IDENTIFICATION OF MULTIPLE BIOLOGICAL (MICRO) ORGANISMS BY DETECTION OF THEIR NUCLEOTIDE SEQUENCES ON ARRAYS
First Claim
1. A method for identifying and/or quantifying an organism or part of an organism in a sample by detecting a nucleotide sequence specific for said organism, among at least 4 other sequences from other organisms or from parts of the organisms comprising the steps of:
- producing derived sequences from said organism nucleotide sequences by incorporation of at least one common sequence in said organism nucleotide sequences in order to obtain a partial homology between the said specific derived nucleotide sequences;
amplifying said specific derived nucleotide sequences by PCR into double stranded target nucleotide sequences using a unique pair of primers, which recognize the common sequence of the derived sequences and which are capable of amplifying at least 4 of said other derived nucleotide sequences as to produce full-length target nucleotide sequences having between about 60 and about 800 bases;
contacting said full length target nucleotide sequences resulting from the amplifying step with at least 5 different single-stranded capture nucleotide sequences having between about 55 and about 800 bases, said single-stranded capture being covalently bound in an microarray to insoluble solid support(s) and said capture nucleotide sequences comprising a nucleotide sequence of at least 15 bases which is able to specifically bind to said full-length target nucleotide sequence without binding to said at least 4 other derived nucleotide sequences, and said specific sequence is separated from the surface of the solid support by a spacer comprising a nucleotide sequence of at least 40 bases in length; and
detecting specific hybridization of said target nucleotide sequence to said capture nucleotide sequences.
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Abstract
The present invention is related to a method for identifying and/or quantifying an organism or part of an organism in a sample by detecting a nucleotide sequence specific for said organism, among at least 4 other nucleotide sequences from other organisms or from parts of the organisms. The method includes the steps of: producing derived sequences from the organism nucleotide sequences by incorporation of at least one common sequence in said organism nucleotide sequences in order to obtain a partial homology between the said specific derived nucleotide sequences; amplifying said specific derived nucleotide sequences by PCR into double stranded target nucleotide sequences using a unique pair of primer(s), which recognize the common sequence of the derived sequences and which are capable of amplifying at least 4 other derived nucleotide sequences as to produce full-length target nucleotide sequences having between 60 and 800 bases; contacting said full-length target nucleotide sequences resulting from the amplifying step with at least 5 different single-stranded capture nucleotide sequences having between 55 and 800 bases, preferably between about 60 and about 450 bases, said single-stranded capture nucleotide sequences being covalently bound in an microarray to insoluble solid support(s) and said capture nucleotide sequences comprising a nucleotide sequence of at least 15 bases which is able to specifically bind to said full-length target nucleotide sequence without binding to said at least 4 other derived nucleotide sequences, and said specific sequence is separated from the surface of the solid support by a spacer comprising a nucleotide sequence of at least 40 bases in length; and detecting specific hybridization of said target nucleotide sequence to said capture nucleotide sequences present at specific locations.
43 Citations
60 Claims
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1. A method for identifying and/or quantifying an organism or part of an organism in a sample by detecting a nucleotide sequence specific for said organism, among at least 4 other sequences from other organisms or from parts of the organisms comprising the steps of:
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producing derived sequences from said organism nucleotide sequences by incorporation of at least one common sequence in said organism nucleotide sequences in order to obtain a partial homology between the said specific derived nucleotide sequences;
amplifying said specific derived nucleotide sequences by PCR into double stranded target nucleotide sequences using a unique pair of primers, which recognize the common sequence of the derived sequences and which are capable of amplifying at least 4 of said other derived nucleotide sequences as to produce full-length target nucleotide sequences having between about 60 and about 800 bases;
contacting said full length target nucleotide sequences resulting from the amplifying step with at least 5 different single-stranded capture nucleotide sequences having between about 55 and about 800 bases, said single-stranded capture being covalently bound in an microarray to insoluble solid support(s) and said capture nucleotide sequences comprising a nucleotide sequence of at least 15 bases which is able to specifically bind to said full-length target nucleotide sequence without binding to said at least 4 other derived nucleotide sequences, and said specific sequence is separated from the surface of the solid support by a spacer comprising a nucleotide sequence of at least 40 bases in length; and
detecting specific hybridization of said target nucleotide sequence to said capture nucleotide sequences. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42)
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33. The method of claim 62, wherein the amplification is a real time PCR.
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43. A method for identifying and/or quantifying at least 5 transcripts of a cell in a sample comprising the steps of:
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producing derived sequences from the parts of the transcript sequences present in the cell extract by incorporation of at least one common sequence in said parts of transcript sequences in order to obtain a partial homology between the said derived nucleotide sequences;
amplifying said derived nucleotide sequences as to produce full-length target nucleotide sequences having between 60 and 800 bases;
contacting said full-length target nucleotide sequences resulting from the amplifying step with at least 5 different single-stranded capture nucleotide sequences having between about 55 and about 800 bases, said single-stranded capture nucleotide sequences being covalently bound in an microarray to insoluble solid support(s) and said capture nucleotide sequences comprising a nucleotide sequence of at least 15 bases which is able to specifically bind to said full-length target nucleotide sequence, and said specific sequence is separated from the surface of the solid support by a nucleotide sequence of at least 40 bases in length; and
detecting specific hybridization of said target nucleotide sequence to said capture nucleotide sequences and quantifying the transcript expression level in the cell. - View Dependent Claims (44, 45, 46, 47, 48, 49, 50)
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51. A diagnostic and/or quantification kit which comprises
an insoluble solid support upon which single stranded capture nucleotide sequences are bound in an array, said single stranded capture nucleotide sequences containing a sequence of between about 10 and about 600 bases specific for a target nucleotide sequence to be detected and/or quantified and having a total length comprised between about 30 and about 800 bases comprising a spacer having a nucleotide sequence of at least 40 bases, said single stranded capture nucleotide sequences being disposed upon the surface of the solid support and an amplification (PCR) solution that comprises at least 5 different target specific primers and a universal primer pair, a thermostable DNA polymerase, a plurality of dNTPs and a buffered solution having a pH comprised between 7 and 9 for containing the primers.
Specification