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Zweitzig D.R.,ZEUS Scientific Incorporated | Riccardello N.M.,ZEUS Scientific Incorporated | Sodowich B.I.,ZEUS Scientific Incorporated | O'Hara S.M.,ZEUS Scientific Incorporated
Nucleic Acids Research | Year: 2012

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 -11U 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. © The Author(s) 2012.


Sodowich B.I.,ZEUS Scientific Incorporated | Zweitzig D.R.,ZEUS Scientific Incorporated | Riccardello N.M.,ZEUS Scientific Incorporated | O'Hara S.M.,ZEUS Scientific Incorporated
BMC Microbiology | Year: 2013

Background: Antimicrobial Susceptibility Testing (AST) is a methodology in which the sensitivity of a microorganism is determined via its inability to proliferate in the presence of an antimicrobial agent. Results are reported as minimum inhibitory concentrations (MICs). The present study demonstrates that measurement of DNA polymerase activity via Enzymatic Template Generation and Amplification (ETGA) can be used as a novel means of determining the MIC of a microbe to an antibiotic agent much sooner than the current standardized method. Methods. Time course analysis of ETGA is presented from bacterial cultures containing antibiotic agents and compared to the end-point results of standard macrobroth method AST. Results: MIC determinations from ETGA results at 4, 6, and 22 hours are compared to the MICs from the standard method and the results are shown to be in agreement. Additionally, reliable AST analysis using ETGA can be performed on bacteria harvested directly from spiked blood cultures. Conclusions: AST analysis with ETGA is shown to be equivalent to AST analysis using gene-specific qPCR assays against the measured microbe. Future development of this novel method for performing AST in a clinical setting is discussed. © 2013 Sodowich et al.; licensee BioMed Central Ltd.

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