FECAL-COLIFORMS IN SOIL DETECTED BY POLYMERASE CHAIN-REACTION AND DNA-DNA HYBRIDIZATIONS

The application of sewage sludge on agricultural lands is a common practice in the USA. The survival of pathogens and indicator organisms added to soil via sludge needs to be determined. A rapid method utilizing polymerase chain reaction (PCR) and a gene-specific probe was used to detect low numbers of fecal coliforms in soil. Two 23-base primers were designed from a region of the lamB gene that codes for an outer-membrane protein present specifically in fecal coliforms. Use of these primers for PCR amplification of a 179-bp fragment of lamB allowed specific detection of fecal-coliform indicator organisms. Sensitivity of amplification from whole-cell lysates of pure cultures of Escherichia coli using detection via ethidium bromide was 10(4) colony-forming units (CFUs). When detection of these cell lysates was via a lamB gene-specific probe, a total of 4 x 10(2) CFUs could be detected from these pure cultures. When genomic deoxyribonucleic acid (DNA) preparations were made from these pure cultures, 100 ag (10(-18)) of DNA was detectable. Following the inoculation of E. coli into soil, cells were extracted with CaCl2, and concentrated and purified by sucrose density centrifugation. The DNA obtained by lysing the cells was amplified using a new "double PCR" 50-cycle protocol. Escherichia coli were detected in Brazito sandy loam (mixed, thermic Typic Torripsamment) and Pima silty clay loam (fine-silty, mixed (calcareous), thermic Typic Torrifluvent). Using a 5' end-labeled lamB-specific probe to detect PCR products, one CFU of E. coli was detectable in 1 g of soil. One PCR-amplificable CFU g-1 would be equivalent to many copies of amplificable target DNA, due to the presence of dead and lysed cells along with cells containing more than one copy of the genome. Sample processing and PCR amplification can be completed within 7 to 8 h. Thus, the use of PCR for sensitive detection of introduced bacteria in environmental soil samples is possible.