DNA AND RNA EXTRACTIONFROM FECES FOR DNA PROBE ANALYSIS

NUCLEIC ACIDS CAN BE RAPIDLY AND EFFICIENTLY EXTRACTED FROM AQUEOUS SOLUTIONS INTO BUTANOL CONTAINING A CATIONIC DETERGENT. SUBSEQUENTLY, THESE NUCLEIC ACIDS CAN BE TRANSFERRED FROM THE CATIONIC DETERGENT/BUTANOL PHASE INTO A SECOND AQUEOUS PHASE CONTAINING HIGH CONCENTRATIONS OF SALT. NUCLEIC ACIDS TRANSFERRED BETWEEN THE PHASES ARE STABLE AND ACCESSIBLE, AND THEY CAN BE ANALYZED DIRECTLY IN THE SECOND AQUEOUS PHASE USING DNA PROBES. THE PURPOSE OF THIS PROJECT IS TO DEMONSTRATE: (1) THE RAPID AND EFFICIENT EXTRACTION OF TOTAL NUCLEIC ACIDS FROM FECAL SAMPLES USING THE CATIONIC DETERGENT/BUTANOL SYSTEM, AND (2) THE DETECTION AND SPECIFIC IDENTIFICATION OF CAMPYLOBACTER JEJUNI AND C. COLI NUCLEIC ACIDS IN THE FECAL EXTRACTS USING DNA PROBES. THE LONG-TERM OBJECTIVES OF THISPROJECT ARE TO DEVELOP: (1) A SERIES OF RAPID DNA PROBE TESTPRODUCTS FOR THE DETECTION AND SPECIFIC IDENTIFICATION OF IMPORTANT DIARRHEAL PATHOGENS IN FECES; AND (2) ADDITIONAL HIGHLY SENSITIVE DNA PROBE TEST PRODUCTS FOR THE DETECTION AND SPECIFIC IDENTIFICATION OF PATHOGENS IN OTHER COMPLEX SAMPLES, SUCH AS SPUTUM, BLOOD, FOODS, OR WATER SUPPLIES.