Amplification and detection process

• US 5,849,544 A
• Filed: 01/24/1995
• Issued: 12/15/1998
• Est. Priority Date: 07/24/1992
• Status: Expired due to Term

• Current Assignee: Adelaide Children'S Hospital, Eppendorf AG
• Original Assignee: Adelaide Children'S Hospital, University Of Australia
• Inventors: Morris, Charles Phillip, Harris, Raymond John
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/374,764
• Time in Patent Office: 1,421 Days
• Field of Search: 435/5, 435/6, 435/91.2, 935/77, 935/78
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

This method for detecting a target nucleic acid sequence involves amplification and detection in the same vessel and comprises: (a) amplification of the target nucleic sequence in a vessel which is provided with a solid phase capture probe comprising a nucleic acid sequence capable of hybridizing to at least a portion of said amplified target nucleic acid sequence, said capture probe being incapable of participating or not participating in standard nucleic acid sequence amplification processes, (b) bringing a sample suspected of comprising said target nucleic acid sequence into contact with said capture probe under conditions which allow said amplified target nucleic acid sequence to be bound by said capture probe, and (c) detecting the presence of bound target nucleic acid sequence. In a further aspect, the present invention provides an assay system or kit, for detecting a target nucleic acid sequence in a sample suspected of comprising said target nucleic acid sequence, comprising: (a) a capture probe comprising a nucleic acid sequence capable of hydridizing to at least a portion of said amplified target nucleic acid sequence, said capture probe being immobilized on a solid phase support which forms a part of or is insertable into a container for the sample, and said capture probe being incapable of participating in standard nucleic acid sequence amplification processes, (b) reagents for amplification of said target nucleic acid sequence, and (c) means for detecting said target nucleic acid sequence, when bound by said capture probe.

Claims

1. A method for detecting a target nucleic acid sequence, which method involves amplification and detection in the same reaction vessel and comprises:

(a) amplification of the target nucleic acid sequence in a vessel which is provided with a solid phase capture probe comprising a nucleic acid sequence which hybridises to at least a portion of the said amplified target nucleic acid sequence, said capture probe not participating in nucleic acid sequence amplification processes, and said vessel being thermally stable, chemically derivatizable and thin-walled;

(b) bringing a sample suspected of comprising said amplified target nucleic acid sequence into contact with said capture probe under conditions which allow said amplified target nucleic acid sequence to be bound by said capture probe; and

(c) detecting the presence of bound amplified target nucleic acid sequence.

2. The method of claim 1, wherein the capture probe is attached to an inside surface of a reaction vessel or to a solid support which is immersible into a reaction vessel.

3. The method of claim 2, wherein the capture probe is attached to a dipstick, paramagnetic or non-paramagnetic beads, or an inside surface of a test tube, a well of a microtitre tray or modified microtitre tray lid with sections which are immersed or immersible into the reaction vessel.

4. The method of claim 1, wherein the target nucleic acid sequence is subjected to an amplification process.

5. The method of claim 4, wherein said amplification process is polymerase chain reaction;

ligase chain reaction;

nucleic acid sequence based amplification Q replicase dependent amplification or strand displacement amplification.

6. The method of claim 1, wherein said capture probe comprises an amplification blocking group or groups.

7. The method of claim 6, wherein the blocking group or groups comprise a 3'"'"'-sequence of a 5'"'"'-sequence which does not hybridise to the amplified target nucleic acid sequence;

a 3'"'"'-dideoxyribonucleotide or a 3'"'"'-terminus linked to the solid support.

8. The method of claim 1, wherein the 3'"'"'-terminus of the said capture probe is attached to the solid support.

9. The method of claim 1, wherein the 5'"'"'-terminus of the said capture probe is attached to the solid support.

10. The method of claim 1, wherein the 5'"'"'-terminus of the said capture probe does not comprise a 5'"'"'-phosphate group.

11. The method of claim 1, wherein the capture probe is linked through one terminus to a solid support and modified at the other terminus by any one of the preceding claims.

12. The method of claim 1, wherein said capture probe comprises a nucleic acid sequence which hybridises to at least a portion of said amplified target nucleic acid sequence a nucleic acid spacer arm between the point of attachment to a solid phase linker which is, in turn, attached to the solid support.

13. The method of claim 1, wherein said bound amplified target nucleic acid sequence is detected by means of a label incorporated therein.

14. The method of claim 13, wherein said detectable label is selected from the group consisting of florescent labels;

time resolved fluorescent labels;

biotin;

avidin;

streptavidin;

radiolabels;

enzymes;

dyes and rare earth metals.

15. The method of claim 1, wherein the amplified target nucleic acid sequence is selected from the group consisting of nucleic acid sequences specific to species of microorganisms;

nucleic acid sequences characteristic of or associated with individuals, genetic disorders or diseases and nucleic acid sequences determinant of tissue type, sex or taxonomic classification.

16. The method of claim 1, when utilised in a forensic application;

for maternity or parental testing;

or in quality control in agriculture, horticulture or the food or pharmaceutical industries.

20. The assay system or kit of claim 1, further comprising means for incorporating a detectable label in the bound target nucleic acid sequence.

21. The assay system or kit of claim 20, said detectable label is selected from the group consisting of fluorescent labels, time resolved fluorescent labels, biotin, avidin, streptavidin, radiolabels, enzymes, dyes, intercalators and rare earth metals.

22. The assay system or kit of claim 1, wherein the target nucleic acid sequence is selected from the group consisting of nucleic acid sequences specific to species of microorganisms, nucleic acid sequences characteristic of or associated with individuals, genetic disorders or disease and nucleic acid sequences determinant of tissue type, sex or taxonomic classification.

23. The assay system or kit of claim 1, adapted for use in a forensic application, for maternity or paternity testing, or in quality control in agriculture, horticulture or the food or pharmaceutical industry.

17. An assay system or kit, for detecting a target nucleic acid sequence in a sample suspected of comprising said target nucleic acid sequence, comprising:

(a) a capture probe comprising a nucleic acid sequence which hybridises to at least a portion of said amplified target nucleic acid sequence, said capture probe being immobilized on a solid phase support which forms a part of or is insertable into a container for the sample, said container being thermally stable, chemically derivatizable and thin-walled, and wherein said capture probe does not participate in standard nucleic acid sequence amplification processes,

(b) reagents for amplification of said target nucleic acid sequence, and

(c) means for detecting said target nucleic acid sequence, when bound by said capture probe in said container.

18. The assay system or kit of claim 17, wherein said solid phase support is a dipstick;

paramagnetic or non-paramagnetic beads;

or an inside surface of a well of a microtitre tray, a test tube or a microtitre tray lid with sections that are immersed or immersible into the reaction vessel.

19. The assay system or kit of claim 17, wherein the reagents for amplification of said target nucleic acid sequence are those for use in polymerase chain reaction, ligase chain reaction, nucleic acid sequence based amplification, Q replicase dependent amplification or strand displacement amplification.