Dr. Miriam Jauset-Rubio and Prof. Ciara o’sullivan published in collaboration Salah Kortli, Herbert Tomaso, Mohammed Nooredeen Abbas, Abdulaziz S. Bashammakh, Mohammad S. El-Shahawi, Abdulrahman O. Alyoubie and Mounir Ben-Ali the following article:

Yersinia pestis detection using biotiny lated dNTPs for signal enhancement in lateral flow assays

Analytica Chimica Acta 1112 (2020)

Abstract:

Due to the extreme infectivity ofYersinia pestisit poses a serious threat as a potential biowarfare agent,which can be rapidly and facilely disseminated. A cost-effective and specific method for its rapiddetection 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 amplifi-cation with a tailed forward primer and biotinylated dNTPs, which is performed in less than 30 min. Thepolymerase chain reaction (PCR) and enzyme linked oligonucleotide assay (ELONA) were used to opti-mise assay parameters for implementation on the LFA, and achieved detection limits of 45 pM and940 fM using SA-HRP and SA-polyHRP, respectively. Replacing PCR with isothermal amplification, namelyrecombinase polymerase amplification, similar signals were obtained (314 fM), with just 15 min ofamplification. The lateralflow 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 detectionof a nucleic acid target has been demonstrated and this generic platform can be exploited for a multitudeof diverse real life applications.