RESUMO
This study highlights the development of a multiplex real-time loop-mediated isothermal amplification assay. The developed assay employed a dual-function oligonucleotide (DFO) which simultaneously monitors the emitted amplification signals and accelerates the amplification process. The DFO was a modification of loop primer (LP); the 5'-end and 3'-end of the LP was tagged with fluorophore and quencher, respectively. The DFO was quenched in its unbound state and fluoresces only when it anneals to the specific target during the amplification process. With the same working mechanism as LP, DFO allowed the detection of target genes in less than 1 h in a real time monitoring system. We demonstrated this detection platform with Burkholderia pseudomallei, the causative agent of melioidosis. An internal amplification control (IAC) was incorporated in the assay to rule out false negative result and to demonstrate that the assay was successfully developed in a multiplex system. The assay was 100% specific when it was evaluated against 96 B. pseudomallei clinical isolates and 48 other bacteria species. The detection limit (sensitivity) of the developed assay was 1 fg/µl of B. pseudomallei genomic DNA and 18.2 CFU/ml at the bacterial cell level. In spiked blood samples, the assay's detection limit was 14 CFU/ml. The assay's diagnostic evaluation showed 100% diagnostic sensitivity, diagnostic specificity, positive predictive value, and negative predictive value. An integrated multiplex LAMP and real-time monitoring system was successfully developed, simplifying the workflow for the rapid and specific nucleic acid diagnostic test.
