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Ultra Rapid and Sensitive Detection of Neisseria gonorrhoeae and Antimicrobial Resistance Susceptibility Charlotte A. 2 Gaydos , 1 Geddes * and Chris D. of Fluorescence and Department of Chemistry and Biochemistry, UMBC, Baltimore, MD 2Johns Hopkins Medical Institutions, Division of Infectious Diseases, Baltimore, MD *[email protected] Background Gonorrhea (GC) is the second most commonly reported bacterial sexually-transmitted infection (STI) worldwide. In 2012, 334,826 cases of GC were reported to the CDC. This infection has serious sequelae among women: PID, tubal factor infertility, chronic pelvic pain, and ectopic pregnancy. Gonorrhea infections can also increase HIV transmission 3-5 fold. The most vulnerable groups for STIs and their sequelae are women and infants, adolescents, racial and ethnic minorities, and persons in correctional facilities. These “Special Focus Populations” bear most of the burden of morbidity from gonorrhea. Often persons who present to a clinic for STI testing never return to the clinic to receive their STI test results. Thus the availability of a point-of-care test (POCT), which can be resulted to them immediately is highly desirable. In the present study, we report the development of an ultra rapid and sensitive assay for detection of GC. The detection assay is based on microwaveaccelerated metal-enhanced fluorescence (MAMEF), which combines the principles of MEF (Figure 1) with low power microwave irradiation to accelerate the kinetic processes of target-probe recognition and binding (Figure 2). Results 1. Ultra-rapid lysis of bacterial cells 105 104 103 102 3. MAMEF-based detection of GC DNA 101 MAMEF NAAT - + 5 1 - 1 13 Figure 4. Rapid lysis of GC bacterial cells following a 10-second microwave irradiation using the lysing chamber (Figure 3A). Colony counts are displayed as colony forming units (CFU/mL) For DNA sensing, a 200 µL solution of the target DNA or unknown sample is dropped onto the assay surface, which contains the anchor probe, combined with an excess of the fluorescent probe. The application of low power microwaves for 3 minutes, drives the hybridization to completion. Figure 2. MAMEF-based GC detection Figure 3. A)lysing chamber. B) Silvered 96-well plate. -S-AGTCTATAGCAGT 7 Microfluidic lysing device 1 2 3 3 4 1 Microfluidic-based cell lysis and DNA fragmentation Original position of dark line 8 1 – Laser 2 – Prism 3 – Thin gold film 4 – Camera 5 – Computer 6 – Sample 7 – Anchor probe 8 – Fully complementary sequence 5 6 2 100 bp Detection of GC target DNA is mediated by a fluorescent probe-based approach, which is enhanced by the intrinsic properties of the MAMEF approach. 4. Detection of resistance-associated mutations Figure 1. Metal-enhanced fluorescence (MEF) Rapid lysis of bacterial cells and fragmentation of bacterial genomic DNA is carried out in a lysing chamber (Figure 3A) using conventional microwave irradiation. The lysing chambers are composed of gold triangles deposited on glass slides, and a self-adhesive silicon isolators (D = 30 mm) placed over the gold triangles to create a lysing chamber. Immediately following the lysis step, detection of GC genomic DNA is carried out in silvered plate (Figure 3B), which have been shown to enhance the fluorescence signal. Detection of target genomic DNA is mediated by the complementary binding of two probes to the target sequence as shown in figure 2. The assay is highly sensitive and no signal was detected in the absence of target DNA. Testing of 20 individual dry vaginal swabs by MAMEF 2. Fragmentation of genomic DNA Methods Rapid lysis of cells, fragmentation, and detection of target DNA can be carried out in less than 10 minutes. + TCAGATATCGTCA 1Institute Johan H. 1 Melendez , θ Angular shift 4 Fragmented DNA 1 – DNA ladder 2 – Unlysed GC cells 3 – Lysed GC – lysing chamber 4 – Lysed GC – microfluidic lysing Conclusions Surface Plasmon Resonance (SPR) for detection of mutant sequences Reference and acknowledgements Our lysing approach can effectively lyse cell and fragment DNA. Zang, Y.; Agreda, P.; Kelly, S.; Gaydos, C.; Geddes, C. D. Development of a microwave-accelerated metal –enhanced fluorescence 40 second, 100 cfu/mL point of care assay for the detection of Chlamydia trachomatis. IEEE Trans BioMed Eng. 2011. 58:781-784. Dry vaginal swabs are an effective collection method for MAMEF testing. Melendez, J. H.; Huppert, J. S.; Jett-Goheen, M.; Hesse, E. A.; Quinn, N.; Gaydos, C. A.; Geddes CD. Blind evaluation of the microwave-accelerated metal-enhanced fluorescence ultra-rapid and sensitive Chlamydia trachomatis test by use of clinical samples. J Clin Microbiol. 2013, 51, 2913-20. Sensitivity and specificity of the MAME assay is high. The authors would like to thank The Meyerhoff Graduate Fellows Program, The Institute of Fluorescence and the Department of Chemistry and Biochemistry, University of Maryland Baltimore County for support. Financial support from the NIH / NIAID MARCE – Mid-atlantic Regional Center of Excellence for Biodefense and Emerging Infectious Diseases (NIAID/NIH) 2 U54 AI057168-06 and NIH/NIBIB 1U54 EB007958-4 and NIH/NIGMS T32GM066706 are also gratefully acknowledged. MAMEF is an ultra-fast and sensitive approach for detection of gonorrhea.