Polymorphism genotyping based on loop-mediated isothermal amplification and smartphone detection

Abstract

The genotyping of a single-nucleotide polymorphism (SNP) is addressed through methods based on loop-mediated isothermal amplification (LAMP) combined with user-friendly optical read-outs to cover the current demand for point-of-care DNA biomarker detection. The modification of primer design and reaction composition improved the assay selectivity yielding allele-specific results and reducing false-positive frequency. Furthermore, the reduced cost, ease of use and effectiveness of colorimetric detection (solution and hybridisation chip formats) were availed for the image capture by a smartphone, reching high sensitivity. In order to evaluate their discriminating capacities, LAMP-based methods were applied to human samples to genotype a SNP biomarker (rs1954787) located in the GRIK4 gene and related to the treatment response to anti-depressants drugs. Sensitive (limit of detection: 100 genomic DNA copies), reproducible (< 15% error), fast (around 70 min) and low-cost assays were accomplished. Patient subgroups were correctly discriminated, agreeing with reference sequencing techniques. The achieved analytical performances using the developed amplification-detection principles confirmed the approach potential for point-of-care optical DNA testing.

A duplex probe-directed recombinase amplification assay for detection of single nucleotide polymorphisms on 8q24 associated with prostate cancer

“…These isothermal amplification techniques include loop-mediated amplification (LAMP), nucleic acid sequence-based amplification (NASBA), smart-amplification process version 2 (SMAP2), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA), etc. Some studies have also applied these techniques to SNP detection (22)(23)(24)(25)(26)(27)(28). However, each of these methods has its own shortcomings and limits its application in SNP assay.…”

One-step colorimetric genotyping of single nucleotide polymorphism using probe-enhanced loop-mediated isothermal amplification (PE-LAMP)

“…Furthermore, the loop primer has been introduced to LAMP, leading to a significant enhancement of reaction rate 11. In the last few years, several LAMP-based SNP detection methods have been reported, such as allele-specific LAMP (AS-LAMP) 12-15, the one-step strand displacement coupled LAMP (LAMP-OSD) 16, and the LAMP combined with allele-selective oligonucleotide hybridization (LAMP-ASO) 17. The strategy of AS-LAMP is placing the site of interest at the 3' or 5' end of the initial primers such that the specific amplification could be realized by mesophilic DNA polymerases with the ability of mismatch discrimination.…”

“…The methods used for monitoring LAMP reaction have been drawing attention from diverse fields of science, but a suitable colorimetric tool for SNP detection is still a challenge. Except for the real-time fluorescence assay that depends on the bulky instrument, currently described reporting systems in LAMP-based SNP detection include several visualized methods such as enzyme-linked immunosorbent assay 17, nanoparticles 24 and lateral flow dipstick 13. These visualized reporting systems, though costly and laborious, are promising in POCT.…”

A review of smartphone point‐of‐care adapter design

“…It is evident that smartphones will dominate the POC field, enabling an easy‐to‐use mobile application that can perform complicated laboratory tests without the need for very deep knowledge about the instrumentation and the complicated test procedures used by specialists in a large laboratory environment . Recent innovations verified that smartphone‐based applications are very promising demonstrating precise measurements, a low limit of detection (LOD), a wide dynamic range, low power consumption, easy‐to‐use smart applications, a low coefficient of variation (CV%), a high regression coefficient (R 2 ), high specificity, reliable sensitivity, rapid testing, cost‐effective adapter designs, and high selectivity . These innovations include the major parts of POC applications, such as surgical treatment application (Section 3.2), surgical diagnosis (Section 3.1), ophthalmic applications (Section 3.3), biochemical applications (Section 3.4), environmental monitoring applications, biomedical applications, electrochemical applications (Section 3.5), colorimetric applications, imaging applications, and spectrometry applications .…”

“…13 Recent innovations verified that smartphone-based applications are very promising demonstrating precise measurements, [14][15][16][17][18] a low limit of detection (LOD), [19][20][21][22][23] a wide dynamic range, [24][25][26][27] low power consumption, easy-to-use smart applications, a low coefficient of variation (CV%), a high regression coefficient (R 2 ), high specificity, reliable sensitivity, rapid testing, cost-effective adapter designs, and high selectivity. [28][29][30][31][32] These innovations include the major parts of POC applications, such as surgical treatment application (Section 3.2), surgical diagnosis (Section 3.1), ophthalmic applications (Section 3.3), biochemical applications (Section 3.4), [33][34][35] environmental monitoring applications, [36][37][38][39][40][41][42] biomedical applications, [43][44][45] electrochemical applications (Section 3.5), colorimetric applications, [46][47][48][49] imaging applications, [50][51][52][53][54] and spectrometry applications. …”