Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection

[EN] The costs of current genotyping methods limit their application to personalized therapy. The authors describe an alternative approach for the detection of single-point-polymorphisms using recombinant polymerase amplification as an allele-specific technique. The use of short and chemically modified primers and locked nucleic acids allowed for a selective isothermal amplification of wild-type or mutant variants at 37 °C within 40 min. An amplification chip platform containing 100 wells was manufactured with a 3D printer and using thermoplastic polylactic acid. The platform reduces reagent consumption and allows parallelization. As a proof of concept, the method was applied to the genotyping of four SNPs that are related to the treatment of tobacco addiction. The target polymorphisms included rs4680 (COMT gene), rs1799971 (OPRM1 gene), rs1800497 (ANKK1 gene), and rs16969968 (CHRNA5 gene). The genotype populations can be well discriminated.The authors acknowledge the financial support received from the Generalitat Valenciana (GVA-PROMETEOII/2014/040 project and GRISOLIA/2014/024 PhD grant) and the Spanish Ministry of Economy and Competitiveness (MINECO CTQ2013-45875-R project).Yamanaka, E.; Tortajada-Genaro, LA.; Maquieira, A. (2017). Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection. Microchimica Acta. 184(5):1453-1462. https://doi.org/10.1007/s00604-017-2144-0S145314621845Manolio TA, Chisholm RL, Ozenberger B, Roden DM, Williams MS, Wilson R et al (2013) Implementing genomic medicine in the clinic: the future is here. Genitourin Med 15:258–267Scott SA (2013) Clinical pharmacogenomics: opportunities and challenges at point-of-care. Clin Pharmacol Ther 93:33Limaye N (2013) Pharmacogenomics, Theranostics and personalized medicine-the complexities of clinical trials: challenges in the developing world. Appl Transl Genomics 2:17–21Abul-Husn NS, Owusu Obeng A, Sanderson SC, Gottesman O, Scott SA (2014) Implementation and utilization of genetic testing in personalized medicine. Pharmacogenomics Pers Med 7:227–240Knez K, Spasic D, Janssen KP, Lammertyn J (2014) Emerging technologies for hybridization based single nucleotide polymorphism detection. Analyst 139:353–370Shen W, Tian Y, Ran T, Gao Z (2015) Genotyping and quantification techniques for single-nucleotide polymorphisms. TrAC Trends Anal Chem 69:1–13Milbury CA, Li J, Makrigiorgos GM (2009) PCR-based methods for the enrichment of minority alleles and mutations. Clin Chem 55:632–640Asari M, Watanabe S, Matsubara K, Shiono H, Shimizu K (2009) Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA. Anal Biochem 386:85–90Taira C, Matsuda K, Yamaguchi A, Sueki A, Koeda H, Takagi F, Kobayashi Y, Sugano M, Honda T (2013) Novel high-speed droplet-allele specific-polymerase chain reaction: application in the rapid genotyping of single nucleotide polymorphisms. Clin Chim Acta 424:39–46Tortajada-Genaro LA, Mena S, Niñoles R, Puigmule M, Viladevall L, Maquieira A (2016) Genotyping of single nucleotide polymorphisms related to attention-deficit hyperactivity disorder. Anal Bioanal Chem 408:2339–2345Woolley CF, Hayes MA (2014) Emerging technologies for biomedical analysis. Analyst 139:2277–2288Craw P, Balachandran W (2012) Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review. Lab Chip 12:2469–2486Zhang L, Zhang Y, Wang C, Feng Q, Fan F, Zhang G, Kang X, Qin X, Sun J, Li Y, Jiang X (2014) Integrated microcapillary for sample-to-answer nucleic acid pretreatment, amplification, and detection. Anal Chem 86:10461–10466Chen F, Zhao Y, Fan C, Zhao Y (2015) Mismatch extension of DNA polymerases and high-accuracy single nucleotide polymorphism diagnostics by gold nanoparticle-improved isothermal amplification. Anal Chem 87:8718–8723Li J, Macdonald J (2015) Advances in isothermal amplification: novel strategies inspired by biological processes. Biosens Bioelectron 64:196–211Santiago-Felipe S, Tortajada-Genaro LA, Morais S, Puchades R, Maquieira A (2014) One-pot isothermal DNA amplification–hybridisation and detection by a disc-based method. Sens Actuator B-Chem 204:273–281Santiago-Felipe S, Tortajada-Genaro LA, Puchades R, Maquieira Á (2016) Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc. Microchim Acta 183:1195–1202Tortajada-Genaro LA, Santiago-Felipe S, Amasia M, Russom A, Maquieira A (2015) Isothermal solid-phase recombinase polymerase amplification on microfluidic digital versatile discs (DVDs). RSCAdv 5:29987–29995Li Z, Liu Y, Wei Q, Liu Y, Liu W, Zhang X, Yu Y (2016) Picoliter well Array Chip-based digital recombinase polymerase amplification for absolute quantification of nucleic acids. PLoS One 11:e0153359Daher RK, Stewart G, Boissinot M, Boudreau DK, Bergeron MG (2015) Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology. Mol Cell Probes 29:116–121Shin Y, Perera AP, Kim KW, Park MK (2013) Real-time, label-free isothermal solid-phase amplification/detection (ISAD) device for rapid detection of genetic alteration in cancers. Lab Chip 13:2106–2114NgePN RCI, Woolley AT (2013) Advances in microfluidic materials, functions, integration, and applications. Chem Rev 113:2550–2583Bhattacharjee N, Urrios A, Kang S, Folch A (2016) The upcoming 3D-printing revolution in microfluidics. Lab Chip 16:1720–1742Waheed S, Cabot JM, Macdonald NP, Lewis T, Guijt RM, Paull B, Breadmore MC (2016) 3D printed microfluidic devices: enablers and barriers. Lab Chip 16:1993–2013Bierut LJ, Madden PA, Breslau N, Johnson EO, Hatsukami D, Pomerleau OF, Swan GE, Rutter J, Bertelsen S, Fox L, Fugman D, Goate AM, Hinrichs AL, Konvicka K, Martin NG, Montgomery GW, Saccone NL, Saccone SF, Wang JC, Chase GA, Rice JP, Ballinger DG (2007) Novel genes identified in a high-density genome wide association study for nicotine dependence. Hum MolGen 16:24–35Carpenter MJ, Jardin BF, Burris JL, Mathew AR, Schnoll RA, Rigotti NA, Cummings KM (2013) Clinical strategies to enhance the efficacy of nicotine replacement therapy for smoking cessation: a review of the literature. Drugs 73:407–426Moody C, Newell H, Viljoen H (2016) FA mathematical model of recombinase polymerase amplification under continuously stirred conditions. Biochem Eng J 112:193–201Dimitrov RA, Zuker M (2004) Prediction of hybridization and melting for double-stranded nucleic acids. Biophys J 87:215–226Zhang C, Xing D (2007) Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends. Nucleic Acids Res 35:4223–4237Liu B, Huang PJJ, Zhang X, Wang F, Pautler R, IpACF LJ (2013) Parts-per-million of polyethylene glycol as a non-interfering blocking agent for homogeneous biosensor development. 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Consumer electronics devices for DNA genotyping based on loop-mediated isothermal amplification and array hybridisation

[EN] 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-mu m 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 (sic)/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.The authors acknowledge the financial support received from the Generalitat Valenciana, Spain (GVA-PROMETEOII/2014/040 Project and GRISOLIA/2014/024 Ph.D. grant) and the Spanish Ministry of Economy and Competitiveness, Spain (MINECO CTQ2016-75749-R project) by U. E. FEDER funds. The authors also thank J. Carrascosa for supporting the DVD reader measurements.Tortajada-Genaro, LA.; Yamanaka, ES.; Maquieira Catala, A. (2019). Consumer electronics devices for DNA genotyping based on loop-mediated isothermal amplification and array hybridisation. Talanta. 198:424-431. https://doi.org/10.1016/j.talanta.2019.01.124S42443119

One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method

[EN] An integrated sensor comprising isothermal DNA amplification and in situ detection is presented. The method principle is based on recombinase polymerase amplification (RPA) and detection in the microarray format by compact disc technology as a high-throughput sensing platform. Primers were immobilised on the polycarbonate surface of digital versatile discs (DVD) and, after hemi-nested amplification, multiplexing identification of each tethered product was achieved by optical scanning with a 650 nm-laser of the DVD drive. The efficiency of one-pot hybridisation/elongation/detection depended strongly on probedensity and other factors such as the concentration of the unbound primers present in solution. The optimised conditions provided equivalent amplification factors (7.3 x 10(8) -8.9 x 10(8) fold) to those obtained by conventional reactions performed in vials. The proposed method was applied to Salmonella detection (generic by hns and oriC genes, and specific for subspecies I by STM4507 gene). A triplex assay was satisfactorily compared to the non-integrated protocols. Food and vaccine samples were analysed in a shorter time with less handling. The results indicate that the multiplex DVD assay is a simple, competitive, isothermal, portable system that is particularly useful for microbiological routine analysis. (C) 2014 Elsevier B.V. All rights reserved.This research has been funded through Projects GVA-PROMETEO/2010/008 (Generalitat Valenciana) and CTQ/2013/ 45875-R (MINECO). The Spanish Ministry of Education and Science provided S.S.F. with a grant for her PhD studies.Santiago Felipe, S.; Tortajada-Genaro, LA.; Morais, S.; Puchades, R.; Maquieira Catala, Á. (2014). One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method. Sensors and Actuators B: Chemical. 204:273-281. https://doi.org/10.1016/j.snb.2014.07.073S27328120

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.This research has been funded through projects FEDER MINECO INNPACTO IPT-2011-1132-010000, CTQ/2013/45875R, and PrometeoII/2014/040 (GVA).Tortajada-Genaro, LA.; Mena-Mollá, S.; Niñoles Rodenes, R.; Puigmule, M.; Viladevall, L.; Maquieira Catala, Á. (2016). Genotyping of single nucleotide polymorphisms related to attention-deficit hyperactivity disorder. Analytical and Bioanalytical Chemistry. 408(9):2339-2345. https://doi.org/10.1007/s00216-016-9332-3S233923454089Cortese S. The neurobiology and genetics of Attention-Deficit/Hyperactivity Disorder (ADHD): what every clinician should know. Eur J Paediatr Neurol. 2012;16:422–33.Contini V, Rovaris DL, Victor MM, Grevet EH, Rohde LA, Bau CH. Pharmacogenetics of response to methylphenidate in adult patients with attention-deficit/hyperactivity disorder (ADHD): a systematic review. Eur Neuropsychopharmacol. 2013;23:555–60.Gardiner SJ, Begg EJ. Pharmacogenetics, drug-metabolizing enzymes, and clinical practice. Pharmacol Rev. 2006;58(3):521–90.Abul-Husn NS, Obeng AO, Sanderson SC, Gottesman O, Scott SA. Implementation and utilization of genetic testing in personalized medicine. Pharmacogenomics Pers Med. 2014;7:227.Altman RB, Flockhart D, Goldstein DB, editors. Principles of pharmacogenetics and pharmacogenomics. Cambridge: Cambridge University Press; 2012.Hawi Z, Cummins TDR, Tong J, Johnson B, Lau R, Samarrai W, et al. The molecular genetic architecture of attention deficit hyperactivity disorder. Mol Psychiatry. 2015;20:289–97.Limaye N. Pharmacogenomics, Theranostics and Personalized Medicine-the complexities of clinical trials: challenges in the developing world. Appl Transl Genomics. 2013;2:17–21.Manolio TA, Chisholm RL, Ozenberger B, Roden DM, Williams MS, Wilson R, et al. Implementing genomic medicine in the clinic: the future is here. Genet Med. 2013;15:258–67.Kim S, Misra A. PharmGKB: the Pharmacogenomics Knowledge Base. Annu Rev Biomed Eng. 2007;9:289–320.Lucarelli F, Tombelli S, Minunni M, Marrazza G, Mascini M. Electrochemical and piezoelectric DNA biosensors for hybridisation detection. Anal Chim Acta. 2008;609:139–59.Knez K, Spasic D, Janssen KP, Lammertyn J. Emerging technologies for hybridization based single nucleotide polymorphism detection. Analyst. 2014;139:353–70.Choi JY, Kim YT, Byun JY, Ahn J, Chung S, Gweon DG, et al. Integrated allele-specific polymerase chain reaction–capillary electrophoresis microdevice for single nucleotide polymorphism genotyping. Lab Chip. 2012;12:5146–54.Ragoussis J. Genotyping Technologies for Genetic Research. Annu Rev Genomics Hum Genet. 2009;10:117–33.Sethi D, Gandhi RP, Kuma P, Gupta KC. Chemical strategies for immobilization of oligonucleotides. Biotechnol J. 2009;4:1513–29.Bañuls MJ, Morais SB, Tortajada-Genaro LA, Maquieira A. Microarray Developed on Plastic Substrates. Microarray Technology: Methods and Applications, 2016; 37-51.Tortajada-Genaro LA, Rodrigo A, Hevia E, Mena S, Niñoles R, Maquieira A. Microarray on digital versatile disc for identification and genotyping of Salmonella and Campylobacter in meat products. Anal Bioanal Chem. 2015;407:7285–94.Kieling C, Genro JP, Hutz MH, Rohde LA. A current update on ADHD pharmacogenomics. Pharmacogenomics. 2010;11:407–19.Kim BN, Kim JW, Cummins TD, Bellgrove MA, Hawi Z, Hong SB, et al. Norepinephrine genes predict response time variability and methylphenidate-induced changes in neuropsychological function in attention deficit hyperactivity disorder. J Clin Psychopharmacol. 2013;33:356–62.Carpentier PJ, Arias Vasquez A, Hoogman M, Onnink M, Kan CC, Kooij JJS, et al. Shared and unique genetic contributions to attention deficit/hyperactivity disorder and substance use disorders: A pilot study of six candidate genes. Eur Neuropsychopharmacol. 2013;23:448–57.Zhang Y, Haraksingh R, Grubert F, Abyzov A, Gerstein M, Weissman S, et al. Child development and structural variation in the human genome. Child Dev. 2013;84:34–48.Asari M, Watanabe S, Matsubara K, Shiono H, Shimizu K. Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA. Anal Biochem. 2009;386:85–90.Choi JY, Kim YT, Ahn J, Kim KS, Gweon DG, Seo TS. Integrated allele-specific polymerase chain reaction–capillary electrophoresis microdevice for single nucleotide polymorphism genotyping. Biosens Bioelectron. 2012;35:327–34.Konstantou JK, Ioannou PC, Christopoulos TK. Dual-allele dipstick assay for genotyping single nucleotide polymorphisms by primer extension reaction. Eur J Hum Genet. 2009;17:105–11.Sebastian T, Cooney CG, Parker J, Qu P, Perov A, Golova JB, et al. Integrated amplification microarray system in a lateral flow cell for warfarin genotyping from saliva. Clin Chim Acta. 2014;429:198–205

Development of a gas chromatography-mass spectrometry method for the determination of pesticides in gaseous and particulate phases in the atmosphere

[EN] A reliable multi-residue method for determining gaseous and particulate phase pesticides in atmospheric samples has been developed. This method, based on full scan gas chromatography-mass spectrometry (GC-MS), allowed the proper determination of sixteen relevant pesticides, in a wide range of concentrations and without the influence of interferences. The pesticides were benfluralin, bitertanol, buprofezin, chlorfenvinphos, chlorpyrifos, chlorpyrifos-methyl, ethalfluralin, fenthion, lindane, malathion, methidathion, propachlor, propanil, pyriproxifen, tebuconazol and trifluralin. Comparisons of two types of sampling filters (quartz and glass fibre) and four types of solid-phase cartridges (XAD-2, XAD-4, Florisil and Orbo-49P) showed that the most suitable supports were glass fibre filter for particulate pesticides and XAD-2 and XAD-4 cartridges for gaseous pesticides (> 95% recovery). Evaluations of elution solvents for ultrasonic-assisted extraction demonstrated that isooctane is better than ethylacetate, dichloromethane, methanol or a mixture of acetone:hexane (1:1). Recovery assays and the standard addition method were performed to validate the proposed methodology. Moreover, large simulator chamber experiments allowed the best study of the gas-particle partitioning of pesticides for testing the sampling efficiency for the validation of an analytical multiresidue method for pesticides in air. Satisfactory analytical parameters were obtained, with a repeatability of 5 +/- 1%, a reproducibility of 13 +/- 3% and detection limits of 0.05-0.18 pg m(-3) for the particulate phase and 26-88 pg m(-3) for the gaseous phase. Finally, the methodology was successfully applied to rural and agricultural samples in the Mediterranean area. (C) 2011 Elsevier B.V. All rights reserved.The research leading to these results received funding from the Spanish Ministry of Science and Innovation (projects ECOPEST: CGL2007-65223/CLI and IMPESTAT: CGL2010-18474), the CONSOLIDER-INGENIO Programme (GRACCIE, CSD2007-00067). ECOPEST project is cofunded by Generalitat Valenciana (ACOMP 2010/261). Fundacion CEAM is supported by Generalitat Valenciana. The authors also thank J.T.B. for his contribution.Borrás García, EM.; Sánchez, P.; Muñoz, A.; Tortajada Genaro, LA. (2011). Development of a gas chromatography-mass spectrometry method for the determination of pesticides in gaseous and particulate phases in the atmosphere. ANALYTICA CHIMICA ACTA. 69:57-65. https://doi.org/10.1016/j.aca.2011.05.009S57656