title: Multiplex isothermal helicase-dependent amplification assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae.
abstract: Thermophilic helicase dependent amplification (tHDA), which employs helicase to unwind double-stranded DNA at constant temperature, is a relatively new isothermal nucleic acid amplification technology. In this study, the development and optimization of a 4-plex tHDA assay for detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are described. tHDA is combined with sequence-specific sample preparation on magnetic beads and homogeneous endpoint fluorescence detection using dual-labeled probes. This 4-plex tHDA assay was applied to the detection of 2 genes on CT and a multicopy gene on NG in the presence of an internal control. The assay showed high analytical sensitivity and specificity of simultaneous CT/NG detection and is compatible with a wide variety of sample types and media. The isothermal reaction conditions and homogeneous endpoint detection utilized in this assay are well suited for laboratory automation and high-throughput screening applications as well as for point-of-care testing.
authors: QIAGEN North America, Research and Development, QIAGEN Inc., Gaithersburg, MD 20878, USA. victoria.doseeva@qiagen.com
authors: Victoria
authors: V
authors: Doseeva
authors: Thomas
authors: T
authors: Forbes
authors: John
authors: J
authors: Wolff
authors: Yuri
authors: Y
authors: Khripin
authors: Dominic
authors: D
authors: O'Neil
authors: Thomas
authors: T
authors: Rothmann
authors: Irina
authors: I
authors: Nazarenko
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods; D002690:Chlamydia Infections / Q000175:diagnosis* / Q000382:microbiology; D002692:Chlamydia trachomatis / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D004265:DNA Helicases / Q000378:metabolism; D006069:Gonorrhea / Q000175:diagnosis* / Q000382:microbiology; D006801:Humans; D008863:Microspheres; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009344:Neisseria gonorrhoeae / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D019095:Point-of-Care Systems; D012680:Sensitivity and Specificity
chemical_list: D015345:Oligonucleotide Probes; D004265:DNA Helicases