Method for discriminating between live and dead microbes in a sample

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.

1 - 14 . (canceled) 
     
     
         15 . 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.   
     
     
         16 . The method according to  claim 15 , wherein the RNA-labelling agent is a thiol-labelled RNA precursor. 
     
     
         17 . The method according to  claim 16 , wherein the thiol-labelled RNA precursor is selected from the group consisting of 4-thiouridine, 2-thiouridine, 2,4-dithiouridine, 2-thio-4-deoxyuridine, 5-carbethoxy-2-thiouridine, 5-carboxy-2-thiouridine, 5-(n-propyl)-2-thiouridine, 6-methyl-2-thiouridine and 6-(n-propyl)-2-thiouridine, thereby obtaining thiouridine-labelled RNAs. 
     
     
         18 . The method according to  claim 16 , wherein the thiol-labelled RNA precursor is 4-thiouridine, thereby obtaining thiouridine-labelled RNAs. 
     
     
         19 . The method according to  claim 15 , wherein conditions promoting nucleotide substitution comprise chemically modifying the labelled RNAs, thereby obtaining chemically-modified RNAs; and further reverse-transcribing said chemically-modified RNAs. 
     
     
         20 . The method according to  claim 15 , wherein conditions promoting nucleotide substitution comprise chemically modifying the labelled RNAs by alkylation, oxidative-nucleophilic-aromatic substitution or osmium-mediated transformation, thereby obtaining chemically-modified RNAs; and further reverse-transcribing said chemically-modified RNAs. 
     
     
         21 . The method according to  claim 15 , wherein conditions promoting nucleotide substitution comprise chemically modifying the labelled RNAs by alkylation, thereby obtaining chemically-modified RNAs; and further reverse-transcribing said chemically-modified RNAs. 
     
     
         22 . The method according to  claim 21 , wherein alkylation of the labelled RNAs is carried out using an alkylating agent selected from the group consisting of iodoacetamide, iodoacetic acid, N-ethylmaleimide and 4-vinylpyridine. 
     
     
         23 . The method according to  claim 21 , wherein alkylation of the labelled RNAs is carried out using iodoacetamide. 
     
     
         24 . The method according to  claim 15 , wherein the step of sequencing the first and the second set of RNAs comprises:
 (i) reverse-transcribing RNAs,
 thereby obtaining a cDNA library, 
   (ii) optionally, amplifying said cDNA library, and   (iii) sequencing said cDNA library.   
     
     
         25 . The method according to  claim 24 , wherein reverse-transcribing RNAs at step (i) converts uridine (U) in the labelled RNA to cytosine (C) instead of thymidine (T) in the cDNA library when the sample was cultured in presence of an RNA-labelling agent. 
     
     
         26 . The method according to  claim 24 , wherein reverse-transcribing RNAs converts uridine (U) in the labelled RNA to cytosine (C) instead of thymidine (T) in the cDNA library when the sample was cultured in presence of a thiol-labelled RNA precursor. 
     
     
         27 . The method according to  claim 24 , wherein, during reverse-transcription at step (i), RNAs undergo a first-strand synthesis with adenine (A)-to-guanosine (G) substitutions and a second-strand synthesis leading to thymidine (T)-to-cytidine (C) substitutions in the cDNA library when the sample was cultured in presence of an RNA-labelling agent. 
     
     
         28 . The method according to  claim 24 , wherein, during reverse-transcription at step (i), RNAs undergo a first-strand synthesis with A-to-G substitutions and a second-strand synthesis leading to T-to-C substitutions in the cDNA library when the sample was cultured in presence of a thiol-labelled RNA precursor. 
     
     
         29 . The method according to  claim 24 , wherein sequencing said cDNA library at step (iii) is performed by Next-Generation Sequencing (NGS), deep sequencing or targeted sequencing of custom sequences. 
     
     
         30 . The method according to  claim 15 , wherein the at least one microbial nucleic acid sequence hit is identified through:
 (i) optionally, filtering the first and/or second set of sequence reads,   (ii) optionally, assembling the sequence reads into contigs,   (iii) aligning the sequence reads or contigs onto a database comprising microbial nucleic acid sequences,   (iv) identifying the at least one microbial nucleic acid sequence hit mapped against at least one sequence read or contig, and   (v) optionally, re-aligning the sequence reads or contigs onto the microbial nucleic acid sequence hit identified in step (iv), thereby determining a consensus microbial nucleic acid sequence,   wherein the consensus microbial nucleic acid sequence corresponds to the microbial nucleic acid sequence hit.   
     
     
         31 . The method according to  claim 15 , wherein the at least one microbial nucleic acid sequence hit belongs to a live microbe if the number and/or rate of T-to-C substitutions in the sequence reads mapping against the at least one microbial nucleic acid sequence hit in the first set of sequence reads is greater than the number and/or rate of T-to-C substitutions in the sequence reads mapping against the at least one microbial nucleic acid sequence hit in the second set of sequence reads. 
     
     
         32 . The method according to  claim 15 , wherein the microbe is selected from the group consisting of viruses, bacteria, archaea, fungi and protozoans. 
     
     
         33 . A method of treating a subject affected with a microbial infection, comprising:
 (a) providing a sample from the subject,   (b) performing the method according to  claim 15  on said sample,   (c) diagnosing the subject as having a microbial infection if the at least one identified microbial nucleic acid sequence hit belongs to a live microbe, and   (d) treating the subject if said subject was diagnosed as having a microbial infection in step c).   
     
     
         34 . A method for assessing the risk of microbial contamination in a non-biological sample, comprising:
 (a) providing a non-biological sample,   (b) performing the method according to  claim 15  on said non-biological sample, and   (c) concluding that the non-biological sample is at risk of being contaminated if the at least one identified microbial nucleic acid sequence hit belongs to a live microbe.