Nucleotide-selective amplification and array-based detection for identifying multiple somatic mutations.

In the context of personalized and cost-effective treatment, knowledge of the mutational status of specific genes is advantageous to predict which patients are responsive to therapies. As an alternative to one-by-one detection or massive sequencing, the presented genotyping tool determines multiple polymorphic sequences that vary a single nucleotide. The biosensing method includes an effective enrichment of mutant variants and selective recognition by colorimetric DNA arrays. The proposed approach is the hybridization between sequence-tailored probes and products from PCR with SuperSelective primers to discriminate specific variants in a single locus. A fluorescence scanner, a documental scanner, or a smartphone captured the chip images to obtain spot intensities. Hence, specific recognition patterns identified any single-nucleotide change in the wild-type sequence overcoming qPCR methods and other array-based approaches. Studied mutational analyses applied to human cell lines provided high discrimination factors, the precision was 95%, and the sensitivity was 1% mutant of total DNA. Also, the methods showed a selective genotyping of the KRAS gene from tumorous samples (tissue and liquid biopsy), corroborating results by NGS. The developed technology supported on low-cost robust chips and optical reading provides an attractive pathway toward implementing fast, cheap, reproducible discrimination of oncological patients.

Reversed-phase allergen microarrays on optical discs for multiplexed diagnostics of food allergies.

A high percentage of the population suffers from multiple food allergies justifying  the importance of reliable diagnostic methods. Single-analyte solutions based on the determination of specific immunoglobulins E (sIgE) are safe and fast but are generally time-consuming and expensive. Thus sustainable microanalytical methods that provide multianalyte profiling information are highly demanded. This work presents the in vitro biosensing of specific IgE levels based on a reversed-phase allergen array. The approach consists of optical biosensing supported by direct multiplex immunoassays and on-disc technology. It identifies 12 sIgE associated with food allergies in a single analysis with a low serum sample volume (25 µL). After processing captured images, specific signals for each target biomarker correlate to their concentration. The assay analytically performs well with 0.3 IU/mL and 0.41 IU/mL as the detection and quantification limits in serum, respectively. This novel method achieves excellent clinical specificity (100%) and high sensitivity (91.1%), considering the diagnosis obtained by clinical history and ImmunoCAP analysis. The results demonstrate that microanalytical systems based on allergen arrays can potentially diagnose multiple food allergies and are easily implemented in primary care laboratory settings.

A genosensor for detecting single-point mutations in dendron chips after blocked recombinase polymerase amplification.

A biosensing system was developed to accurately detect a single nucleotide change in the target organism genome, integrating a selective isothermal amplification and a sensitive dendron-mediated DNA hybridization assay in the array format. The novelty arises from the coupling reactions of the dendron and its use as a crosslinker. The allele-specific probes were oligonucleotide-dendron conjugates prepared by fast and clean click-chemistry (thiol-yne reaction) and coupled onto the photo-activated surface of polycarbonate substrates (carbodiimide reaction). The output was forest-array chips with multipoint-site crosslinkers and compatible with current microarray fabrication technologies. The products of blocked recombinase polymerase amplification (blocked RPA), formed at 37 °C, were hybridized with attached probes for specific nucleotide genotyping. The developed approach exhibited sensitive recognition of DNA variants compared to chips based on linear crosslinkers (10-100 fold), showing excellent analytical performances for planar chip and fluidic formats. The methodology was successfully applied to detect the H1047R mutation in the PIK3CA gene (c.3140A > G) from clinical samples of human cancer tissues, the results being consistent with sequencing techniques. The colorimetric biosensing method was reliable, versatile, low cost, sensitive (detection limit genomic DNA: 0.02 ng µL-1), and specific (accuracy >95%).

Discrimination of Single-Nucleotide Variants Based on an Allele-Specific Hybridization Chain Reaction and Smartphone Detection.

Massive DNA testing requires novel technologies to support a sustainable health system. In recent years, DNA superstructures have emerged as alternative probes and transducers. We, herein, report a multiplexed and highly sensitive approach based on an allele-specific hybridization chain reaction (AS-HCR) in the array format to detect single-nucleotide variants. Fast isothermal amplification was developed before activating the HCR process on a chip to work with genomic DNA. The assay principle was demonstrated, and the variables for integrating the AS-HCR process and smartphone-based detection were also studied. The results were compared to a conventional polymerase reaction chain (PCR)-based test. The developed multiplex method enabled higher selectivity against single-base mismatch sequences at concentrations as low as 103 copies with a limit of detection of 0.7% of the mutant DNA percentage and good reproducibility (relative error: 5% for intra-assay and 17% for interassay). As proof of concept, the AS-HCR method was applied to clinical samples, including human cell cultures and biopsied tissues of cancer patients. Accurate identification of single-nucleotide mutations in KRAS and NRAS genes was validated, considering those obtained from the reference sequencing method. To conclude, AS-HCR is a rapid, simple, accurate, and cost-effective isothermal method that detects clinically relevant genetic variants and has a high potential for point-of-care demands.

Enhanced asymmetric blocked qPCR method for affordable detection of point mutations in KRAS oncogene.

An accurate genetic diagnostic is key for adequate patient management and the suitability of healthcare systems. The scientific challenge lies in developing methods to discriminate those patients with certain genetic variations present in tumor cells at low concentrations. We report a method called enhanced asymmetric blocked qPCR (EAB-qPCR) that promotes the blocker annealing against the primer-template hybrid controlling thermal cycling and reaction conditions with nonmodified oligonucleotides. Real-time fluorescent amplification curves of wild-type alleles were delayed by about eight cycles for EAB-qPCR, compared to conventional blocked qPCR approaches. This method reduced the amplification of native DNA variants (blocking percentage 99.7%) and enabled the effective enrichment of low-level DNA mutations. Excellent performance was estimated for the detection of mutated alleles in sensitivity (up to 0.5% mutant/total DNA) and reproducibility terms, with a relative standard deviation below 2.8%. The method was successfully applied to the mutational analysis of metastatic colorectal carcinoma from biopsied tissues. The determined single-nucleotide mutations in the KRAS oncogene (codon 12-13) totally agreed with those obtained from next-generation sequencing. EAB-qPCR is an accurate cheap method and can be easily incorporated into daily routine to detect mutant alleles. Hence, these features are especially interesting to facilitate the diagnosis and prognosis of several clinical diseases.

Magnetic concentration of allele-specific products from recombinase polymerase amplification.

The studied challenge is the specific detection of low-abundant genomic variants that differ by a single nucleotide from the wild type. The combination of blocked recombinase polymerase amplification (RPA) and selective capture by probes immobilised on magnetic-core particles integrated into a flow system is presented. The sensing principle was demonstrated as the effective concentration-detection of the specific generated products was achieved. The analytical performance of resulting assay was successfully compared to PCR-based methods or array formats, providing faster effective detection of the selective products. As proof of concept, the single-nucleotide substitutions of the KRAS gene at codon 12 were studied in chip with parallel microchambers and permanent magnets. The blocked RPA products (generated at 37 °C) from tumour biopsies (extracted DNA 4 ng) provided a specific fluorescent bead-line that depends on the present mutation. The results agree with those reported by next-generation sequencing and provide new opportunities for in vitro diagnostic and personalised medicine.

Consumer electronics devices for DNA genotyping based on loop-mediated isothermal amplification and array hybridisation.

Consumer electronic technologies offer practical performances to develop compact biosensing systems intended for the point-of-care testing of DNA biomarkers. Herein a discrimination method for detecting single nucleotide polymorphisms, based on isothermal amplification and on-chip hybridisation, was developed and integrated into user-friendly optical devices: e.g., USB digital microscope, flatbed scanner, smartphone and DVD drive. In order to adequately identify a single base change, loop-mediated isothermal amplification (LAMP) was employed, with high yields (8 orders) within 45 min. Subsequently, products were directly hybridised to the allele-specific probes attached to plastic chips in an array format. After colorimetric staining, four consumer electronic techniques were compared. Sensitive precise measurements were taken (high signal-to-noise ratios, 10-µm image resolution, 99% scan-to-scan reproducibility). These features confirmed their potential as analytical tools, are a competitive alternative to fluorescence scanners, and incorporate additional advantages, such as user-friendly interface and connectivity for telemedicine needs. The analytical performances of the integrated platform (assay and reader) in the human samples were also excellent, with a low detection limit (100 genomic DNA copies), and reproducible (<15%) and cheap assays (< 10 €/test). The correct genotyping of a genetic biomarker (single-nucleotide polymorphism located in the GRIK4 gene) was achieved as the assigned genotypes agreed with those determined by using sequencing. The portability, favourable discriminating and read-out capabilities reveal that the implementation of mass-produced low-cost devices into minimal-specialised clinical laboratories is closer to becoming a reality.

Digital versatile discs as platforms for multiplexed genotyping based on selective ligation and universal microarray detection.

The development of a high-performance assay readout using integrated detectors is a current challenge in the implementation of DNA tests in diagnostic laboratories, particularly for supporting pharmacogenetic tests. A method for allelic discrimination, associated with single nucleotide polymorphisms (SNPs), is presented. Genomic DNA is extracted from blood and buccal swab samples. The procedure comprises fast multiplex ligation-dependent probe amplification, PCR amplification using universal primers and subsequent barcode hybridization. In this last step, each product is recognized by the specific probes immobilized on the surface of an optical disc. Assay results can be obtained with a disc reader. The optical sensing method in a DNA microarray format was optimized and evaluated for the simultaneous identification of 28 polymorphisms associated with psychiatric pharmacogenomics. The target biomarkers were located in the genes related to drug-metabolizing enzymes and drug transporters. The multiplexing capability and assay selectivity strongly depended on correct design (ligation probes, tails and barcodes). The discriminant analysis of reader outputs (spot intensities) led to patients being classified into different allelic populations. The obtained assignations correlated properly with the results provided by the reference technique (bead arrays), and the assay ended in an 8-fold shorter time using affordable equipment. The combination of a highly selective genotyping reaction as array-MLPA and the compact disc technology provides a reliable point-of-care approach. This genotyping tool is useful for the selection of personalized drug therapies in decentralized clinical laboratories.

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

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.

Blocked recombinase polymerase amplification for mutation analysis of PIK3CA gene.

A blocked recombinase polymerase amplification (blocked-RPA) approach has been developed for the enrichment of mutated templates in heterogeneous specimens as tumor tissues. This isothermal amplification technique opens alternative solutions for meeting the technological demand of physician office laboratories. Herein, the detection of mutations in PIK3CA gene, such as p.E545K, and p.H1047L, is presented. The main element was an oligonucleotide (dideoxycytidine functionalized at 3'-end) which matched with wild-type sequence in the target locus. The amplification was performed operating at 37 °C during 40 min. The results demonstrated that the competition between the upstream primer and the blocker reduced the percentage of amplified wild-type allele, making the detection of the present mutation easier. For mutation discrimination, a fast hybridization assay was performed in microarray format on plastic chip and colorimetric detection. This approach enabled the reliable discrimination of specific mutations against a background of up to 95% wild-type DNA. The applicability of the method, based on the combination of blocked-RPA and low-cost chip hybridization, was successfully proven for the genotyping of various cancer cell lines as well as tumor tissues. The assignations agreed with those provided by next-generation sequencing. Therefore, these investigations would support a personalized approach to patient care based on the molecular signature of human cancers.

Primer design for SNP genotyping based on allele-specific amplification-Application to organ transplantation pharmacogenomics.

Diagnostic methods based on single nucleotide polymorphism (SNP) biomarkers are essential for the real adoption of personalized medicine. Allele specific amplification in a homogeneous format or combined to microarray hybridization are powerful approaches for SNP genotyping. However, primers must be properly selected to minimize cross-reactivity, dimer formation and nonspecific hybridization. This study presents a design workflow diagram for the selection of required oligonucleotides for multiplex assays. Based on thermodynamic restrictions, the oligonucleotide sets are chosen for a specific amplification of wild- and mutant-type templates. Design constraints include the structural stability of primer-template duplexes, template-probe duplexes and self-annealing complexes or hairpins for each targeted gene. The performance of the design algorithm was evaluated for the simultaneous genotyping of three SNPs related to immunosuppressive drugs administered after solid organ transplantation. The assayed polymorphisms were rs1045642 (ABCB1 gene), rs1801133 (MTHFR gene) and rs776746 (CYP3A5 gene). Candidates were confirmed by discriminating homozygote and heterozygote populations using a fluorescence solution method and two colorimetric microarray methods on polycarbonate chips. The analysis of patient samples provided excellent genotyping results compared to those obtained by a reference method. The study demonstrates that the development of the allele-specific methods as pharmacogenetic tools can be simplified.

Genotyping of single nucleotide polymorphisms related to attention-deficit hyperactivity disorder.

Pharmacological treatment of several diseases, such as attention-deficit hyperactivity disorder (ADHD), presents marked variability in efficiency and its adverse effects. The genotyping of specific single nucleotide polymorphisms (SNPs) can support the prediction of responses to drugs and the genetic risk of presenting comorbidities associated with ADHD. This study presents two rapid and affordable microarray-based strategies to discriminate three clinically important SNPs in genes ADRA2A, SL6CA2, and OPRM1 (rs1800544, rs5569, and rs1799971, respectively). These approaches are allele-specific oligonucleotide hybridization (ASO) and a combination of allele-specific amplification (ASA) and solid-phase hybridization. Buccal swab and blood samples taken from ADHD patients and controls were analyzed by ASO, ASA, and a gold-reference method. The results indicated that ASA is superior in genotyping capability and analytical performance.

Microarray Developed on Plastic Substrates.

There is a huge potential interest to use synthetic polymers as versatile solid supports for analytical microarraying. Chemical modification of polycarbonate (PC) for covalent immobilization of probes, micro-printing of protein or nucleic acid probes, development of indirect immunoassay, and development of hybridization protocols are described and discussed.

Real-time loop-mediated isothermal DNA amplification in compact disc micro-reactors.

An integrated device composed of micro-reactors embedded onto compact discs is proposed for real-time targeted DNA determination. The method principle is based on in-disc loop-mediated isothermal amplification (iD-LAMP) and quantitative optical read-out by a disc drive. In the presence of a target, the turbidimetric or colorimetric properties of reaction solution change, and the transmitted intensity of the disc drive laser modifies according to reaction yield. Monitoring real-time curves allowed the quantitative determination of DNA template amounts. The best amplification/detection results were obtained with micro-reactors (2mm diameter and 1.1mm in depth) drilled on a digital video disc (DVD) and detection based on the colorimetric mode. As proof-of-concept, the assay was applied to detect pathogenic bacteria Salmonella spp. and to identify bovine meat in food samples. Ninety-six samples were simultaneously analysed in 15 min, with high selectivity and sensitivity (5 CFU/mL and 10 µg/g for bacteria and meat, respectively). The in-disc results were comparable to those obtained by conventional LAMP or qPCR approaches. The developed device allows low sample and reagent consumption (3 µL of reaction), portability, ease-of-use, and rapid low-cost high-throughput analyses.

Multiplex DNA detection of food allergens on a digital versatile disk.

The development of a DNA microarray method on a digital versatile disk (DVD) is described for the simultaneous detection of traces of hazelnut ( Corylus avellana L.), peanut ( Arachis hypogaea ), and soybean ( Glycine max ) in foods. After DNA extraction, multiplex PCR was set up using 5'-labeled specific primers for Cor a 1, Ar h 2, and Le genes, respectively. Digoxin-labeled PCR products were detected by hybridization with 5'-biotinylated probes immobilized on a streptavidin-modified DVD surface. The reaction product attenuates the signal intensity of the laser that reached the DVD drive used as detector, correlating well with the amount of amplified sequence. Analytical performances showed a detection limit of 1 µg/g and good assay reproducibility (RSD 8%), suitable for the simultaneous detection of the three targeted allergens. The developed methodology was tested with several commercially available foodstuffs, demonstrating its applicability. The results were in good agreement, in terms of sensitivity and reproducibility, with those obtained with ELISA, PCR-gel agarose electrophoresis, and RT-PCR.

Multiplexed microimmunoassays on a digital versatile disk.

Multiplexed microimmunoassays for five critical compounds were developed using a digital versatile disk (DVD) as an analytical support and detecting technology. To this end, coating conjugates were adsorbed on the polycarbonate face of the disk; a pool of specific antibodies, gold labeled secondary antibodies, and silver amplification were addressed for developing the assays. The detection principle is based on the capture of attenuated analog signals with the disk drive that were proportional to optical density of the immunoreaction product. The multiplexed assay achieved detection limits (IC10) of 0.06, 0.25, 0.37, 0.16, and 0.10 microg/L, sensitivities of (IC50) 0.54, 1.54, 2.62, 2.02, and 5.9 microg/L, and dynamic ranges of 2 orders of magnitude for atrazine, chlorpyrifos, metolachlor, sulfathiazole, and tetracycline, respectively. The features of the methodology were verified by analyzing natural waters and compared with reference chromatographic methods, showing its potential for high-throughput multiplexed screening applications. Analytes of different chemical nature (pesticides and antibiotics) were directly quantified without sample treatment or preconcentration in a total time of 30 min with similar sensitivity and selectivity to the ELISA plate format using the same immunoreagents. The multianalyte capabilities of immunoassaying methods developed with digital disk and drive demonstrated the competitiveness to quantify targets that require different sample treatment and instrumentation by chromatographic methods.