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

• US 20070099204A1
• Filed: 05/24/2006
• Published: 05/03/2007
• Est. Priority Date: 03/24/2000
• Status: Active Grant

• Current Assignee: Eppendorf Array Technologies SA (Eppendorf Se)
• Original Assignee: Eppendorf Array Technologies SA (Eppendorf Se)
• Inventors: Remacle, Jose, Zammatteo, Nathalie, Husar, Dieter, Margaine, Sylvain, Koehn, Heinz, Alexandre, Isabelle
• Granted Patent: US 7,829,313 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: B01L 2200/0621 Control of the sequence of chambers filled or emptied
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0806 Standardised forms, e.g. compact disc [CD] format
• CPC Class Codes: B01L 2300/0822 Slides
• CPC Class Codes: B01L 2300/0832 cylindrical, tube shaped
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

Disclosed is a system and method conducting real-time PCR. 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 comprising the steps of:

a) conducting at least one PCR cycle to form amplicons;

b) hybridizing the amplicons to immobilized capture molecules;

c) detecting the hybridized amplicons; and

d) repeating steps a) b) c) at least once.

2. The process of claim 1, wherein the PCR cycle comprises the steps of denaturation, annealing, and elongation, wherein the PCR solution is in constant contact with the capture molecules so that the amplicons are hybridized to the capture molecules during the annealing step.

3. The process of claim 1, wherein the capture molecules are in intermittent contact with the PCR solution.

4. The process of claim 3 comprising the steps of

a) denaturation;

b) annealing;

c) elongation;

d) denaturation;

e) hybridization; and

f) detection;

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

5. The process of claim 3 comprising the steps of

a) denaturation;

b) annealing;

c) elongation;

d) denaturation;

e) hybridization; and

f) detection;

wherein the capture molecules are in contact with the PCR solution only during steps a) and e).

6. The process of claim 3 comprising the steps of

a) denaturation;

b) annealing;

c) elongation;

d) denaturation;

e) hybridization; and

f) detection,

wherein the capture molecules are in contact with the PCR solution during every step except step f).

7. The process of claim 1, wherein the capture molecule comprises a spacer portion being a polynucleotide chain having a length of at least 20 nucleotides and a capture portion containing from 10 to 100 nucleotides specific of the amplicons produced during the PCR.

20. The process of claim 7, 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.

21. The process of claim 7, wherein capture molecules are immobilized in specifically localized areas of a solid support in the form of a micro-array of at least 4 capture molecules per cm².

25. The process of claim 1, wherein the at least one PCR cycle comprises labeling said nucleotide sequence specific to said organism to form labeled target nucleotide sequences.

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

34. The process of claim 33, wherein said polymerase enzyme is a Thermus aquaticus DNA polymerase enzyme.

35. The process of claim 1, wherein the capture molecules are immobilized in an array, and detection of the amplicons is performed by monitoring a signal 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.

38. The process of claim 1, wherein the capture molecules are unlabeled.

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

41. The process of claim 1, wherein the capture molecules bear both a fluorescent quencher or acceptor and a fluorescent donor.

42. The process of claim 41, wherein the capture molecules are cut by an enzymatic activity.

43. The process of claim 1, wherein the step of detecting the hybridized amplicons and the at least one PCR cycle are conducted in one chamber.

45. The process of claim 43, wherein the solution contained in the chamber is moved away from the array during the step of detecting the hybridized amplicons.

47. The process of claim 45, wherein changing the position of the chamber comprises turning the chamber upside down.

53. The process of claim 1, wherein the PCR cycle comprises an annealing step, and a signal is measured within 5 minutes after the beginning of the annealing step.

54. The process of claim 1, wherein the PCR cycle comprises 3 temperature steps, and a signal is measured at the end of at least one of the 3 temperature steps of the PCR cycle.

55. The process of claim 54, wherein a signal is monitored with time by performing at least two measurements during at least one of the 3 temperature steps of the PCR cycle.

56. The process of claim 54, 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.

57. The process of claim 3, wherein the detected signal is the result of the accumulation of the amplicons on the capture molecules during the hybridization steps related to different PCR cycles.

60. The process of claim 54, 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 step.

64. The process of claim 1, wherein quantification of the biological organism or part of an organism in a sample is obtained by comparing the number of PCR cycles necessary to reach a fixed value (CT) with the CT of a reference nucleotide molecule.

66. The process of claim 1, wherein the quantification of the biological organism or part of an organism in a sample is obtained by comparing the number of PCR cycles necessary to reach a fixed value (CT) with a standard curve wherein CT values are plotted against standard concentrations.

68. The process of claim 1, wherein quantification of the biological organism or part of an organism in a sample is performed in terms of copy number in the sample, by comparing signal data of the target specific to the organism to a predetermined number of standard copies added to the analyzed solution.

69. The process of claim 68, wherein amplification of the different targets are performed by PCR having tailed primers, and using second primer(s) identical or complementary to the tail(s) of the tailed primers.

73. The process of claim 1, applied to a sample containing a nucleotide sequence having a homology higher than 30% with at least four other homologous nucleotide sequences that are potentially also present in the sample.

74. The process of claim 73, wherein the nucleotide sequence present in the sample differs by one nucleotide from other homologous nucleotide sequences that are potentially also present in the sample.

78. The process of claim 1, wherein the hybridization of an amplicon specific of an organism to the said capture molecules forms a single spot signal at a predetermined location, whereby the detection of said single spot signal allows the discrimination of the specific amplicon from homologous amplicons from other organisms.

86. The process of claim 1, wherein the step of detecting the hybridized amplicons comprises use of a method selected from the group consisting of:

colorimetry, fluorescence, time-resolved fluorescence, photothermal interference contrast, Rayleigh scattering, Raman scattering, surface plasmon resonance, change of mass, quartz crystal microbalances, cantilevers, differential pulse voltametry, chemical cartography by non linear generation frequency spectroscopy, optical change, resistivity capacitance, anisotropy, refractive index and/or counting nanoparticles.

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

89. The process of claim 87, wherein the lower 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 immobilized on the capture molecule.

90. The process of claim 87, wherein the lower 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 immobilized on the capture molecule.

92. A process for identifying and/or quantifying a gene of an organism in a sample potentially containing at least four nucleotide sequences that are homologous to said gene, said process being based on detecting the mRNA of the gene and comprising the step of:

a) copying the mRNA into a cDNA;

b) amplifying said cDNA or part of it into double-stranded target nucleotide sequences by at least two PCR cycles using a primer pair which is capable of amplifying at least two homologous nucleotide sequences from the same organism;

c) contacting said target nucleotide sequences with a single-stranded capture molecule, said single-stranded capture molecule being covalently bound in a location of an array to an insoluble solid support, and wherein said capture molecule comprises a capture portion of between 10 and 600 bases able to specifically bind to said target nucleotide sequence without binding to said at least four homologous nucleotide sequences; and

d) detecting specific hybridization of the said target nucleotide sequence to the said capture molecule, wherein the hybridization on capture molecules is combined in one process with the real time PCR process of claim 1.

113. An apparatus for use in the process of claim 1, said apparatus comprising:

a) capture molecules bound to an insoluble solid support surface at specific locations according to an array;

b) a device for thermal regulation;

c) a device for detecting a signal formed at the location of the binding between an amplicon and a capture molecule; and

d) a computer program for transforming the signal into digital data.

115. The apparatus of claim 113, further comprising a reaction chamber for PCR amplification, such that amplification and detection on the array are integrated into the same apparatus in order to detect the hybridized amplicons during the PCR cycles of amplification.

118. The apparatus of claim 113, further comprising a thermal cycler for carrying out an automated PCR amplification of nucleotide sequences obtained from an organism or part of an organism into double-stranded target nucleotide sequences, said thermal cycler being capable of alternately heating and cooling said support for producing labeled target nucleotides.

122. The apparatus of claim 113, comprising a detector selected from the method group consisting of:

colorimetry, fluorescence, time-resolved fluorescence, photothermal interference contrast, Rayleigh scattering, Raman scattering, surface plasmon resonance, change of mass, quartz crystal microbalances, cantilevers, differential pulse voltametry, chemical cartography by non linear generation frequency spectroscopy, optical change, resistivity, capacitance, anisotropy, refractive index and/or counting nanoparticles.

124. The apparatus of claim 113, further comprising:

a storage system for storing data from different measurements for at least 5 different locations of the support at a defined timing of a thermal cycle;

a controller repeating the steps of detection and storage at least one time in at least one thermal cycle for each location of array; and

a computer program for processing the data obtained in at least one thermal cycle in order to detect and/or quantify the amount of nucleotide molecule present in a sample before amplification.

125. The apparatus of claim 113, further comprising:

a) laser source;

b) a focusing device for a laser beam produced by said laser source;

c) a photomultiplier; and

d) a pin hole.

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93. A diagnostic and/or quantification kit of a biological organism or part of an organism comprising:

an insoluble solid support surface upon which single-stranded capture molecules are covalently bound, said capture molecules being disposed upon the surface of the solid support according to an array, wherein said capture molecules comprise a capture portion of between 10 and 600 bases which is able to specifically bind to a nucleotide sequence of said organism or part of it; and

a reaction chamber for performing a genetic amplification together with the identification and/or quantification of amplified nucleotide sequences from said organism or part of it wherein the detection for the presence of any amplified sequences of an organism and the genetic amplification are performed in real time.

98. The diagnostic and/or quantification kit of claim 93, wherein the reaction chamber is composed of two compartments being in fluidic contact with each other, one compartment being provided with an array of bound capture molecules.

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