Multiplex Assay for Rapid Detection and Analysis of Nucleic Acid Using Barcode Receptor Encoded Particle (BREP)

Abstract

Several multiplex nucleic acid assay platforms have been developed in response to the increasing importance of nucleic acid analysis, but these assays should be optimized as per the requirements of point-of-care for clinical diagnosis. To achieve rapid and accurate detection, involving a simple procedure, we propose a new concept in the field of nucleic acid multiplex assay platforms using hydrogel microparticles, called barcode receptor-encoded particles (BREPs). The BREP assay detects multiple targets in a single reaction with a single fluorophore by analyzing graphically encoded hydrogel particles. By introducing sets of artificially synthesized barcode receptor and barcode probes, the BREP assay is easily applicable in multiplexing any genetic target; sets of barcode receptors and barcode probes should be designed delicately for universal application. The performance of the BREP assay was successfully verified in a multiplex assay for the identification of different malaria species with high sensitivity, wide dynamic range, fast detection time, and multiplexibility.

Keywords: DNA; hydrogel microparticle; molecular diagnosis; multiplex.

Figure 1

(a) Schematic illustration of Barcode Receptor Encoding Particle (BREP) assay. (b) Particle image and graphical encoding created with combinations of holes on square shape particles with a protruding bar.

Figure 2

Optimization of BREP assay conditions. (a) Optimization of assay duration conducted at 45 °C with 10 pmol/mL of synthetic barcode for positive sample, 10 pmol/mL of barcode probe for negative sample, and deionized water as blank. Fluorescence signal intensity at 10, 20, and 30 min. (b) Temperature optimization was conducted with 10 pmol/mL of synthetic barcode for positive sample, 10 pmol/mL of barcode probe for negative sample, and deionized water as blank, with the assay duration being 20 min. Fluorescence signal intensity at 37, 40, 42, 45, and 50 °C. Bar graph showing mean values of fluorescence signal intensity. Error bars represent standard deviation (n = 3).

Figure 3

(a) PAGE to detect the size of the amplicon (16 nt). Lane 1: Barcode probe (38 nt); Lane 2: Barcode (16 nt); Lanes 3 and 5: amplicons of positive DNA template; Lanes 4 and 6: amplicons of negative DNA template. (b) Standard curve fitted by fluorescence signal intensity with respect to barcode concentrations of 0.3125, 0.625, 1.25, 2.5, 5, and 10 μM (standard deviation (SD) of each value was 1.93, 0.39, 0.13, 2.92, 0.20, and 2.73, respectively; n = 3) (c) Fluorescence signal intensity with respect to DNA template concentrations of 15.38, 1.53, 0.15, 0.015, and 0.0015 pM (standard deviation (SD) of each value was 4.51, 3.84, 3.61, 1.62, 7.68, 1.00, and 0.77, respectively; n = 3).

Figure 4

Multiplex BREP assay. (a) Validation of amplification using agarose gel electrophoresis (Multiplex) Lane 1: 50 bp DNA ladder; Lanes 2–5: amplification step product with Plasmodium falciparum (P.f), P. vivax (P.v), P. malariae (P.m), and P. ovale (P.o) of 0.2 ng/μL DNA template; Lane 6: negative control, deionized water. (b) 4-plex assay to detect four species of malarial parasite. The + and – signs indicate the presence (+) and absence (−) of the target DNA template. (c) Comparison of the results obtained after singleplex and multiplex assays. Bar graph showing mean values of background subtracted from the fluorescence signal intensity. Error bars represent standard deviation (n = 3).

Figure 5

BREP assay with clinical samples. (a) Singleplex BREP assay with clinical sample of four patients with malaria. (b) Comparison of the results obtained after singleplex and multiplex assays with both synthetic DNA template and clinical samples from patients with malaria. Bar graph showing mean values of background subtracted from fluorescence signal intensity. Error bars represent standard deviation (n = 3).