Visual detection of gene mutations based on isothermal strand-displacement polymerase reaction and lateral flow strip

Abstract

Here, we describe a simple and sensitive approach for visual detection of gene mutations based on isothermal strand-displacement polymerase reactions (ISDPR) and lateral flow strip (LFS). The concept was first demonstrated by detecting the R156H-mutant gene of keratin 10 in Epidermolytic hyperkeratosis (EHK). In the presence of biotin-modified hairpin DNA and digoxin-modified primer, the R156H-mutant DNA triggered the ISDPR to produce numerous digoxin- and biotin-attached duplex DNA products. The product was detected on the LFS through dual immunoreactions (anti-digoxin antibody on the gold nanoparticle (Au-NP) and digoxin on the duplex, anti-biotin antibody on the LFS test zone and biotin on the duplex). The accumulation of Au-NPs produced the characteristic red band, enabling visual detection of the mutant gene without instrumentation. After systematic optimization of the ISDPR experimental conditions and the parameters of the assay, the current approach was capable of detecting as low as 1-fM R156H-mutant DNA within 75 min without instrumentation. Differentiation of R156H- and R156C-mutant DNA on the R156 mutation site was realized by using fluorescein- and biotin-modified hairpin probes in the ISDPR process. The approach thus provides a simple, sensitive, and low-cost tool for the detection of gene mutations.

Figure 1

Typical photo images of LFSs in the presence of 1-nM R156H-mutant DNA(A), 0-nM R156H-mutant DNA(B), 50-nM noncomplementary DNA(C), 1-nM wild-type DNA(D), 1-nM R156H-mutant DNA+50-nM noncomplementary DNA(E), and 1-nM R156H-mutant DNA+1-nM wild-type DNA(F). ISDPR reactions were performed at 42°C for 60 min.

Figure 2

(A) The optical responses of 1-nM R156H-mutant DNA on the LFSs with different ISDPR solutions. Solution 1: 5.0×10⁻⁸ M biotin-modified hairpin probe, 5.0×10⁻⁸ M digoxin-modified primer, 3-U polymerase Klenow fragment exo⁻, 50-µM dNTPs, 6% DMSO, 0.1% BSA, 1-mM DTT, and 5 mM MgCl₂ in 50-mM Tris–HCl (pH 8.0) buffer; Solution 2: solution 1 without polymerase Klenow fragment exo⁻; Solution 3: solution 1 without dNTP; Solution 4: solution 1 without biotin-modified hairpin probe; and Solution 5: solution 1 without digoxin-modified primer. ISDPRs were performed 1 h at 42°C. Corresponding photo images of the LFSs are shown in the inset. (B) The effect of ISDPR time on the S/N ratio of assays. (C) Gel image of ISDPR products in the absence (lane a) and presence of 1-nM R156H-mutant DNA. Lane a: product generated in the absence of R156H-mutant DNA with an ISDPR time of 60 min. Lanes b–d: products generated in the presence of 1-nM R156H-mutant DNA at different ISDPR time intervals: (b) t = 0 min, (c) t = 15 min, (d) t = 30 min, and (e) t = 60 min. Lane e: DNA ladder markers (10, 20, 30, 40, 50, 60, 70, 80, and 90 bp from the bottom). ISDPRs were performed with solution 1 in (A) at 42°C.

Figure 3

Calibration curve of the assay with different concentrations of R156H-mutant DNA. The inset is photo images of LFSs after applying the ISDPR product of 0-nM and 1-fM R156H-mutant DNA.

Figure 4

Typical photo images of the LFSs in the presence of 1-nM R156H+1-nM R156C-mutant DNA, 1-nM R156C-mutant DNA, 1-nM R156H-mutant DNA, 0-nM mutant DNA (control), and 1-nM R156 wild-type DNA. Experimental conditions are the same as in Figure 3.

Scheme 1

(A) Schematic illustration of the formation of digoxin- and biotin-attached duplex DNA complexes based on isothermal strand-displacement polymerase reactions. (B) Schematic illustration for the visual detection of ISDPR products on a lateral flow strip.