Molecular detection of Mycobacterium tuberculosis by tHDA-ELISA DIG detection system
Abstract number: 1733_1479
Gill P., Abdul-Tehrani H., Ghaemi A., Hashempour T., V-p Amiri M., Ghalami M., Eshraghi N., Noori-Daloii M.
Objectives: The recent resurgence in tuberculosis (TB) cases poses a serious public health problem. Effective TB management require the simple detection and rapid identification of the aetiologic agent. The nucleic acid amplification methods have proven to be very useful tools in the rapid diagnosis of Mycobacterium tuberculosis. Several PCR-based colorimetric methods such as PCR-ELISA have been previously described for molecular diagnosis of tuberculosis. However this study, we designed and developed a novel non-PCR-based colorimetric assay for specific and sensitive detection of rrs gene of Mycobacterium tuberculosis, is named enzyme-linked immunosorbent assay of thermophilic helicase-dependent DNA amplification (tHDA-ELISA).
Methods: In this procedure, like PCR-ELISA, the tHDA reaction selectively amplified a target sequence defined by two primers and simultaneously incorporated digoxigenin-deoxyuridine triphosphate (DIG-dUTP) in the resulting amplified DNAs. However, unlike PCR-ELISA, the tHDA uses an additional enzyme called a thermophilic helicase to separate DNA rather than heat. Thus the entire tHDA reaction can be performed at isothermal temperature which is optimised for synthesis and it needs no an expensive and power-hungry thermocycler. Then the DIG-labeled amplicons were detected using species-specific probe (biotin-modified), and colorimetric ELISA method.
Results: Our results were shown an equal sensitivity and specificity between this method and ELISA colorimetric assay of PCR products.
Conclusions: THDA-ELISA DIG detection system can be used more cost-effectively than PCR-ELISA for molecular detection of Mycobacterium tuberculosis in developing countries. The other formats of this technology are under study in real-time format and mobile format.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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