Identification of biological (micro) organisms by detection of their homologous 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 of said organism, wherein said specific nucleotide sequence presents a homology higher than 30% with at least 4 other homologous nucleotide sequences from other organisms comprising:
- amplifying said specific nucleotide sequence by PCR into double-stranded target nucleotide sequence using primer pairs which are capable of amplifying at least two of said homologous nucleotide sequences from other organisms so as to produce a full-length target nucleotide sequence having between 100 and 800 bases;
contacting said target nucleotide sequence resulting from the amplifying step with single-stranded capture nucleotide sequences, said single-stranded capture nucleotide sequences being covalently bound in an array to an insoluble solid support via a spacer comprising a nucleotide sequence of at least 40 bases in length, wherein said array comprises at least 4 different bound single-stranded capture nucleotide sequences/cm2 of solid support surface and wherein said capture nucleotide sequences comprise a nucleotide sequence of about 15 to about 40 bases which is able to specifically bind to said full-length target nucleotide sequence without binding to said at least 4 homologous nucleotide sequences; and
detecting specific hybridization of said target nucleotide sequence to said capture nucleotide sequences.
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Abstract
A method for identifying or quantifying an organism by a detecting its nucleotide sequence among at least 4 other homologous sequences comprising amplifying nucleic acids from the organism to generate target nucleotide sequences to be detected; contacting the target nucleotide sequences with single stranded capture nucleotide sequences bound by a single predetermined link to an insoluble solid support and discriminating the binding of a target nucleotide sequence specific of an organism with a signal resulting from a hybridization by complementary base pairing between the target nucleotide sequence and its corresponding capture nucleotide sequence is disclosed. The capture nucleotide sequence is bound to the insoluble solid support at a specific location on an array having a density of at least 4 different bound single stranded capture nucleotide sequences/cm2. The location of the signal on the array allows identification or quantification of the organism.
33 Citations
22 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 of said organism, wherein said specific nucleotide sequence presents a homology higher than 30% with at least 4 other homologous nucleotide sequences from other organisms comprising:
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amplifying said specific nucleotide sequence by PCR into double-stranded target nucleotide sequence using primer pairs which are capable of amplifying at least two of said homologous nucleotide sequences from other organisms so as to produce a full-length target nucleotide sequence having between 100 and 800 bases; contacting said target nucleotide sequence resulting from the amplifying step with single-stranded capture nucleotide sequences, said single-stranded capture nucleotide sequences being covalently bound in an array to an insoluble solid support via a spacer comprising a nucleotide sequence of at least 40 bases in length, wherein said array comprises at least 4 different bound single-stranded capture nucleotide sequences/cm2 of solid support surface and wherein said capture nucleotide sequences comprise a nucleotide sequence of about 15 to about 40 bases which is able to specifically bind to said full-length target nucleotide sequence without binding to said at least 4 homologous nucleotide sequences; 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)
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Specification