Multiplex isothermal helicase-dependent amplification assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae

• Published in: Diagnostic microbiology and infectious disease 2011-12, Vol.71 (4), p.354-365
• Main Authors: Doseeva, Victoria, Forbes, Thomas, Wolff, John, Khripin, Yuri, O'Neil, Dominic, Rothmann, Thomas, Nazarenko, Irina
• Format: Article
• Language: English
• Subjects: Bacteriological Techniques - methods; Bacteriology; Biological and medical sciences; Chlamydia Infections - diagnosis; Chlamydia Infections - microbiology; Chlamydia trachomatis; Chlamydia trachomatis - enzymology; Chlamydia trachomatis - genetics; Chlamydia trachomatis - isolation & purification; DNA Helicases - metabolism; Endpoint fluorescence detection; Fundamental and applied biological sciences. Psychology; Gonorrhea - diagnosis; Gonorrhea - microbiology; Humans; Infectious Disease; Infectious diseases; Internal Medicine; Medical sciences; Microbiology; Microspheres; Miscellaneous; Molecular Diagnostic Techniques - methods; Multiplex helicase-dependent amplification; Neisseria gonorrhoeae; Neisseria gonorrhoeae - enzymology; Neisseria gonorrhoeae - genetics; Neisseria gonorrhoeae - isolation & purification; Nucleic Acid Amplification Techniques - methods; Oligonucleotide Probes - genetics; Point-of-Care Systems; Sensitivity and Specificity; Sequence-specific sample preparation
• ISSN: 0732-8893; 1879-0070
• DOI: 10.1016/j.diagmicrobio.2011.08.021

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.