Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

Abstract

During the past 50 years, in vitro measurement of DNA polymerase activity has become an essential molecular biology tool. Traditional methods used to measure DNA polymerase activity in vitro are undesirable due to the usage of radionucleotides. Fluorescence-based DNA polymerase assays have been developed; however, they also suffer from various limitations. Herein we present a rapid, highly sensitive and quantitative assay capable of measuring DNA polymerase extension activity from purified enzymes or directly from microbial lysates. When tested with purified DNA polymerase, the assay detected as little as 2 × 10 −11 U of enzyme (∼50 molecules), while demonstrating excellent linearity ( R 2 = 0.992). The assay was also able to detect endogenous DNA polymerase extension activity down to less than 10 colony forming units (cfu) of input Gram-positive or Gram-negative bacteria when coupled to bead mill lysis while maintaining an R 2 = 0.999. Furthermore, preliminary evidence presented here suggests that DNA polymerase extension activity is an indicator of microbial viability, as demonstrated by the reproducibly strong concordance between assay signal and bacterial colony formation. Together, the innovative methodology described here represents a significant advancement toward sensitive detection of potentially any microorganism containing active DNA polymerase within a given sample matrix.

Feasibility study demonstrating that enzymatic template generation and amplification can be employed as a novel method for molecular antimicrobial susceptibility testing

“…The bead-mill tubes were subsequently milled for bacteria lysis, incubated at 37°C for 20 minutes followed by 95°C for 5 minutes (to terminate the reaction), spun down, and stored at -20°C prior to analysis. At the final time point, ETGA reagent and positive controls [21] were performed alongside the samples.…”

“…The subsequent qPCR detection signal is directly proportional to the amount of substrate extended, which is proportional of the amount of microbial DNA polymerase extension activity present, and this is proportional to the amount of viable proliferating bacteria present from culture. Complete details regarding the ETGA assay have been previously described [21] a hyperlink is provided [http://nar.oxfordjournals.org/content/40/14/e109.full.pdf+html?sid=ea56a354-4e91-4515-aec8-ccdc5acfb438]. …”

“…ETGA readout by qPCR was performed by adding 4 μL of each sample into a reaction well containing 27.2 μL of qPCR reaction mix which has been previously described [21]. …”

“…The gene targets for the S. aureus and E. coli -specific qPCR assays are nuc and uidA respectively. The primer and probe sequences for these assays have been previously reported [21]. All qPCR analysis was performed on a Roche LightCycler 480 II system (Roche Applied Science, Indianapolis, IN).…”

“…Our group has previously reported a novel methodology termed Enzyme Template Generation and Amplification (ETGA) that enables universal, sensitive and quantitative measurement of bacterial proliferation via measurement of endogenous DNA polymerase activity [21]. In this report, we demonstrate that molecular AST and MIC determination can be performed via ETGA-mediated monitoring of DNA polymerase activity.…”