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Microwave-Accelerated Metal-Enhanced Fluorescence (MAMEF) Point-ofCare Test for the Detection of Chlamydia trachomatis Melendez JH1, Huppert J2, Hesse E2, Jett-Goheen M3, Quinn, N3, Gaydos CA3, Geddes CD1* 1Institute of Fluorescence and Department of Chemistry and Biochemistry, UMBC, Baltimore, MD, USA 2Cincinnati children’s hospital Medical Center, Cincinnati, OH, USA 3Johns Hopkins Medical Institutions, Division of Infectious Diseases, Baltimore, MD, USA *[email protected] Background Results Chlamydia trachomatis (CT) is the most commonly reported sexually-transmitted infections (STI). In 2011, there were over 1.2 million cases of chlamydia reported to the CDC. This infection has serious sequelae among women including 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 the results of a blinded study evaluating the performance of two microwave-accelerated metal-enhanced fluorescence (MAMEF) assays for detection of CT DNA. MAMEF combines the benefits of low power microwave acceleration (MA) to rapidly accelerate biological reactions to completion within seconds with those of metal-enhanced fluorescence (MEF) for amplification of the fluorescence signal. The overall prevalence of CT in this sample set was 17.7% 257 vaginal swabs from 245 subjects ranging in age from 14 to 22 years of age 88.6% of subjects were African American and the majority of subjects with a Chlamydia infection were African American (95.6%) Methods In the present study, detection of CT DNA is mediated by a two-step process. First, CT cells from vaginal samples are rapidly lysed and the DNA fragmented using lysing chambers composed of gold triangles deposited on glass slides and heated using conventional microwave irradiation (Figure 1). After a centrifugation step, detection of CT genomic DNA is carried in silver-covered wells known as fire-in-the-hole (Figure 1), 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 2. For increased sensitivity and specificity, two sets of probes targeting the 16S rRNA gene and the cryptic plasmid of CT were developed and tested. The flow chart showing the sequence of events and timeline for a MAMEF-based CT test are shown in Figure 1. Table 1. MAMEF results vs. NAATs using vaginal swabs MAMEF Assay Elution of vaginal swab in distilled water Microwave lysing (35 seconds) Centrifugation (3 minutes) Detection by MAMEF (< 5 minutes) Data analysis Sensitivity (%) Specificity (%) 16S rRNA and cryptic plasmid combined 73.3 92.9 16S rRNA 75.5 92.9 Cryptic plasmid 82.2 92.9 Table 2. Sensitivity and specificity of the MAMEF assays Lysing chamber Conclusions Silver-covered disposable wells for MAMEF-based CT detection Our lysing approach can effectively lyse cells and fragment DNA in 35 seconds Figure 1. Sequence of events and timeline for MAMEF-based CT detection Dry vaginal swabs are an effective collection method for MAMEF testing The cryptic plasmid MAMEF-based assay is more sensitive than the 16S rRNA assay Specificity of both MAMEF assays is very high Our CT assay can specifically detect over 80% of cases in less than 10 minutes MAMEF is an ultra-fast and sensitive approach for detection of CT Reference and acknowledgements 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. Figure 2. Probe-based CT detection using MAMEF 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. www.postersession.com