Nanoparticles-based lateral flow biosensor coupled with multiple cross displacement amplification Plus for simultaneous detection of nucleic acid and prevention of carryover contamination

Abstract

Abstract Isothermal amplification techniques, which avoid thermal cycling, are often confounded by non-specific amplification arising from off-target hybrids, interactions between (hetero-dimer) or within (self-dimerization) primers and carryover contaminants. Here, we developed a new method, termed multiple cross displacement amplification Plus (MCDA Plus), which uses self-avoiding molecular recognition system (SAMRS) components and antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) enzyme to improve the specificity of MCDA and prevent carryover contamination, respectively. In the presence of SAMRS components, the false positive results arising from off-target hybrids and undesired interactions between or within primers are completely eliminated, which increase the reproducibility of the assay. In the presence of AUDG, the false positive results generating from carryover contaminants are removed, thus the genuine positive results are obtained from the amplification of target sequences. Moreover, we also devised a new analysis strategy, which was intended for the detection of amplified products using lateral flow biosensor (LFB). The analysis system only required a single labeled primer, thus eliminated the false positive result yielding from hybridization (the labeled primer and probe, or between two labeled primers). Therefore, the SAMRS components, AUDG and LFB convert standard MCDA from an assay suited only for the research laboratory into one that has applicable value at points-of-care testing, in the field environments and in poor-resource settings. For demonstration purpose, Mycobacterium tuberculosis complex (MTC), which is the causes of human Tuberculosis (TB), are detected by the novel MCDA Plus technique to demonstrate the capability of target analysis. The performance of MCDA Plus assay in identifying MTC from pure culture and clinical samples is successfully evaluated. The proof-of-concept technique can be reconfigured to detect various nucleic acid sequences by redesigning the specific MCDA Plus primers.