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A multiplexed isothermal amplification assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae

Abstract number: P665

Objectives: Our objective was to develop a sensitive, highly specific, multiplexed assay for the detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). We have combined Qiagen's proprietary Hybrid Capture® (HC) technology for sample processing with isothermal helicase dependent amplification (tHDA) and endpoint fluorescence detection to develop a multiplex assay for the detection of CT and NG in clinical samples. This product is currently at a research prototype stage and is not yet commercially available.

Methods: Hybrid Capture sample preparation involved hybridization of sequence-specific oligoribonucleotide probes to target DNA. RNA:DNA hybrids were captured by HC antibodies conjugated to magnetic beads, which can be placed directly into the amplification reaction without prior elution.

Captured target is amplified by tHDA, an isothermal technology which utilizes a thermostable helicase to unwind double-stranded DNA, followed by priming of the single-stranded target and extension by DNA polymerase. This amplification technology eliminates the need for thermocycling. tHDA products are detected in a closed-tube format by endpoint fluorescence detection with dual-labeled probes.

Our model assay has two CT amplification targets, including the cryptic plasmid and the outer membrane protein (omp) gene. Dual targets ensures against deletion or mutation of the target sequence causing false negative results. NG amplification target is the outer membrane opacity protein (opa), a multi-copy gene.

Results: Using this research prototype test system, we detected as little as two CT elementary bodies, and less than ten NG cells per mL of sample. Targets were detectable in multiplex, and each target was detectable in the presence of an excess (105) of the other. All CT serovars A-K, and L1-L3 were amplified and detected at equivalent sensitivity. The method is suitable for the processing of samples in many different media, among them: urine, PreservCyt®, STM and SurePath. The clinical utility of this assay was demonstrated on a limited number of clinical samples.

Conclusions: The combination of sequence-specific sample preparation and isothermal target amplification allows for a multiplex CT/NG assay which delivers high analytical sensitivity and specificity. The combination of short turn-around time (under three hours), isothermal reaction conditions, and closed-tube format make the assay well suited to adaptation for future high-throughput automation.

Session Details

Date: 10/04/2010
Time: 00:00-00:00
Session name: Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Vienna, Austria, 10 - 13 April 2010
Presentation type:
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