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Nucleic acid detection system and method for detecting influenza

  • US 8,980,561 B1
  • Filed: 08/22/2007
  • Issued: 03/17/2015
  • Est. Priority Date: 08/22/2006
  • Status: Active Grant
First Claim
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1. A stepwise method for detecting the presence of an influenza virus target nucleic acid in a clinical sample, comprising:

  • (a) isolating influenza virus particles which contain the target nucleic acid from the clinical sample using magnetic affinity capture;

    (b) releasing the target nucleic acid from the influenza virus particles;

    (c) amplifying influenza target nucleic acid using reverse transcriptase in combination with helicase dependent amplification, using amplification primers specific to the influenza target nucleic acid, to generate a solution containing a DNA amplification product corresponding to the influenza target nucleic acid sequence;

    (d) hybridizing a detection oligonucleotide complementary to a first sequence of the influenza target nucleic acid to the DNA amplification product, which first sequence does not overlap with the amplification primer binding regions on the influenza target nucleic acid, which detection oligonucleotide was previously coupled to a visually detectable label, to generate a solution containing labeled DNA amplification product;

    (e) applying an aliquot of the solution containing the labeled DNA amplification product to a sample receiving zone of a lateral flow chromatographic device, wherein the lateral flow chromatographic device comprises a lateral flow matrix which defines a flow path and which comprises in series;

    (i) a sample receiving zone for receiving an aliquot of a fluid sample; and

    ,(ii) a capture zone in lateral flow contact with said receiving zone, said capture zone comprising a microporous membrane, at least a portion of which contains at least one capture oligonucleotide immobilized thereto, which capture oligonucleotide is complementary to a second and distinct sequence of the influenza target nucleic acid;

    (f) the capture oligonucleotide hybridizing directly to the second and distinct sequence of the influenza target nucleic acid; and

    ,(g) detecting the presence of the influenza target nucleic acid by visually detecting the label at the site of the immobilized capture oligonucleotide;

    wherein the isolating, releasing, and amplifying steps are performed in a single chamber; and

    wherein the method achieves a sensitivity comparable to that of a typical homogeneous fluorescence assay while the detecting step is performable by the unaided human eye without the use of instrumentation.

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