Effect of lysing methods and their variables on the yield of Escherichia coli O157:H7 DNA and its PCR amplification

A number of cell lysing methods were quantitatively compared for the detection of Escherichia coli O157.H7 DNA by the polymerase chain reaction. These methods included heating cell suspensions at 94.5 or 99.5 degrees C in distilled HsO, PCR buffer, sodium dodecyl sulfate, Triton X-100 and the use of lysozyme, proteinase K, and lysozyme in combination with proteinase K. The yield of DNA was affected by the age of the culture. A 4 h old culture resulted in a higher yield of released DNA than did a 24 h old culture. The maximum yield of DNA was obtained after treatment of cells with lysozyme (2 mg/ml) at 37 degrees C for 15 min followed by treatment with proteinase K (0.8 mg/ml) at 55 degrees C for 45 min with subsequent heating at 99.5 degrees C for 10 min. The highest amplification of released DNA was obtained by lysing cells either by heating at 99.5 degrees C for 10 min in PCR buffer or treating the cells with only proteinase K (0.8 mg/ml) at 55C for 15 min followed by heating at 99.5 degrees C for 10 min. These results reflect the fact that the presence of either lysozyme or proteinase K in PCR reactions even after their denaturation at 99.5 degrees C partially inhibits the PCR reaction. Maximum amplification can therefore be expected to occur following enzymatic treatment of cells only after the target DNA has undergone purification. (C) 1998 Elsevier Science B.V. All rights reserved.