Multiplex Assay Optimized for Sexually Transmitted Bacteria

A relatively new isothermal nucleic acid-amplification technology has been developed to detect the most insidious of all sexually transmitted diseases of bacterial etiology.

Thermophilic helicase dependent amplification (tHDA) employs helicase to unwind double-stranded DNA at constant temperature, has now been optimized to detect Chlamydia trachomatis and Neisseria gonorrhoeae.

Scientists at the Research and Development department at QIAGEN Inc., (Gaithersburg, MD, USA) combined tHDA with sequence-specific sample preparation on magnetic beads and homogeneous endpoint fluorescence detection using dual-labeled probes. This four-plex tHDA assay was applied to the detection of two genes on C. trachomatis and a multicopy gene on N. gonorrhoeae in the presence of an internal control.

Gene targets for multiplex tHDA assay included C. trachomatis genomic DNA olfactory marker protein (omp) gene, C. trachomatis cryptic plasmid gene, and NG multicopy opacity protein (opa) gene. Several different strand displacement polymerases were evaluated during the tHDA assay optimization. The assay showed high analytical sensitivity and specificity of simultaneous detection of both bacteria and is compatible with a wide variety of sample types and media.

The performance of a tHDA system may be further improved as tHDA technology currently has some limitations. Nonspecific amplification and primer-dimer formation are more pronounced in tHDA than in polymerase chain reaction (PCR), making multiplexing with the tHDA system more challenging. Nonspecific amplification of tHDA products tends to occur during tHDA reaction setup at room temperature.

The isothermal reaction conditions and homogeneous endpoint detection utilized in the assay were well suited for laboratory automation and high-throughput screening applications as well as for point-of-care testing. The system is still under development but has already proved that the multiplex isothermal amplification assay with endpoint fluorescent detection is a simple and economic alternative to the real-time PCR. The study was published in the December 2011 edition of the journal Diagnostic Microbiology and Infectious Disease.

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