Method for discriminating between live and dead microbes in a sample
AssigneePathoquest
InventorsMarc ELOIT, Justine CHEVAL, Erika MUTH, Pascale BEURDELEY, Stéphane CRUVEILLER
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Abstract
A method for discriminating between live and dead microbes in a sample, by discriminating between transcriptionally-active and inert microbial nucleic acid sequences in the sample. In particular, the method is based on the comparison of levels of nucleotide substitution in a sample cultured in presence of an RNA-labelling agent. Also, a diagnosis method of microbial infections in a subject; and to methods of assessing the risk of contamination of a sample, implementing the method for discriminating between live and dead microbes in a sample.
Claims21
1-14. (canceled)
A method for discriminating between live and dead microbes in a sample, comprising discriminating between transcriptionally-active and inert microbial nucleic acid sequences in the sample, wherein the method comprises the steps of:
- (a) sequencing a first and a second set of RNAs extracted from the sample, wherein the first set of RNAs is obtained by culturing the sample in presence of an RNA-labelling agent and further by submitting the extracted RNAs to conditions promoting nucleotide substitution;
- and the second set of RNAs is obtained by culturing the sample in absence of an RNA-labelling agent;
- thereby obtaining a first and a second set of sequence reads;
- (b) comparing the number and/or rate of substituted nucleotides in the sequence reads mapping against at least one microbial nucleic acid sequence hit in the first and in the second set of sequence reads;
- and
- (c) concluding that the at least one microbial nucleic acid sequence hit belongs to a live microbe if the number and/or rate of substituted nucleotides in the sequence reads mapping against said at least one microbial nucleic acid sequence hit in the first set of sequence reads is greater than the number and/or rate of nucleotides randomly substituted in the second set of sequence reads.
Dependencies are inferred from the claim text.