Abstract
Multiplex polymerase chain reaction (PCR) is an effective tool for simultaneous detection of target genes. Nevertheless, their use has been restricted due to the intrinsic interference between primer pairs. Performing several single PCRs in an array format instead of a multiplex PCR is a simple way to overcome this obstacle. However, there are still major technical challenges in designing a new generation of single PCR microreactors with a small sample volume, rapid thermal cycling, and no evaporation during amplification. We report a simple and robust core-shell bead array for a series of single amplifications. Four core-shell beads with a polymer coating and PCR mixture were synthesized using liquid marble formation and subsequent photo polymerization. Each bead can detect one target gene. We constructed a customised system for thermal cycling of these core-shell beads. Phylogrouping of the E. coli strains was carried out based on the fluorescent signal of the core-shell beads. This platform can be a promising alternative for multiplex nucleic acid analyses due to its simplicity and high throughput. The platform reported here also reduces the cycling time and avoids evaporation as well as contamination of the sample during the amplification process.
Highlights
Escherichia coli (E. coli) is a bacterium that is frequently found in the environment and the intestine of humans and some animals
AUsssinaygParcinocriep-lsehell bead as a microreactor (Figure 1), we demonstrate here an amplification-based assay for tracing marker genes and phylogrouping E. coli strains
The resulting core-shell beads were subsequently located on a customised polymerase chain reaction (PCR) device for thermal cycling
Summary
Escherichia coli (E. coli) is a bacterium that is frequently found in the environment and the intestine of humans and some animals. Phylogenetic studies of E. coli strains identified four main groups comprising A, B1, B2, and D [3,4]. The distribution within these groups is related to the host origin of the strains. Phylogroups B2 and D demonstrated to include virulent strains, expressing several virulence factors [5,6]. Phylogroups A and B1 are the most predominant and commensal groups in human and animal [7,8,9]
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