Yersinia pestis detection using biotinylated dNTPs for signal enhancement in lateral flow assays
Autor: | Mounir Ben-Ali, Abdulrahman O. Al-Youbi, Miriam Jauset-Rubio, Abdulaziz S. Bashammakh, Herbert Tomaso, Mohammed Nooredeen Abbas, Ciara K. O'Sullivan, Salah Kortli, Mohammad S. El-Shahawi |
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Rok vydání: | 2020 |
Předmět: |
Yersinia pestis
Deoxyribonucleotides Loop-mediated isothermal amplification Recombinase Polymerase Amplification 02 engineering and technology Polymerase Chain Reaction 01 natural sciences Biochemistry Analytical Chemistry law.invention law Environmental Chemistry Biotinylation Spectroscopy Polymerase chain reaction Chemistry Oligonucleotide 010401 analytical chemistry Amplicon 021001 nanoscience & nanotechnology Molecular biology 0104 chemical sciences genomic DNA Nucleic acid 0210 nano-technology Nucleic Acid Amplification Techniques |
Zdroj: | Analytica Chimica Acta. 1112:54-61 |
ISSN: | 0003-2670 |
Popis: | Due to the extreme infectivity of Yersinia pestis it poses a serious threat as a potential biowarfare agent, which can be rapidly and facilely disseminated. A cost-effective and specific method for its rapid detection at extremely low levels is required, in order to facilitate a timely intervention for containment. Here, we report an ultrasensitive method exploiting a combination of isothermal nucleic acid amplification with a tailed forward primer and biotinylated dNTPs, which is performed in less than 30 min. The polymerase chain reaction (PCR) and enzyme linked oligonucleotide assay (ELONA) were used to optimise assay parameters for implementation on the LFA, and achieved detection limits of 45 pM and 940 fM using SA-HRP and SA-polyHRP, respectively. Replacing PCR with isothermal amplification, namely recombinase polymerase amplification, similar signals were obtained (314 fM), with just 15 min of amplification. The lateral flow detection of the isothermally amplified and labelled amplicon was then explored and detection limits of 7 fM and 0.63 fg achieved for synthetic and genomic DNA, respectively. The incorporation of biotinylated dNTPs and their exploitation for the ultrasensitive molecular detection of a nucleic acid target has been demonstrated and this generic platform can be exploited for a multitude of diverse real life applications. |
Databáze: | OpenAIRE |
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