Method and kit for the detection and/or quantification of homologous nucleotide sequences on arrays
First Claim
1. A method for the identification and/or quantification of the presence of one or several among at least 7 organisms or part thereof in a biological sample, comprising the steps of:
- -amplifying a target nucleotide sequence specific for said organisms using at least 2 different primer pairs, each primer pair being capable of amplifying nucleotide sequences from at least 4 different organisms and having a nucleotide sequence homology higher than 85% with each of the specific nucleotide sequences to be amplified and wherein each primer of a pair is chosen in a sequence coding for a different protein;
-providing an array onto which single-stranded capture nucleotide sequences are arranged at pre-determined locations at a density of at least 10 different capture nucleotide sequences/cm2, said single-stranded capture nucleotide sequences being covalently bound to an insoluble support via a spacer which is at least 20 nucleotides in length, and comprising a nucleotide sequence of about 10 to 50 bases specific for a given target sequence of a specific organism and showing a homology of less than 85% to the other capture nucleotide sequences;
-contacting all of said target amplified sequences with the array in one solution under conditions allowing hybridization of the target amplified sequences to complementary capture nucleotide sequences present on the array;
-detecting and quantifying signals present on specific locations on the array;
wherein the intensities of the signals at specific locations allows identification and quantification of the presence in the biological sample of one or several among said at least 7 organisms.
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Abstract
The invention relates to a method and a kit for the specific identification and/or quantification of one or several among at least 7 organisms or parts thereof, in a biological sample being possibly contaminated by at least 4 other organisms, by detecting at least one nucleotide sequence specific of each of the organisms possibly present in said biological sample, wherein said nucleotide sequence is homologous with at least 4 other nucleotide sequences. The method comprises the steps of: amplifying the nucleotide sequences specific of said organisms into target amplified nucleotide sequences using at least 2 different primer pairs, each one being capable of amplifying at least 4 of said homologous nucleotide sequences from other organisms and having an homology higher than 85% with each of the said amplified homologous nucleotide sequences to be amplified; providing an array onto which single-stranded capture nucleotide sequences are arranged at pre-determined locations, said single-stranded capture nucleotide sequences being covalently bound to an insoluble support, via a spacer which is at least 6.8 nm in length, and wherein said capture nucleotide sequences comprise a nucleotide sequence of about 10 to 50 bases which is able to specifically bind to one target amplified sequence without binding to said other amplified homologous nucleotide sequences and presenting an homology lower than 85% with the other capture nucleotide sequences of the said other amplified homologous sequences, contacting said target amplified sequences with the array in one solution under conditions allowing hybridization of the target amplified sequences to complementary capture nucleotide sequences present on the array; and detecting and quantifying signals present on specific locations on the array; wherein the intensities of the signals in specific locations allows identification of the organisms.
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Citations
24 Claims
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1. A method for the identification and/or quantification of the presence of one or several among at least 7 organisms or part thereof in a biological sample, comprising the steps of:
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-amplifying a target nucleotide sequence specific for said organisms using at least 2 different primer pairs, each primer pair being capable of amplifying nucleotide sequences from at least 4 different organisms and having a nucleotide sequence homology higher than 85% with each of the specific nucleotide sequences to be amplified and wherein each primer of a pair is chosen in a sequence coding for a different protein; -providing an array onto which single-stranded capture nucleotide sequences are arranged at pre-determined locations at a density of at least 10 different capture nucleotide sequences/cm2, said single-stranded capture nucleotide sequences being covalently bound to an insoluble support via a spacer which is at least 20 nucleotides in length, and comprising a nucleotide sequence of about 10 to 50 bases specific for a given target sequence of a specific organism and showing a homology of less than 85% to the other capture nucleotide sequences; -contacting all of said target amplified sequences with the array in one solution under conditions allowing hybridization of the target amplified sequences to complementary capture nucleotide sequences present on the array; -detecting and quantifying signals present on specific locations on the array; wherein the intensities of the signals at specific locations allows identification and quantification of the presence in the biological sample of one or several among said at least 7 organisms. - 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)
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Specification