Label-Free Cross-Priming Amplification Coupled With Endonuclease Restriction and Nanoparticles-Based Biosensor for Simultaneous Detection of Nucleic Acids and Prevention of Carryover Contamination

Abstract

Here, we reported on a label-free cross-priming amplification (CPA) scheme that utilized endonuclease restriction for simultaneous detection of nucleic acids and elimination of carryover contamination. Reaction mixtures were detected in a nanoparticle-based lateral flow biosensor (LFB). The assay exhibited attractive traits in that it did not require the use of labeled primers or labeled probes, and thus, the technique could prevent undesired results arising from unwanted hybridization between labeled primers or between a probe and labeled primer. Isothermal amplification and endonuclease restriction digestion were conducted in a single pot, and the use of a closed-tube amplification removed false-positive results due to contaminants. To validate the assay's applicability, we employed the novel technique to detect the pathogen Staphylococcus aureus in pure cultures and artificial blood samples. The assay could detect target bacterium in pure culture with a 100 fg.μL^-1 detection limit, and in spiked blood samples with a 700 cfu.mL^-1 detection limit. The whole process, including sample procedure (20-min), isothermal amplification (60-min), endonuclease digestion (10-min) and result reporting (within 2-min), could be finished within 95-min. As a poof-of-concept assay, the technique devised in the current report could be employed for detecting various other sequences if the specific CPA primers were available.

Keywords: S. aureus; cross-priming amplification; endonuclease restriction; lateral flow biosensor; limit of detection.

Figure 1

Outline of label-free CPA coupled with lateral flow biosensor (LFB). (A) Outline of label-free CPA with biotin-14-dATP and FITC-aha-dUTP. (B) The detailed structure of LFB. (C) Schematic illustration of the principle of LFB for visualization of CPA products. (D) Interpretation of the LFB results: (I), positive (two crimson red bands, including test line and control line, appeared on the NC regions of the biosensor); (II), negative (only the control line zone showed a crimson red line).

Figure 2

Sensitivity of label-free CPA-LFB assay using serially diluted S. aureus (ATCC 43300) templates. Real-time turbidity (A), Colorimetric indicator (MG, B) and biosensor (C) were applied for analyzing the CPA reactions products. Signals/Tubes/biosensors 1, 2, 3, 4, 5, 6, and 7 represent the genomic DNA levels of 1 ng.μL⁻¹, 100 pg.μL⁻¹, 10 pg.μL⁻¹, 1 pg.μL⁻¹, 100 fg.μL⁻¹, 10 fg.μL⁻¹, and 1 fg.μL⁻¹. Tube/biosensor 8, blank control (DW).

Figure 3

Schematic illustration of CPA-ER pollution elimination by a primer enzymatic method. (A) CPA amplification with the core primer (As) carrying the recognition site of endonuclease restriction BpuEI. (B) Schematic presentation of digesting carryover CPA amplicons. CPA amplification products were cut into several kinds of DNA strands by endonuclease restriction BpuEI. As was the core primer in the CPA system, which consisted of the reverse complementary sequence of 1a region and 2s. E-As was the core primer in the CPA-ER assay, which consisted of the reverse complementary sequence of 1a, recognition site, and 2s.

Figure 4

Control of carryover contamination in CPA-ER assay. Sensitivity of conventional CPA-ER (A) and CPA (B) using serial dilution of simulated carryover contamination (1 × 10⁻¹², 1 × 10⁻¹³, 1 × 10⁻¹⁴, 1 × 10⁻¹⁵, 1 × 10⁻¹⁶, 1 × 10⁻¹⁷, 1 × 10⁻¹⁸, and 1 × 10⁻¹⁹ g.μL⁻¹) as determined using colorimetric indicator (Top row) and biosensor (Bottom row).

Figure 5

CPA-ER method prevent false-positive result due to carryover contamination. Sensitivity of conventional CPA-ER (A) and CPA technique (B) using serial dilutions (1 ng.μl⁻¹, 100 pg.μl⁻¹, 10 pg.μl⁻¹, 1 pg.μl⁻¹, 100 fg.μl⁻¹, 10 fg.μl⁻¹, and 1 fg.μl⁻¹) of ATCC 43300 and 1 × 10⁻¹⁸ g.μL⁻¹ of simulated carryover contamination as determined using LFB (Top row) and colorimetric indicator (Bottom row).

Figure 6

Sensitivity of label-free CPA-ER-LFB for detecting S. aureus in blood samples. Monitoring techniques, including biosensor (A) and colorimetric indicator (MG, B), were applied for reporting label-free CPA-ER-LFB results. Ten-fold serial dilutions of target bacterium were subjected to label-free CPA-ER-LFB reaction. Biosensor (A)/Tubes (B) 1–8 represented the cell levels of 140,000 CFU, 14,000 CFU, 1,400 CFU, 140 CFU, 14 CFU, 1.4 CFU, 0.14 CFU per reaction, negative control (non-contaminated sputum samples). The cell levels of 140,000 CFU, 14,000 CFU, 1,400 CFU, 140 CFU, and 14 CFU per reaction yielded the positive amplifications.