Self-contained device integrating nucleic acid extraction, amplification and detection

• US 6,649,378 B1
• Filed: 11/02/2000
• Issued: 11/18/2003
• Est. Priority Date: 07/13/1995
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Xtrana Inc. (Bio-Techne Corporation)
• Inventors: Kozwich, Diane L., Gerdes, John C.
• Primary Examiner(s): SISSON, BRADLEY L
• Application Number:
  US09/705,043
• Time in Patent Office: 1,111 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/287.2, 536/23.1, 536/24.3, 536/24.31, 536/24.37, 475/287.2, 422/68.1, 422/69, 422/70
• US Class Current: 435/91.2
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/047 Additional chamber, reservoir
• CPC Class Codes: B01L 2300/049 Valves integrated in closure
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2400/0644 rotary valves
• CPC Class Codes: B01L 3/502 with fluid transport, e.g. in multi-compartment structures centrifugal-type cuvettes G01N21/07; analysis by separation into components G01N30/00; automatic analysers G01N35/00
• CPC Class Codes: B01L 3/5082 Test tubes per se
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/6867 Replicase-based amplification, e.g. using Q-beta replicase
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: C12Q 2547/101 by confinement to a single tube/container
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads

Abstract

Self-contained devices are described that integrate nucleic acid extraction, specific target amplification and detection into a single device. This integration permits rapid and accurate nucleic acid sequence detection. The invention may be used, for example, in the screening for nucleic acid sequences which may be indicative of genetic defects or contagious diseases, as well as for monitoring efficacy in the treatment of contagious diseases.

Claims

1. A method of detecting a target nucleic acid in a sample, wherein all of said method steps take place within a multi-chambered self-contained device having at least two chambers, said method comprising:

a) extracting said target nucleic acid, wherein said extraction includes binding said target nucleic acid to a solid phase or silica slurry;

b) amplifying said target nucleic acid on said solid phase or silica slurry with a first primer having a first label and a second primer having a second label under conditions that preferentially amplify said target nucleic acid to produce an amplification reaction mixture comprising target amplicons having said first label and said second label, wherein prior to amplification said nucleic acid present in a double-stranded form are rendered single-stranded;

c) transferring said reaction mixture to a detection chamber of said device; and

d) detecting said target amplicons within said detection chamber by a lateral flow assay, wherein said assay comprises wicking said target amplicons onto a membrane, wherein said target amplicons bind to at least a first and second reagent and at least one of said reagents is bound to said membrane, whereby when said at least two reagents are both bound to said target amplicons a visible signal is detected, wherein said amplification and said extraction take place within a first chamber of said device that is in fluid communication with said detection chamber, or said extraction takes place within a first chamber of said device and said amplification takes place in a second chamber of said device that is in fluid communication with both said first chamber and said detection chamber.

2. The method of claim 1, wherein said first reagent comprises colored microparticles having first receptors bound thereto, wherein said first receptors bind specifically to said first label on said target amplicons to form microparticle-bound amplicons and said second reagent comprises membrane-bound second receptors that bind specifically to said second label on said amplicons.

3. The method of claim 1, wherein said amplification methodology is polymerase chain reaction (PCR), ligase chain reaction, Qβ

replicase, strand displacement assay (SDA), nucleic acid sequence-based amplification (NASBA), or cascade rolling circle amplification (CRCA).

4. A method of detecting a target nucleic acid in a sample, wherein all of said method steps take place within a multi-chambered self-contained device having at least two chambers, said method comprising:

a) extracting said target nucleic acid, wherein said extraction includes binding said target nucleic acid to a solid phase or silica slurry;

b) amplifying said target nucleic acid on said solid phase or silica slurry with a first primer having a first label and to a second primer under conditions that preferentially amplify said target nucleic acid to produce an amplification reaction mixture comprising singly labeled target amplicons, wherein prior to amplification said nucleic acid present in a double-stranded form are rendered single-stranded;

c) transferring said reaction mixture to a detection chamber of said device; and

d) detecting said target amplicons within said detection chamber by a lateral flow assay, wherein said assay comprises wicking said target amplicons onto a membrane, wherein said singly labeled target amplicons bind to at least a first, a second and a third reagent and at least one of said reagents is bound to said membrane, whereby when said at least three reagents are all bound to said target amplicons a visible signal is detected, wherein said amplification and extraction take place within a first chamber of said device that is in fluid communication with said first chamber, or said extraction takes place within a first chamber of said device and said amplification takes place in a second chamber that is in fluid communication with both said first chamber and said detection chamber.

5. The method of claim 4, wherein said first reagent comprises colored microparticles having first receptors bound thereto, wherein said first receptors bind specifically to said label on said target amplicons, said second reagent comprises a labeled oligonucleotide capable of specifically hybridizing to said target amplicons, and said third reagent comprises receptors bound to said membrane and specific for said label on said oligonucleotide.

6. The method of claim 4, wherein said amplification methodology is polymerase chain reaction (PCR), ligase chain reaction, Qβ

replicase, strand displacement assay (SDA), nucleic acid sequence-based amplification (NASBA), or cascade rolling circle amplification (CRCA).

7. A method of detecting a target nucleic acid in a sample, wherein all of said method steps take place within a multi-chambered self-contained device having at least two chambers, said method comprising:

a) extracting said target nucleic acid, wherein said extraction includes binding said target nucleic acid to a solid phase or silica slurry;

b) amplifying said target nucleic acid on said solid phase or silica slurry with first and second complementary primers under conditions that preferentially amplify said target nucleic acid to produce an amplification reaction mixture comprising target amplicons, wherein prior to amplification said nucleic acid present in a double-stranded form are rendered single-stranded;

c) transferring said reaction mixture to a detection chamber of said device; and

d) detecting said target amplicons within said detection chamber by a lateral flow assay, wherein said assay comprises wicking said target amplicons onto a membrane, wherein said target amplicons bond to at least a first reagent, a second reagent, a third reagent, and a fourth reagent, and at least one of said reagents is bound to said membrane, whereby when said at least four reagents area all bound to said target amplicons a visible signal is detected, wherein said amplification and extraction take place within a first chamber of said device that is in fluid communication with said first chamber, or said extraction takes place within a first chamber of said device and said amplification takes place within a second chamber that is in fluid communication with both said first chamber and said detection chamber.

8. The method of claim 7, wherein said first reagent comprises an oligonucleotide comprising a first label and said second reagent comprises a second oligonucleotide comprising a second label, wherein said first and second oligonucleotides are capable of hybridizing to said target amplicon to form hybridized target amplicons comprising said first and second labels, said third reagent comprises first receptors specific for said first label on said hybridized target amplicon and having colored microparticles bound thereto, and said fourth reagent comprises membrane-bound second receptors specific for said second label on said hybridized target amplicon.

9. The method of claim 7, wherein said amplification methodology is polymerase chain reaction (PCR), ligase chain reaction, Qβ

replicase, strand displacement assay (SDA), nucleic acid sequence-based amplification (NASBA), or cascade rolling circle amplification (CRCA).

10. A method of detecting a target nucleic acid in a sample, wherein all of said method steps take place within a multi-chambered self-contained device having at least two chambers, said method comprising:

a) extracting said target nucleic acid, wherein said extraction includes binding said target nucleic acid to a solid phase or silica slurry;

b) hybridizing said target nucleic acid to a DNA-RNA-DNA probe having a first label on the 5′

end and a second label on the 3′

end to form a reaction mixture comprising a hybridized product having a first and second label, wherein prior to hybridization said nucleic acid present in a double-stranded form are rendered single-stranded;

c) transferring said reaction mixture to a detection chamber of said device; and

d) detecting said target nucleic acid within said detection chamber by a lateral flow assay, wherein said assay comprises;

i) contacting said hybridized product with colored microparticles having first receptors bound thereto, wherein said first receptors on said microparticles are specific for and bind to said first label;

ii) contacting said microparticle-bound hybridized product with RNase H to form a mixture, wherein said RNase H cleaves said probe only if said probe is hybridized to said target nucleic acid; and

iii) contacting said mixture from step ii) with a membrane having a capture zone, wherein said capture zone comprises second receptors specific for said second label, wherein said mixture wicks along said membrane to said capture zone and a visible line does not form at said capture zone if said target nucleic acid is present in said sample.

11. A method of detecting a target nucleic acid in a sample, comprising:

i) extracting said target nucleic acid, wherein extraction takes place in a first tube comprising a solid phase matrix that binds said target nucleic acid, said matrix tube being positioned inside a second tube having a closed end and an open end and lid for closing the open end, said second tube further containing a reagent cell positioned within said second tube and over said first tube and containing nucleic acid amplification reagents;

ii) amplifying said target nucleic acid bound on said solid phase matrix by releasing said amplification reagents comprising a first primer having a first label and a second primer having a second label from said reagent cell into said first tube under conditions that preferentially amplify said target nucleic acid to produce a reaction mixture comprising target amplicons having said first label and said second label, wherein prior to amplification said nucleic acid present in a double-stranded form are rendered single-stranded;

iii) inserting a result stick comprising a membrane having first and second reagents disposed thereon through a seal in said cap and into said reaction mixture, wherein when said target amplicons bind to both said first and second reagents a visible signal is detected, and wherein said extraction, amplification, and detection occur while said second tube open end is closed by said lid.

12. The method of claim 11, wherein said reagent cell further comprises at least one pouch containing a diluent.

13. The method of claim 11, wherein said first reagent comprises colored microparticles coated with first receptors specific for said first label and said second reagent comprises second receptors specific for said second label.

14. The method of claim 11, wherein said solid phase matrix is aluminum oxide or silica.

15. A method of detecting a target nucleic acid in a sample, wherein all of said method steps take place within a multi-chambered self-contained device having at least two chambers, said method comprising:

a) extracting said target nucleic acid, wherein said extraction includes binding said target nucleic acid to a solid phase or silica slurry;

b) amplifying said target nucleic acid on said solid phase or silica slurry with a first primer having a first label and to a second primer under conditions that preferentially amplify said target nucleic acid to produce an amplification reaction mixture comprising singly labeled target amplicons, wherein prior to hybridization said nucleic acid present in a double-stranded form are rendered single-stranded;

c) transferring said reaction mixture to a detection chamber of said device; and

d) detecting said target amplicons within said detection chamber by a lateral flow assay, wherein said assay comprises wicking said singly labeled target amplicons onto a membrane, wherein said singly labeled target amplicons bind to at least a first and a second reagent and at least one of said reagents is bound to said membrane, whereby when said at least two reagents are all bound to said target amplicons a visible signal is detected, wherein said amplification and extraction take place within a first chamber of said device that is in fluid communication with said first chamber, or said extraction takes place within a first chamber of said device and said amplification takes place in a second chamber that is in fluid communication with both said first chamber and said detection chamber.

16. The method of claim 15, wherein said first reagent comprises an oligonucleotide having a colored microparticle bound thereto and capable of specifically hybridizing to said target amplicon, and said second reagent comprises a receptor bound to said membrane and specific for said label on said target amplicon.