Probe-lengthening amplification-assisted microchip electrophoresis for ultrasensitive bacteria screening

Abstract

Probe-lengthening amplification (PLA) is a target-specific nucleic acid amplification method that realizes good selectivity by effectively avoiding nonspecific amplification. Here, we propose a probe-lengthening amplification-assisted microchip electrophoresis (MCE) strategy for sensitive analysis of 16S rRNA genes of five bacteria. In this assay, four specific short probes were designed for a target bacterium to recognize its bacterial 16S rRNA gene, integrated into longer DNA ligation duplexes using Ampligase, and subsequently separated and detected by MCE. Along with the rapid generation of ligation duplexes, this approach provides exponential amplification of nucleic acid signals that are useful for sensitive bacterial quantification. Through tactfully combining PLA and MCE, the detection sensitivity of bacterial genes was significantly improved, and a limit of detection (LOD) of 30 fM was realized for the artificial target DNA. This approach was also applied to detect actual bacterial genomic samples with excellent results, demonstrating the potential application of this methodology in infection diagnosis.