcome the currently missing knowledge gap between viability and direct PCR detection of DNA targets. Furthermore, this project will provide evidence that the chemical selected can be applied to a wide range of food-borne pathogens. The output of this novel approach will be extremely useful for the detection of only viable food-borne pathogens by PCR assay.
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