Specific and sensitive nucleic acid detection method

• US 7,105,318 B2
• Filed: 12/17/2002
• Issued: 09/12/2006
• Est. Priority Date: 11/04/1997
• Status: Expired due to Fees

• Current Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Original Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Inventors: Haberhausen, Gerd, Kessler, Christoph, Bartl, Knut, Orum, Henrik
• Primary Examiner(s): STRZELECKA, TERESA E
• Application Number:
  US10/322,138
• Publication Number:
  US 20030175765A1
• Time in Patent Office: 1,365 Days
• Field of Search: None
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2561/101 Taqman

Abstract

Method for the detection of a nucleic acid comprising the production of a plurality of amplificates of a section of this nucleic acid with the aid of two primers, one of which can bind to a binding sequence A of the nucleic acid and the other can bind to a binding sequence C′ which is complementary to a sequence C which is located in the 3′ direction from A and does not overlap with A, contacting the amplificates with a probe having a binding sequence D which can bind to a sequence B which is located between the sequences A and C or to the complement thereof, and detecting the formation of a hybrid of the amplificate and probe where the sequence located between the binding sequences A and C contains no nucleotides that do not belong to the binding sequence D of the probe or its complement D′.

Claims

1. A method for the detection of several nucleic acids comprising the steps:

(a) simultaneously producing in a multiplex reaction a plurality of amplificates of sections of the nucleic acids with the aid of pairs of two primers, of which in each case one can bind to a binding sequence (A′

), which is essentially complementary to a sequence A of a strand of the nucleic acid, and of which the other can bind to a binding sequence C which is located in the 3′

direction from A and does not overlap A, wherein the primers are selected such that the amplificates that are formed do not differ by more than 20% in length and are not longer than 100 nucleotides;

(b) contacting the respective amplifcates with a probe that in each case has a binding sequence D or D′

, which can bind either to the sequence B located between the sequences A and C or to the complement thereof; and

(c) detecting the formation of a hybrid of the amplificate and a probe;

wherein the sequence located between the binding sequences A and C contains no nucleotides or less than 3 nucleotides that do not belong to the sequence section E formed from the binding sequence D of the probe and the sequence of the amplificate bound thereto and the amplificates are shorter than 100 nucleotides.

2. The method of claim 1, wherein amplificates of nucleic acids of HIV, HBV, and HCV are produced simultaneously.

3. The method of claim 1, wherein one of the pairs of two primers is selected from the group consisting of CK10 and CK20 (SEQ ID NOs:

17 and

     18), CK11 and CK20 (SEQ ID NOs;

19 and

     20), CK10-1 and CK20-1 (SEQ ID NOs;

21 and

     22), CK11-1 and CK20-1 (SEQ ID NO;

23 and

     22), CK10-2 and CK20-2 (SEQ ID NOs;

24 and

     25), CK11-2 and CK 20-2 (SEQ ID NOs;

26 and

     25), CK10 and CK21 (SEQ ID NOs;

17 and

     27), CK10-1 and CK21-1 (SEQ ID NOs;

28 and

     29), CK11-1 and CK21-1 (SEQ ID NOs;

30 and

     29), CK10-1 and CK21-2 (SEQ ID NOs;

28 and

     31), CK11-1 and CK21-2 (SEQ ID NOs;

30 and

     31), CK10-2 and CK21-3 (SEQ ID NOs;

24 and

     32), CK11-2 and CK21-3 (SEQ ID NOs;

26 and

     32), CK12 and CK22 (SEQ ID NOs;

33 and

     34), CK12-1 and 22-1 (SEQ ID NOs;

35 and

     36), CK12-1 and 22-2 (SEQ ID NOs;

35 and

     37), CK12-1 and CK22-3 (SEQ ID NOs;

35 and

     38), CK12-2 and CK22-4 (SEQ ID NOs;

39 and

     40), CK12-2 and CK22-5 (SEQ ID NOs;

39 and

     41), CK12 and CK23 (SEQ ID NOs;

33 and

     42), CK12-1 and CK23-1 (SEQ ID NOs;

35 and

     43), CK12-1 and CK23-2 (SEQ ID NOs;

35 and

     44), CK12-2 and CK23-3 (SEQ ID NOs;

39 and

     45), CK12 and CK24 (SEQ ID NOs;

33 and

     46), CK12 and CK24-1 (SEQ ID NOs;

33 and

     47), CK12-1 and CK24-2 (SEQ ID NOs;

35 and

     48), and CK12-2 and CK24-3 (SEQ ID NOs;

39 and

     49).

4. The method of claim 1, wherein one of the pairs of two primers and one of the probes is selected from the group of primer pairs and probes consisting of primer pair SK462 and SK431 (SEQ ID NOs:

50 and

     51), probe SK 102 (SEQ ID NO;

     52);

primer pair RAR1032 and RAR1033 (SEQ ID NOs;

53 and

     54), probe RAR 1034 (SEQ ID NO;

     55);

primer pair GH A1F and GH A1R (SEQ ID NOs;

56 and

     57), probe GH A1P (SEQ ID NO;

     58);

primer pair GH A2F and GH A2R (SEQ ID NOs;

59 and

     60), probe GH A2P (SEQ ID NO;

     61);

primer pair GH A3F and GH A3R (SEQ ID NOs;

62 and

     63), probe GH A3P (SEQ ID NO;

64);

primer pair GH A4F and GH A4R (SEQ ID NOs;

65 and

     66), probe GH A4P (SEQ ID NO;

     67);

primer pair GH A5F and GH A5R (SEQ ID NOs;

68 and

     9), probe GH A5P (SEQ ID NO;

     70); and

primer pair GH A6F and GH A6R (SEQ ID NOs;

71 and

     72), probe GH A6P (SEQ ID NO;

     73).

5. The method of claim 1, wherein one of the pairs of two primers and one of the probes is selected from the group of primer pairs and probes consisting of primer pair HBV-Forward and HBV-Reverse (SEQ ID NOs:

74 and

     75), probe (SEQ ID NO;

     76);

primer pair GHBV-1F and GHBV-1R (SEQ ID NOs;

77 and

     78), probe 1P (SEQ ID NO;

     79);

primer pair GHBV-2F and GHBV-2R (SEQ ID NOs;

80 and

     81), probe 2P (SEQ ID NO;

     82);

primer pair GHBV-3F and GHBV-3R (SEQ ID NOs;

83 and

     84), probe 3P (SEQ ID NO;

     85);

primer pair GHBV-4F and GHBV-4R (SEQ ID NOs;

86 and

     87), probe 4P (SEQ ID NO;

     88); and

primer pair GHBV-5F and GHBV-5R (SEQ ID NOs;

89 and

     90), probe 5P (SEQ ID NO

     91).

6. The method of claim 1, wherein the amplificates are detected by means of mass spectroscopy.

7. The method of claim 1, wherein the step of contacting the respective amplificates with a probe D or D′

is performed concurrently the step of producing the plurality of amplificates.