Here, we devised a novel isothermal technique on the basis of standard multiple cross-displacement amplification (MCDA), which is assisted with self-avoiding molecular recognition system (SAMRS) components and antarctic thermal-sensitive uracil-DNA-glycosylase enzyme (AUDG), termed AUDG-SAMRS-MCDA. To enable product detection on the dipsticks, we firstly developed an analysis strategy, which did not require the labelled primers or probes, and thus, the analysis system avoids the false-positive results arising from undesired hybridization (between two labelled primers, or the labelled probe and primer). The SAMRS components are incorporated into MCDA primers for improve the assay's specificity, which can prevent the false-positive results yielding from off-target hybrids, undesired interactions between (hetero-dimer) or within (self-dimerization) primers. Two additional components (AUDG enzyme and dUTP) were added into the reaction mixtures, which were used for removing the false-positive results generating from carryover contamination, and thus, the genuine positives results were produced from the amplification of target templates. For the demonstration, the label-free AUDG-SAMRS-MCDA technique was successfully applied to detect Pseudomonas aeruginosa from pure culture and blood samples. As a proof-of-concept technique, the label-free AUDG-SAMRS-MCDA method can be reconfigured to detect different target sequences by redesigning the specific primers.
Keywords: AUDG; AUDG–SAMRS–MCDA; LFB; MCDA; P. aeruginosa; SAMRS.