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.
The Agilent 2100 Bioanalyzer can characterize in vitro RNA transcripts for their integrity, purity, concentration, and size. The results are comparable to denatured agarose electrophoresis with ethidium bromide staining and UV spectrophotometry combined. In this report, we describe our strategy for validating this method following the International Conference on Harmonization guidelines. The assay has a linear range of quantitation between 500 and 25 ng/microL. Quantitation accuracy is within +/-20% of measurements produced from spectrophotometry and sizing accuracy is within +/-7% based on theoretical sizes. Concentration and sizing measurements within a single assay produce RSDs that are <10 and <2%, respectively, indicating good precision. The method also maintains a tolerable precision when altering operator, day, and reagent kit lot. The RSD for quantitation is
Bloodstream infections (BSIs) caused by bacteria and fungi are associated with significant morbidity and mortality. Currently, blood culture is the gold standard for confirming a suspected BSI, but has the drawback of lengthy time-to-detection (TTD) required for indicating the presence of microbes. Detection of conserved microbial nucleic acid sequences within blood culture samples via PCR has been demonstrated to offer potential for reducing the TTD of BSI; however, these approaches have various other limitations. We report a novel approach toward rapid detection of microbes from simulated BSI via differential hematopoietic cell lysis followed by enzymatic template generation and amplification (ETGA)-mediated measurement of microbial DNA polymerase extension activity. The differential cell lysis procedure effectively reduced the level of detectable DNA polymerase extension activity associated with human-derived hematopoietic cells present in blood culture samples taken from healthy donors. After treatment with the differential cell lysis procedure, the ETGA assay detected a panel of clinically prevalent bacteria and Candida albicans from spiked blood culture samples. The ETGA blood culture method also reduced by threefold the TTD required for simulated BSI, compared with a continuous-monitoring blood culture instrument. In summary, these findings demonstrate the feasibility of an innovative approach toward a rapid, sensitive, and universal screen for microbes within blood culture samples. Potential for clinical application and automation are also addressed.
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