Nucleic Acid Detection System and Method for Detecting Influenza
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 device for detecting the presence of one or more target nucleic acids in a fluid sample, the device comprising:
an absorbent sample receiving zone for receiving the fluid sample;
a detection zone comprising immobilized capture oligonucleotides, each capture oligonucleotide complementary to a capture sequence of one of the target nucleic acids; and
an absorbent material for transporting the sample from said sample receiving zone to said detection zone;
wherein said detection zone produces a signal visible by the unaided human eye without the use of instrumentation in less than approximately 30 minutes, the signal having a sensitivity comparable to that of a typical homogeneous fluorescence assay.
2 . The device of claim 1 wherein the signal is produced in approximately 5 minutes.
3 . The device of claim 2 wherein the signal is produced in approximately 2 minutes
4 . The device of claim 1 capable of detecting 20 fmoles of one of the target nucleic acids.
5 . The device of claim 1 wherein the target nucleic acids are viral.
6 . The device of claim 1 wherein said absorbent material comprises visible detection particles conjugated to detection oligonucleotides complementary to a detection sequence of one of the target nucleic acids.
7 . The device of claim 6 wherein said detection particles are selected from the group consisting of dyed microspheres, dyed polystyrene microspheres, dyed carboxyl-polystyrene microspheres, dyed microbeads, quantum dots, nano-gold particles, and colloidal gold particles.
8 . The device of claim 6 wherein said detection oligonucleotides comprise peptide nucleic acid (PNA) or locked nucleic acid (LNA).
9 . The device of claim 6 wherein said detection oligonucleotides comprise an inactive 3′.
10 . The device of claim 9 wherein said detection oligonucleotides are modified by an inactive phosphate group or thiolated group instead of an active 3′ hydroxy group, and thus may not be used for primer extension.
11 . The device of claim 6 wherein a concentration of detection particles is approximately 0.01% w/v per assay.
12 . The device of claim 1 wherein said absorbent material comprises a nitrocellulose membrane.
13 . The device of claim 1 wherein said sample receiving zone and/or said absorbent material been impregnated with a substance selected from the group consisting of Triton-X100, SDS, BSA, ficol, polyvinyl pyrolidone, and combinations thereof.
14 . The device of claim 1 wherein said absorbent material comprises a conjugate release pad.
15 . The device of claim 1 wherein a length of said absorbent material is 60 mm.
16 . The device of claim 1 comprising a width of 300 mm.
17 . The device of claim 1 wherein said detection zone comprises a plurality of spots or shapes, each spot or shape comprising a plurality of identical capture oligonucleotides complementary to a capture sequence of one of the target nucleic acids.
18 . The device of claim 17 wherein capture oligonucleotides present at different spots or shapes bind to different capture sequences of one or more of the target nucleic acids.
19 . The device of claim 17 wherein said plurality of spots or shapes comprises a microarray.
20 . The device of claim 1 operable with a sample volume of less than approximately 400 microliters.
21 . The device of claim 20 operable with a sample volume of less than approximately 200 microliters.
22 . The device of claim 21 operable with a sample volume of less than approximately 50 microliters.
23 . The device of claim 22 operable with a sample volume of approximately 20 microliters.
24 . The device of claim 1 wherein said capture oligonucleotides comprise peptide nucleic acid (PNA) or locked nucleic acid (LNA).