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Optimization and Clinical Testing of a Microwave-Accelerated Metal-Enhanced
Fluorescence (MAMEF) Assay for the Detection of Chlamydia trachomatis
Melendez
1
JH ,
Gaydos
2
CA ,
Geddes
1
CD *
1Institute
of Fluorescence and Department of Chemistry and Biochemistry, UMBC, Baltimore, MD 21202, USA
2Johns Hopkins Medical Institutions, Division of Infectious Diseases, Baltimore, MD 21205, USA
*[email protected]
Background
Results
In the present study, detection of CT DNA is mediated by a two-step process. First, CT
cells are rapidly lysed and the DNA fragmented using lysing chambers composed of
gold or aluminum triangles deposited on glass slides and heated using conventional
microwave irradiation (Figure 2). After a centrifugation step, detection of CT genomic
DNA is carried in silver island-covered wells known as fire-in-the-hole plates (Figure 2)
which have been shown to enhance the fluorescence signal. Detection of genomic DNA
is mediated by the complementary binding of two probes to the target sequence as
shown in figure 1. For increased sensitivity and specificity, two sets of probes targeting
the 16S rRNA gene and the Cryptic plasmid of CT has been designed and optimized.
2
3
4
5
Figure 3. A) Fragmentation of CT DNA following 15 seconds microwave irradiation 1) DNA
Ladder; 2) gold triangles (50% power); 3) aluminum triangles (50% power); 4) gold triangles
70% power); 5) aluminum triangles (70% power). B) MAMEF signals of samples # 4 and # 5
from Figure A showing identical fluorescence signal suggestive of similar concentration of DNA.
Au = gold; Al = aluminum.
2. Rapid and sensitive detection of CT DNA
Fluorescence Intensity (AU)
Methods
1
Fluorescence Intensity (AU)
Chlamydia trachomatis (CT) is the most commonly reported sexually-transmitted
infections (STI). In 2009, there were 1.2 million cases of chlamydia reported to the CDC.
This infection has serious sequelae among women: PID, tubal factor infertility, chronic
pelvic pain, and ectopic pregnancy. Chlamydia infections can increase HIV transmission
3-5 fold.
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 which can be resulted to
them immediately is highly desirable. In the present study, we report on the optimization
of an ultra-rapid and sensitive point of care assay for detection of CT, and discuss
clinical testing with vaginal specimens.
1. Efficiency of lysing triangles
10^6 ifu/ml
10^5 ifu/ml
10^4 ifu/ml
10^3 ifu/ml
10^2 ifu/ml
10^1 ifu/ml
0 ifu/ml
800
600
400
200
0
550
575
600
625
1000
800
600
400
200
0
650
0
10^1
10^2
Wavelength (nm)
10^3
10^4
10^5
10^6
ifu/ml
3. Performance of MAMEF in GenProbe Media and using dry swabs
MAMEF
MAMEF
Figure 1. Probe-based detection using rapid MicrowaveAccelerated Metal-Enhanced Fluorescence (MAMEF)
+
-
21
4
+
-
+
19
13
-
7
11
NAAT
NAAT
• Vaginal swabs rehydrated in 2 mL of distilled
water
+
5
15
Dry swabs
Sensitivity = 84%
GenProbe media
Sensitivity = 58%
Testing of two individual sample sets from 95 individuals.
Dry swabs – 45 samples and GenProbe media – 50 samples.
Conclusions
• Lysing chambers used for cell
lysing and DNA fragmentation
 Cell lysis and DNA fragmentation is achieved in 30 seconds, and gold and
aluminum lysing triangles are equally effective.
• 5 minute
centrifugation step
 Dry vaginal swabs are an effective collection method and sensitivity of the
MAMEF assay is very high using dry swabs rehydrated in water.
 Rapid lysis of bacterial cells, fragmentation and detection of genomic DNA can
be carried out in less than 10 minutes.
Reference and acknowledgements
Fire-in-the-hole plate used for DNA
detection. Detection in the wells is
mediated by probes as shown in figure 1.
Figure 2. Workflow for the ultra-rapid and sensitive
detection of CT DNA
RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com
Zang Y, Agreda P, Kelly S, Gaydos C, Geddes CD. 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 58:781-784, 2011.
The authors would like to thank The Meyerhoff Graduate Fellowship 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 - Midatlantic Regional Center of Excellence for
Biodefense and Emerging Infectious Diseases (NIAID/NIH) 2 U54 AI057168-06 and NIH/NIBIB 1U54
EB007958-4 are also gratefully acknowledged.