Sample preparation module for bacterial lysis and isolation of DNA from human urine

Abstract

Silica impregnated polymer monolithic columns may provide a simple method for lysing and extracting DNA from bacteria inside of microfluidic chips. Here we use Escherichia coli as a test organism for a point of care thermoplastic microfluidic module designed to take in a urine sample, mix it with lysis buffer, and perform a hybrid chemical/mechanical lysis and solid phase extraction of nucleic acids from the sample. To demonstrate proof-of-concept, we doped human hematuric urine samples with E. coli at concentrations ranging from 10(1)-10(5) colony-forming units/mL (CFU/mL) to simulate patient samples. We then performed on-chip lysis and DNA extraction. The bacterial DNA was amplified using real-time PCR demonstrating lysis and isolation down to 10(1) CFU/mL. Results were comparable to a commercial kit at higher concentrations and performed better at recovering DNA at lower concentrations.

Fig. 1

(a) SEM Image of a lysis/solid-phase extraction monolith taken at ×10,000 magnification. The spheres in the image are the silica particles, (b) CAD drawing of chip components, and (c) a photograph of an array of monolith filled channels. Only the front most channel has nanoports in place

Fig. 2

Pore size distribution determined using image analysis of scanning electron micrographs of representative monoliths

Fig. 3

Summary of lysis quantification experiments. DNA concentration was measured after samples were run over a column in the presence of both low and high salt buffer solutions. The positive control is the Qiagen kit. The negative control is a sample mixed with NaCl buffer and left on the bench for the duration of the experiment. The experimental samples and the negative controls were gently filtered (0.2 μm filter) and ethanol precipitated to remove any fluorescent cell debris prior to fluorescent quantification. *p<0.001

Fig. 4

Summary of amplification threshold values for E. coli and whole blood spiked urine samples. A lower threshold value indicates more DNA was present in the extracted sample. F1 refers to the first 70 μl sample collected after elution and F2 refers to the second. Elutions that never amplified are not included in this data. 22/27 (81%) of the microchannels produced amplifiable results