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Sensitive and Rapid Detection of Chlamydia trachomatis by Recombinase Polymerase Amplification Directly from Urine Samples  Katrin Krõlov, Jekaterina.

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Presentation on theme: "Sensitive and Rapid Detection of Chlamydia trachomatis by Recombinase Polymerase Amplification Directly from Urine Samples  Katrin Krõlov, Jekaterina."— Presentation transcript:

1 Sensitive and Rapid Detection of Chlamydia trachomatis by Recombinase Polymerase Amplification Directly from Urine Samples  Katrin Krõlov, Jekaterina Frolova, Oana Tudoran, Julia Suhorutsenko, Taavi Lehto, Hiljar Sibul, Imre Mäger, Made Laanpere, Indrek Tulp, Ülo Langel  The Journal of Molecular Diagnostics  Volume 16, Issue 1, Pages (January 2014) DOI: /j.jmoldx Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2 Figure 1 Assay sensitivity determined as the lowest detectable template amount and assay specificity determined as cross-reactivity with gDNA from other species. A: Assay sensitivity was analyzed by performing C. trachomatis CDS2-specific RPA with a pGL3-CDS2 template at the indicated copy number per reaction, with the C. trachomatis gDNA (CT gDNA) template at the indicated amount per reaction and with total DNA purified from the indicated amount of C. trachomatis–positive urine (total DNA CT pos I or II). B: Human GAPDH-specific RPA sensitivity analysis was performed using pure human gDNA (HS gDNA) at the indicated amount per reaction and with total DNA purified from the indicated amount of C. trachomatis–positive urine. C: C. trachomatis CDS2 and human GAPDH detection assay specificity was analyzed using 20 pg of M. genitalium, 20 pg of C. trachomatis, 20 pg of U. urealyticum, 100 pg of N. gonorrhoeae, 100 pg of E. coli, and 1 ng of H. sapiens gDNA. All amplification reactions were performed at 38°C for 30 minutes. The reaction products were analyzed on PCRD-2 lateral flow strips, where T indicates the test band or presence of the biotin-FAM–labeled reaction product and C indicates the lateral flow control band. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3 Figure 2 Minimum reaction time required for product formation at template concentration near detection limit. C. trachomatis–specific assay was performed using 500 or 100 pGL3-CDS2 template copies per reaction. The reaction was terminated after 0, 5, 10, 15, and 20 minutes, respectively, using 10 minutes of incubation at 50°C. The reaction products were analyzed on PCRD-2 lateral flow strips, where T indicates the test band or presence of the biotin-FAM–labeled reaction product and C indicates the control band. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4 Figure 3 C. trachomatis detection directly from heat-treated urine samples. A: CDS2 detection from 5 μL of C. trachomatis–positive or –negative urine samples subjected to 5 minutes of incubation at 90°C. B: The sensitivity of the assay was established as a specific detection of CDS2 and GAPDH using a dilution series of C. trachomatis–positive heat-treated urine (sample 1) as a template. The reaction products were analyzed on PCRD-2 lateral flow strips, where T indicates the test band or presence of the biotin-FAM–labeled reaction product and C indicates the lateral-flow control band. C: General layout of C. trachomatis detection assay with indicated steps, required time, and temperature. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

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