Nucleic acid detection system and method for detecting influenza
View Patent ↗The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.
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.
2. The method according to claim 1 , wherein the isolation of influenza virus particles comprises (a) incubating the clinical sample with magnetic beads functionalized with at least one affinity ligand capable of binding to the influenza virus particles, and (b) separating magnetic bead-bound cells and/or particles from other elements present in the clinical sample by applying a magnetic field to the sample.
3. The method according to claim 2 , wherein the affinity ligand is an antibody.
4. The method according to claim 2 , which further comprises a wash step following separation of magnetic bead-bound influenza virus particles from other elements present in the clinical sample.
5. The method according to claim 1 , wherein releasing the target nucleic acid from the influenza virus particles is achieved by (i) alkaline lysis followed by neutralization or (ii) heating.
6. The method according to claim 1 , in which the amplification primers of step (c) comprise a 5′ poly dA or poly dT sequence of between 5 and 20 bases in length.
7. The method according to claim 1 , wherein labeling the DNA amplification product is achieved concurrently with amplification of target nucleic acid.
8. The method according to claim 7 , in which the amplification primers have the sequences of SEQ ID NOS: 19 and 20.
9. 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 dependant 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) applying an aliquot of the solution containing the 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 capillary flow path and which comprises in series:
(i) a sample receiving zone for receiving an aliquot of a fluid sample;
(ii) a labeling zone in lateral flow contact with said sample receiving zone, wherein the labeling zone comprises a porous material containing at least one detection oligonucleotide diffusively bound thereto, which detection oligonucleotide is complementary to a first sequence of the influenza target nucleic acid and was previously coupled to a visually detectable label; and,
(iii) a capture zone in lateral flow contact with said labeling 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;
(e) allowing the solution to traverse through the labeling and capture zones, under conditions sufficient to enable the hybridization of both the first sequence and the second distinct sequence of the DNA amplification product directly to the detection and capture oligonucleotides respectively; and,
(f) detecting the presence of the 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.
10. The method according to claim 9 , wherein releasing the target nucleic acid from the influenza virus particles is achieved by (i) alkaline lysis followed by neutralization or (ii) heating.
11. The method according to claim 9 , in which the amplification primers of step (c) comprise a poly dA or poly dT sequence of between 5 and 20 bases in length.
12. The method according to claim 11 , in which the amplification primers have the sequences of SEQ ID NOS: 19 and 20.