Core-Shell Beads as Microreactors for Phylogrouping of E. coli Strains

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.

Keywords: PCR; core-shell bead; liquid marble; phylogrouping; simultaneous detection.

Figure 1

Operation of core-shell-bead-based polymerase chain reaction (PCR). (A) (1) Placing a droplet of photopolymer on top of an amphiphobic powder bed. (2) Inserting the PCR solution into the deposited droplet. (3) Covering the droplet with amphiphobic powder. (B) Photo polymerization of the droplet under blue light in a drum rotating at 140 rpm. (C) Transferring the generated core-shell bead to a custom-built thermal cycler for amplification. (D) Discriminating the core shell beads containing amplicons based on fluorescent intensity.

Figure 2

Schematic of the experimental setup.

Figure 3

Amplification plot and phylogrouping of E. coli strains using core shell beads. (I) Positive control; (II–IV) unknown samples; A, B, C, and D beads containing specific set of primers to detect chuA, arpA, TspE4C2, yjaA genes individually; and images of core shell beads, are provided as insets.

Figure 4

Agarose gel electrophoresis of amplicons from the Sybr-Green quadruplex PCR for confirmation. Lane 1: DNA ladder (100–20,000 bp). Lane 2: positive control (EC RBH2). Lane 3: negative control (water). Lane 4 to 6: unknown samples.