Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip

Abstract

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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Figure 1: NAAT platform architecture.
Figure 2: Example structure of DNA probes and melting signals for inverse fluorescence transduction versus conventional fluorescence methods for identifying the L511P mutation in the MTB gene rpoB.
Figure 3: Structure of the CMOS biochip and its individual components.
Figure 4: On-chip detection and quantification of respiratory viruses.
Figure 5: MTB drug-resistance mutation (DRM) panel.
Figure 6: Example MCA probe designs for SNP identification in view of DNA secondary structures.

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Acknowledgements

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.

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Contributions

A.H. conceived the technology. A.H., R.G.K. and G.S. co-supervised the project and wrote the manuscript with input from the other authors. Integrated biochip and optoelectronic components were designed and built by A.M., R. Singh, M.W.M., M.M., M.H., N.W. and E.K. In silico assay design and signal processing algorithms were developed by S.B., J.E., R. Sinha, P.K., B.H. and H.V. Assay implementation and performance validation on clinical samples were executed by P.N., G.D., K.A.J., T.V., G.M., K.B.J., L.P., M.P.S., P.M., B.A.P. and Y.L. Key technical contributions for chemistry, mechanical design and fluidics were provided by P.G., L.B., P.S., N.G., M.T.T., R.B.M. and S.C.

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Correspondence to Arjang Hassibi.

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Competing interests

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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