SYSTEMS AND METHODS FOR POINT-OF-CARE AMPLIFICATION AND DETECTION OF POLYNUCLEOTIDES
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Abstract
Compositions and methods for amplifying and detecting solution-state polynucleotide targets in a single device are described. In one aspect, a method for a coupled isothermal amplification and detection process utilizes a coated solid support, including a solid substrate, a cationic layer, and a plurality of target-specific probes attached to the coated solid support. Polynucleotide targets in the sample are amplified by an isothermal amplification process involving in situ hybridization onto the coated solid support. The entire process can be carried out with a high degree of specificity under low salt conditions in less than one hour. Further aspects of the present invention include methods for coupled hybridization/detection of polynucleotide targets, coated silicon biosensors optimized for use with the coupled detection systems to provide visual detection of polynucleotide targets under visible light conditions, and kits for practicing the above described methods.
158 Citations
152 Claims
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1-39. -39. (canceled)
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40. A method for detecting the presence or absence of a polynucleotide target in a sample, comprising:
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a. providing a coated silicon support comprising a silicon substrate, a cationic layer, and a plurality of target-specific probes attached to the cationic layer; b. applying to the coated solid support the sample and a reaction medium; c. subjecting the sample to an isothermal amplification process capable of amplifying a polynucleotide target; d. subjecting reaction products from the isothermal amplification process to conditions suitable for hybridizing amplified polynucleotide targets to the probes; and e. optically detecting under visible light the presence or absence of polynucleotide targets bound to the probes. - View Dependent Claims (41, 42, 43, 44, 45, 48, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80)
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46-47. -47. (canceled)
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49-51. -51. (canceled)
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62. (canceled)
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81. (canceled)
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82. A method for detecting the presence or absence of a polynucleotide target in a sample, comprising:
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a. providing a coated solid support comprising a solid support and a plurality of target-specific probes attached to the coated solid support; b. applying to the coated solid support the sample and a reaction medium; c. providing conditions and reagents suitable for denaturing polynucleotide targets in the sample by an enzymatic process; d. providing conditions suitable for hybridizing enzymatically denatured polynucleotide targets in the sample to the target-specific probes; e. detecting the presence or absence of polynucleotide targets bound to the probes. - View Dependent Claims (150, 151, 152)
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83-119. -119. (canceled)
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120. A coated biosensor for visual detection of a polynucleotide target in a sample, comprising:
a coated silicon support comprising a silicon substrate, a cationic layer, and a plurality of target-specific probes attached to the silicon substrate, wherein the silicon support does not comprise a layer that produces a thin-film effect and is configured to enable optical detection under visible light of polynucleotide targets in the sample.
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121-149. -149. (canceled)
Specification