Methods for measuring enzyme activity useful in determining cell viability in non-purified samples

A method for detecting the presence of a bacterium or a fungus in a sample, wherein DNA polymerase activity is detected as an indicator of the presence of said bacterium or fungus in said sample, which method comprises:

• (a) preparing the sample using a differential cell lysis sample preparation method, thereby allowing for only bacterium or fungi derived DNA polymerase activity to modify a DNA polymerase substrate;
• (b) contacting the prepared sample of (a) with a nucleic acid molecule which acts as a substrate for DNA polymerase activity in the prepared sample, wherein the substrate is a partially double-stranded DNA substrate consisting of a sense strand DNA oligonucleotide and an antisense strand DNA oligonucleotide, wherein the sense strand DNA oligonucleotide and the antisense strand DNA oligonucleotide overlap to form a double-stranded region and a single-stranded portion of the antisense strand acts as a template with the sense strand of the double-stranded region acting as a primer to create an extension product in the presence of DNA polymerase activity;
• (c) incubating the contacted prepared sample and substrate of (b) under conditions suitable for DNA polymerase activity to generate the extension product from the substrate;
• and
• (d) amplifying at least a portion of the extension product of (c) to generate an amplicon and detecting the amplicon, thereby indicating the presence of the bacterium or fungus.