Firefly luciferase ATP assay as a screening method for bacteriuria

Abstract

A rapid (15 min) test for bacteriuria based on firefly luciferase analysis of bacterial ATP has been evaluated in 2,018 clinical urine specimens. The test procedure involves removal of nonbacterial ATP by treatment of urine with Triton X-100 and apyrase, extraction of bacterial ATP by boiling, and bioluminescent analysis of bacterial ATP by firefly luciferase, using a luminometer. For comparison, the widely used nitrite test was included in the study as an example of an alternative rapid chemical test. The test was set up to distinguish between specimens yielding greater than 10(5) CFU/ml and specimens yielding less than 10(5) CFU/ml. A level of 13.5 nM ATP was chosen to define the limit between negative and positive results. At this discriminatory level, 92% of specimens yielding greater than 10(5) CFU/ml and 88% of specimens yielding less than 10(5) CFU/ml were correctly classified with the luciferase method, whereas corresponding figures for the nitrite test were 55 and 99%, respectively. Of the 12% false luciferase positives, 20% were shown to contain greater than 10(5) CFU/ml on prolonged incubation, thus reducing the false-positive rate to 10%. Of the 8% false luciferase negatives, 65% had low levels of CFU in the range of 10(5) to 10(6).