Rapid detection of urinary tract infections using isotachophoresis and molecular beacons
M. Bercovici, G.V. Kaigala, K.E. Mach, C.M. Han, J.C. Liao, J.G. Santiago
- Novel assay for rRNA focusing directly from cell lysate
- Co-focusing of molecular beacons and rRNA for (amplification free) sequence-specific detection of nucleic acids
- Detection of bacteria from cell lysate in less under 15 min in the range 1E6 to 1E8 cfu/ml.
Urinary tract infections
Infectious diseases caused by bacterial pathogens remain one of the most common causes of mortality worldwide. In the United States, UTI is the second most common infection affecting all patient demographics,with approximately 8 million visits to outpatient clinics and emergency departments, and 100,000 hospitalizations each year. Similarly, in the European Union (EU), UTI is the most common healthcare associated infection. Overall, annual medical expenditures in the EU, from hospital acquired infections alone, is estimated at € 5.48 billion, with UTI accounting for 27% of infections.
The standard culture-based approach for diagnosis of bacteria has a typical delay of 1-3 days from specimen acquisition to results. A rapid, inexpensive, definitive urine test capable of ruling out infection and identifying the pathogen with a high degree of accuracy at the point-of-care would be enormously beneficial in ensuring timely treatment, in eliminating empirical treatment, in relieving patient suffering, and in reducing costs and burden on the health care system.
Principle of the assay
We demonstrated and characterized a new assay for rapid detection of UTI using ITP and molecular beacons. We use on-chip ITP to selectively focus 16S rRNA and molecular beacons directly from bacterial lysate, and presented detection of E. coli in bacteria cultures as well as in patient urine samples in the clinically relevant range 1E6-1E8 cfu/mL.
Figure 1. (a) Schematic showing simultaneous isotachophoretic extraction, focusing, hybridization (with molecular beacons), and detection of 16S ribosomal RNA bound to a molecular beacon. Hybridization of the molecular beacon to 16S rRNA causes a spatial separation of its fluorophore and quencher pair resulting in a strong and sequence-specific increase in fluorescent signal. (b) Raw experimental image showing fluorescence intensity of molecular beacons hybridized to synthetic oligonucleotides using ITP. (c) Detection of oligonucleotides having the same sequence as the target segment of 16S rRNA. Each curve presents the fluorescence intensity in time, as recorded by a point detector at a fixed location in the channel (curves are shifted in time for convenient visualization). 100 pM of molecular beacons and varying concentrations of targets were mixed in the trailing electrolyte reservoir. The total migration (and hybridization) time from the on-chip reservoir to the detector was less than a minute.
Figure 2. Schematic of the lysis procedure. A pellet (from either bacterial culture or infected patient urine) is lysed at room temperature for 5 min. The lysate is diluted 10´ with buffer containing molecular beacons and MgCl2. The mixture of sample and beacons is then incubated at either room temperature or 60°C. 2 mL are introduced into the microfluidic chip reservoir.
Figure 3. Quantitative detection of E. coli 16S rRNA sequence from bacterial cultures, and bacterial detection in patient urine samples. The solid line is presented to aid visualization, and corresponds to a best linear fit on bacteria cultures results. 16S rRNA was extracted and focused with molecular beacons using ITP, and detected on chip. (a) Measured enhancement ratio for cultured bacteria samples (circles) and urine samples (squares) at clinically relevant bacterial concentrations. The top horizontal axis shows estimate molar concentration of target in the initial urine sample. The bacteria concentrations presented on the x-axis were evaluated separately using cell plating.
Bercovici, M., Kaigala, G.V., Mach, K.E., Han, C.M., Liao, J.C. & Santiago, J.G. Rapid Detection of Urinary Tract Infections Using Isotachophoresis and Molecular Beacons. Analytical Chemistry 83, 4110-4117 (2011).