An amplification-free, nanopore-based nucleic acid detection platform has been demonstrated for rapid, rRNA sequence-specific detection of Neisseria gonorrhoeae (N. gonorrhoeae) at concentrations between 10-100 CFU/mL in human urine. Gonorrhea is a very common notifiable communicable disease in the US, and the rate of gonococcal infection continues to increase. Due to its prevalence and the emergence of drug resistant strains, gonorrhea has been listed as a top three public health threat by the CDC. To make timely treatment decisions and reduce medical costs, it is important to engineer rapid, robust, and inexpensive point-of-care (POC) diagnostic devices for pathogens such as N. gonorrhoeae in clinical samples. In our system, an amine-terminated peptide nucleic acid (i.e., an uncharged polyamide analog to DNA/RNA) probe complementary to N. gonorrhoeae 16S rRNA, is covalently bound to carboxyl-functionalized microbeads. When the neutral bead-probe conjugates hybridize with the target rRNA, a negatively charged bead-PNA-target complex is formed that exhibits mobility in an electric field. If the complex is directed to a smaller pore in a glass membrane, it will at least partially block it, resulting in a sustained drop in ionic current thereby signaling the presence of the target rRNA. The ability to detect N. gonorrhoeae over the range of 10 to 100 CFU/mL spiked in human urine was successfully demonstrated with sensitivity and specificity of ~98% and ~100%, respectively. No false positives were observed for the control group of representative background flora (E. coli, K. pneumoniae, and E. faecalis) at 1000 CFU/mL. Finally, N. gonorrhoeae at 50 CFU/mL was successfully detected against 1000 CFU/mL of background flora in urine. These results suggest that this new technology may serve as the basis for POC diagnostic systems for pathogenic bacterial species in clinical samples.

Amplification-free detection of 16S rRNA for point-of-care gonorrhea diagnosis

ACS Spring 2022

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