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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.