Ultra Rapid and Sensitive Detection of Neisseria Gonorrhea and Antimicrobial Resistance Susceptibility

Background: Neisseria gonorrhoeae (GC) is the second most commonly reported notifiable disease in the United States.  Rapid and accurate identification of GC clinical isolates is critical for control of gonorrhea.  Rapid detection of resistance markers is crucial to guide therapeutic options.  While nucleic acid amplification tests (NAATs) are effective, there is still an urgent need for rapid and sensitive molecular methods which could be easily employed in the field as point-of-care (POC) tests.

Methods: A microwave-accelerated metal-enhanced fluorescence (MAMEF) assay targeting the Por A gene of GC has been designed for the rapid and sensitive detection of GC DNA.  Detection is mediated by a two-step process involving the release of GC DNA from bacterial cells through a rapid and low-cost microwave-based approach followed by detection of DNA with an ultra-sensitive and rapid MAMEF assay. Two independent approaches to detect ciprofloxacin-sensitive GC strains have been developed.

Results: Using a microwave-accelerated, microfluidic-based lying approach we have successfully lysed GC cells and fragmented the DNA.  Detection of GC DNA is mediated after a 3 minutes microwave-enhanced fluorescence reaction.  Overall, lysing and detection of GC DNA can be achieved in less than 10 minutes using this microwave-enhanced approach.  Detection of mutations associated with ciprofloxacin resistance has been mediated through a “loss of wildtype” approach by targeting GC GyrA and ParC wildtype sequences; a process which can be carried out in less than 1 minute following DNA extraction. 

Conclusions: We have successfully detected NG DNA in less than 10 minute using an ultra rapid lysing and detection method based on metal-enhanced fluorescence.  The rapid nature of this platform coupled with rapid detection of mutations associated with antimicrobial resistance could be used for rapid detection of antimicrobial-resistant GC in the clinical setting.   Additional studies are ongoing towards the development of this platform as a POC test.