The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. We developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. We detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. We also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing.
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World Health Organization Department of Communicable Disease Surveillance and Response. WHO Global Strategy for Containment of Antimicrobial Resistance (Health Organization, Geneva, 2001).
World Health Organization. Antimicrobial Resistance: 2014 Global Report on Surveillance (World Health Organization, Geneva, 2014).
Baker, S. A return to the pre-antimicrobial era? Science 347, 1064–1066 (2015).
Lushniak, B.D. Antibiotic resistance: a public health crisis. Public Health Rep. 129, 314–316 (2014).
Michael, C.A., Dominey-Howes, D. & Labbate, M. The antimicrobial resistance crisis: causes, consequences, and management. Front. Public Health 2, 145 (2014).
Bartlett, J.G., Gilbert, D.N. & Spellberg, B. Seven ways to preserve the miracle of antibiotics. Clin. Infect. Dis. 56, 1445–1450 (2013).
Gelband, H. & Laxminarayan, R. Tackling antimicrobial resistance at global and local scales. Trends Microbiol. 23, 524–526 (2015).
Caliendo, A.M. et al. Better tests, better care: improved diagnostics for infectious diseases. Clin. Infect. Dis. 57 (Suppl. 3), S139–S170 (2013).
Fakruddin, M. et al. Nucleic acid amplification: alternative methods of polymerase chain reaction. J. Pharm. Bioallied Sci. 5, 245–252 (2013).
Yang, S. & Rothman, R.E. PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect. Dis. 4, 337–348 (2004).
Espy, M.J. et al. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin. Microbiol. Rev. 19, 165–256 (2006).
Rao, A., Fader, B. & Hocker, K. in Molecular Diagnostics Techniques and Applications for the Clinical Laboratory 327–340 (Academic Press, 2010).
Neuzil, P., Zhang, C., Pipper, J., Oh, S. & Zhuo, L. Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes. Nucleic Acids Res. 34, e77 (2006).
Maltezos, G. et al. Exploring the limits of ultrafast polymerase chain reaction using liquid for thermal heat exchange: a proof of principle. Appl. Phys. Lett. 97, 264101 (2010).
Blair, J.M.A., Webber, M.A., Baylay, A.J., Ogbolu, D.O. & Piddock, L.J.V. Molecular mechanisms of antibiotic resistance. Nat. Rev. Microbiol. 13, 42–51 (2015).
Jia, B. et al. CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res. 45, D566–D573 (2017).
Liu, B. & Pop, M. ARDB—Antibiotic Resistance Genes Database. Nucleic Acids Res. 37, D443–D447 (2009).
Hillemann, D., Rüsch-Gerdes, S. & Richter, E. Evaluation of the GenoType MTBDRplus assay for rifampin and isoniazid susceptibility testing of Mycobacteriumtuberculosis strains and clinical specimens. J. Clin. Microbiol. 45, 2635–2640 (2007).
Gaudet, M., Fara, A.G., Beritognolo, I. & Sabatti, M. in Single Nucleotide Polymorphisms: Methods and Protocols 415–424 (Humana Press, 2009).
Mhlanga, M.M. & Malmberg, L. Using molecular beacons to detect single-nucleotide polymorphisms with real-time PCR. Methods 25, 463–471 (2001).
Poritz, M.A. et al. FilmArray, an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection. PLoS One 6, e26047 (2011).
De Wilde, B. et al. Target enrichment using parallel nanoliter quantitative PCR amplification. BMC Genomics 15, 184 (2014).
Spurgeon, S.L., Jones, R.C. & Ramakrishnan, R. High throughput gene expression measurement with real time PCR in a microfluidic dynamic array. PLoS One 3, e1662 (2008).
Toumazou, C. et al. Simultaneous DNA amplification and detection using a pH-sensing semiconductor system. Nat. Methods 10, 641–646 (2013).
Chandler, D.P. et al. Integrated amplification microarrays for infectious disease diagnostics. Microarrays 1, 107–124 (2012).
Popowitch, E.B., O'Neill, S.S. & Miller, M.B. Comparison of the Biofire FilmArray RP, Genmark eSensor RVP, Luminex xTAG RVPv1, and Luminex xTAG RVP fast multiplex assays for detection of respiratory viruses. J. Clin. Microbiol. 51, 1528–1533 (2013).
Goodwin, S., McPherson, J.D. & McCombie, W.R. Coming of age: ten years of next-generation sequencing technologies. Nat. Rev. Genet. 17, 333–351 (2016).
Plummer, J.D., Deal, M.D. & Griffin, P.B. Silicon VLSI Technology: Fundamentals, Practice and Modeling (Prentice Hall, New York, 2000).
Hassibi, A., Vikalo, H., Riechmann, J.L. & Hassibi, B. Real-time DNA microarray analysis. Nucleic Acids Res. 37, e132 (2009).
Manickam, A., Chevalier, A., McDermott, M., Ellington, A.D. & Hassibi, A. A CMOS electrochemical impedance spectroscopy (EIS) biosensor array. IEEE Trans. Biomed. Circuits Syst. 4, 379–390 (2010).
Jang, B., Cao, P., Chevalier, A., Ellington, A. & Hassibi, A. in IEEE International Solid-State Circuits Conference—Digest of Technical Papers 436–437 (IEEE, 2009).
Wang, H., Chen, Y., Hassibi, A., Scherer, A. & Hajimiri, A. in IEEE International Solid-State Circuits Conference—Digest of Technical Papers 438–439 (IEEE, 2009).
Wittwer, C.T. High-resolution DNA melting analysis: advancements and limitations. Hum. Mutat. 30, 857–859 (2009).
Ririe, K.M., Rasmussen, R.P. & Wittwer, C.T. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245, 154–160 (1997).
Sanchez, J.A., Pierce, K.E., Rice, J.E. & Wangh, L.J. Linear-after-the-exponential (LATE)-PCR: an advanced method of asymmetric PCR and its uses in quantitative real-time analysis. Proc. Natl. Acad. Sci. USA 101, 1933–1938 (2004).
Schmittgen, T.D. & Livak, K.J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108 (2008).
Peirson, S.N., Butler, J.N. & Foster, R.G. Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res. 31, e73 (2003).
Thiel, A.J., Frutos, A.G., Jordan, C.E., Corn, R.M. & Smith, L.M. In situ surface plasmon resonance imaging detection of DNA hybridization to oligonucleotide arrays on gold surfaces. Anal. Chem. 69, 4948–4956 (1997).
Guo, X. Surface plasmon resonance based biosensor technique: a review. J. Biophotonics 5, 483–501 (2012).
Fritz, J., Cooper, E.B., Gaudet, S., Sorger, P.K. & Manalis, S.R. Electronic detection of DNA by its intrinsic molecular charge. Proc. Natl. Acad. Sci. USA 99, 14142–14146 (2002).
Xiao, Y., Lai, R.Y. & Plaxco, K.W. Preparation of electrode-immobilized, redox-modified oligonucleotides for electrochemical DNA and aptamer-based sensing. Nat. Protoc. 2, 2875–2880 (2007).
Burg, T.P. et al. Weighing of biomolecules, single cells and single nanoparticles in fluid. Nature 446, 1066–1069 (2007).
Arlett, J.L., Myers, E.B. & Roukes, M.L. Comparative advantages of mechanical biosensors. Nat. Nanotechnol. 6, 203–215 (2011).
Schena, M. DNA Microarrays: A Practical Approach (Oxford University Press, 1999).
Marras, S.A. in Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols 3–16 (Humana Press, 2006).
Ramsay, N. et al. CyDNA: synthesis and replication of highly Cy-dye substituted DNA by an evolved polymerase. J. Am. Chem. Soc. 132, 5096–5104 (2010).
Turner, D.H., Sugimoto, N. & Freier, S.M. Thermodynamics and kinetics of base-pairing and of DNA and RNA self-assembly and helix coil transition. Nucleic Acids 1, 201–227 (1990).
Lee, J.S., Hornsey, R.I. & Renshaw, D. Analysis of CMOS photodiodes. II. Lateral photoresponse. IEEE Trans. Electron Dev. 50, 1239–1245 (2003).
Manickam, A. et al. A fully integrated CMOS fluorescence biochip for DNA and RNA testing. IEEE J. Solid-State Circuits 52, 2857–2870 (2017).
Macleod, H.A. Thin-film Optical Filters (CRC Press, 2001).
Zhang, F. et al. Chemical vapor deposition of three aminosilanes on silicon dioxide: surface characterization, stability, effects of silane concentration and cyanine dye adsorption. Langmuir 26, 14648–14654 (2010).
The authors thank J. SantaLucia and the staff of DNA Software for their technical support and suggestions related to thermodynamic simulations for primer and probe design. Research reported in this publication was supported by the National Human Genome Research Institute of the National Institutes of Health under award R44HG007626. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
All of the authors listed in this paper were employees or contractors of InSilixa, Inc., with the exception of B.H., H.V. and B.A.P., who were academic collaborators on the project.
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Hassibi, A., Manickam, A., Singh, R. et al. Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip. Nat Biotechnol 36, 738–745 (2018). https://doi.org/10.1038/nbt.4179
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