Identification and quantification of a plurality of biological (micro)organisms or their components

• US 7,875,442 B2
• Filed: 05/17/2006
• Issued: 01/25/2011
• Est. Priority Date: 03/24/2000
• Status: Expired due to Fees

• Current Assignee: Eppendorf Array Technologies SA (Eppendorf Se)
• Original Assignee: Eppendorf Array Technologies SA (Eppendorf Se)
• Inventors: Zammatteo, Nathalie, Husar, Dieter, Margaine, Sylvain, Remacle, José , Koehn, Heinz, Alexandre, Isabelle
• Primary Examiner(s): Benzion; Gary
• Assistant Examiner(s): Wilder; Cynthia B
• Application Number:
  US11/436,383
• Publication Number:
  US 20070037187A1
• Time in Patent Office: 1,714 Days
• Field of Search: 435/91.2
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2561/113 Real time assay

Abstract

Disclosed is a system and method conducting real-time PCR. Unlabeled capture molecules of a specific design are immobilized on a solid support, and contacted with amplicons produced in one or more PCR cycles. Detection of amplicons may take place during or between the PCR cycles while the solid support is in fluidic contact with the PCR solution. In an alternate embodiment detection of the amplicons takes place when the solid support is not in fluidic contact with the PCR solution. The method is suitable for the simultaneous detection and quantification of closely homologous target molecules.

Claims

1. A process for real-time PCR of multiple target nucleotide sequences comprising the steps of:

a) providing a closed reaction chamber containing a PCR solution and covalently immobilized unlabeled capture molecules wherein said capture molecules are immobilized in specifically localized areas of a solid support in the form of a micro-array of at least 4 different capture molecules per cm², wherein the PCR solution comprises multiple target nucleotide sequences, primers specific to said target nucleotide sequences, and a fluorescent label and wherein the micro-array is in intermittent contact with the PCR solution;

b) carrying out the following steps without opening the closed reaction chamber;

(i) conducting at least one PCR cycle to form amplicons of the target nucleotide sequence wherein said amplicons arc formed with fluorescent label obtained using either labeled primer or by enzymatic incorporation of labeled nucleotide;

(ii) hybridizing the amplicons to immobilized unlabeled capture molecules; and

(iii) detecting the hybridized amplicons wherein the PCR solution contained in the chamber is moved away from the capture molecules during said detection;

(iv) repeating steps (i)-(iii) at least once

c) performing identification and/or quantification of the target nucleotide sequence corresponding to said capture molecules during the amplification cycles without washing.

2. The process according to claim 1, wherein the PCR cycle comprises the successive steps of:

a) denaturation;

b) annealing;

c) elongation; and

d) denaturation;

and wherein the capture molecules are in contact with the PCR solution only during the step of hybridizing and optionally during step d) of the PCR cycle.

3. The process according to claim 1, wherein the PCR cycle comprises the successive steps of

a) denaturation;

b) annealing;

c) elongation; and

d) denaturation;

and wherein the capture molecules are in contact with the PCR solution during every step except detecting the hybridized amplicons.

4. The process of claim 1, wherein the capture molecule comprises a spacer portion and a capture portion.

5. The process of claim 4 wherein the spacer portion is a polynucleotide chain having a length of at least 20 nucleotides, preferably 40 nucleotides, more preferably at least 90 nucleotides.

6. The process of claim 4, wherein the capture molecule comprises a spacer portion having at least 60% homology, preferably at least 80%, more preferably at least 90%, with SEQ ID NO:

2.

7. The process of claim 4, wherein the capture molecule comprises a spacer portion having at least 60% homology, preferably at least 80%, more preferably at least 90%, with SEQ ID NO:

3.

8. The process of claim 4, wherein the capture portion of the capture molecule contains from 10 to 100 nucleotides, preferably from 15 to 40 nucleotides, more preferably from 20 to 30 nucleotides specific of the amplicons produced during the PCR.

9. The process of claim 4, wherein the capture portion of the capture molecule is comprised between 10 and 600 bases, preferably between 20 and 50 bases, more preferably between 15 and 40 bases.

10. The process of claim 4, wherein the capture molecule is immobilized on a solid support such that the spacer portion is located between the solid support and the capture portion.

11. The process of claim 10 wherein the capture portion of the capture molecule is separated from the surface of the solid support by a spacer portion of at least 6.8 nm.

12. The process of claim 11, wherein said spacer portion is a nucleotide sequence of between about 20 and about 120 bases.

13. The process of claim 10 wherein the capture molecule is immobilized by its 5′

end.

14. The process of claim 10 wherein the capture molecule is immobilized by its 3′

end.

15. The process of claim 10, wherein the distal end of the spacer portion of the capture molecule has a nucleotide containing a free amino group.

16. The process of claim 10, wherein the density of capture molecules on the support is from 20 to 2000 fmoles/cm².

17. The process of claim 10, wherein the capture molecules comprise a capture portion of 10 to 100 nucleotides that is complementary to a specific sequence of the amplicons such that said capture portion define two non-complementary ends of the amplicons and a spacer portion having at least 20 nucleotides, and wherein the two non-complementary ends of the amplicons comprise a spacer end and a non-spacer end, respectively, such that the spacer end is non-complementary to the spacer portion of the capture molecule, and said spacer end exceeds said non-spacer end by at least 50 bases.

18. The process of claim 1, wherein the PCR cycle comprises forming labeled amplicons.

19. The process of claim 1, wherein the detection of the hybridized amplicons is performed by monitoring a signal from the hybridized amplicon, wherein the signal is also present in the solution.

20. The process of claim 1, wherein the PCR solution comprises a thermostable DNA polymerase enzyme that is active at a concentration in salt comprised between 25 and 300 mM.

21. The process of claim 20, wherein said polymerase enzyme is a Thermus aquaticus DNA polymerase enzyme.

22. The process of claim 1, wherein detection of the amplicons is performed by monitoring signals emanating from different locations of the array, with at least two measurements being done per location in at least two PCR cycles, and processing the data obtained in these measurements.

23. The process of claim 1, wherein the PCR solution contained in the chamber is moved away from the capture molecules by changing the position of the chamber and comprises turning the chamber upside down.

24. The process of claim 1, wherein the PCR cycle comprises an annealing step, and the step of detecting the hybridized amplicons is conducted within 5 minutes after the beginning of the annealing step.

25. The process of claim 1, wherein the PCR cycle comprises 3 temperature steps, and the step of detecting the hybridized amplicons is conducted at the end of at least one of the 3 temperature steps of the PCR cycle.

26. The process of claim 25, wherein the step of detecting the hybridized amplicons is conducted by performing at least two measurements during at least one of the 3 temperature steps of the PCR cycle.

27. The process of claim 25, wherein the 3 temperature steps are followed by a step of hybridization to the capture molecules, said step of hybridization being optionally preceded by a denaturation step.

28. The process of claim 19, wherein the monitored signal is the result of the accumulation of the amplicons on the capture molecules during the hybridization steps related to different PCR cycles.

29. The process of claim 25, further comprising a data processing step involving subtracting a first signal, obtained at the denaturation temperature step, from a second signal obtained at the annealing or elongation temperature step.

30. The process of claim 19, further comprising a step of comparing the number of PCR cycles necessary to reach a fixed value of the monitored signal (CT) with the CT of a reference nucleotide molecule, thereby quantifying the target nucleotide sequence in the PCR solution.

31. The process of claim 1, wherein the fluorescence signal of the amplicons in solution is lower than the fluorescence signal of the amplicons hybridized to the immobilized capture molecule.

32. The process of claim 31, wherein the lower fluorescence signal of the amplicons in solution is obtained by a difference in the optimal wavelength of fluorescence excitation between the amplicons present in solution and hybridized to the immobilized capture molecule.

33. The process of claim 31, wherein the lower fluorescence signal of the amplicons in solution is obtained by a difference in the optimal wavelength of fluorescence emission between the amplicons present in solution and hybridized to the immobilized capture molecule.

34. The process of claim 1, wherein the reaction chamber comprises two compartments that are in fluidic contact with each other.

35. The process according to claim 1, wherein said capture molecules are immobilized in specifically localized areas of a solid support in the form of a micro-array of at least 20 different capture molecules per cm².

36. The process according to claim 1, wherein the step of hybridizing the amplicons to immobilized unlabeled capture molecules is conducted during the annealing step of the PCR cycle.