Bacteriophage-based live bacteria detection for rapid infection diagnosis.

Abstract

Bacterial bloodstream infections cause high morbidity and mortality. Although bacteria can be detected by various methods, culture methods are often used for the detection of live, accurate, reproducible, and selective bacterial identification. However, the culture method is time-consuming, and clinicians often start treatment immediately due to the long determination time. This reduces the bacterial density detectable by culture, and in some cases, makes determination difficult. To overcome this challenge, we propose a method that directly combines bacteriophage-based lysis with quantitative PCR (qPCR). This method enables the simple and rapid detection of bacteria without the need for pre-concentration or DNA extraction steps. Escherichia coli K12 (E. coli K12) was used as the model bacterium, and bacteria lysed by the E. coli K12-specific bacteriophage were detected using qPCR. The total analysis time was less than 3 h, and only live bacterial cells were selectively lysed. The method was also used to detect bacteria spiked into reference plasma samples, and bacterial DNA was detected via qPCR. The results obtained from the calibration graph created with cultured bacteria and the one created by spiking bacteria into reference plasma were consistent. The similarities between the calibration graphs from both methods were found to be in the range of 92-102.7 %. The LOD and LOQ values for bacteria spiked into reference plasma were calculated as 14.80 and 3.5x10³ CFU/mL, respectively.