Novel compositions, methods and kits for microorganism detection

Abstract

Methods, compositions and kits for detecting microorganisms and/or profiling microbiota such as for example through use of nucleic acid amplification and detection.

1 .- 28 . (canceled) 
     
     
         29 . A method for detecting the presence of a microorganism nucleic acid in a sample, said method comprising:
 (a) distributing portions of a nucleic acid sample to individual reaction chambers situated within a support;   (b) performing parallel amplification reactions and forming amplification products in individual reaction chambers, wherein each amplification reaction contains a pair of amplification primers configured to produce an amplification product corresponding to said target nucleic acid sequence present within, or derived from, the genome of a microorganism, wherein said amplification product comprises a sequence listed in Table 4 (i.e., SEQ ID NOs:1 through 34); and   (c) determining whether said amplification product has been formed in one or more of said individual reaction chambers.   
     
     
         30 . (canceled) 
     
     
         31 . The method of  claim 29 , wherein said determining includes detecting hybridization of a detectably labeled probe to said amplification product, optionally in real-time. 
     
     
         32 . The method of  claim 29 , wherein at least one pair of said amplification primers configured to produce an amplification product corresponding to said target nucleic acid sequence includes primers containing a nucleic acid sequence that is complementary or identical to a portion of said corresponding target nucleic acid sequence. 
     
     
         33 . (canceled) 
     
     
         34 . The method of  claim 29 , wherein said corresponding target nucleic acid sequence is present within or is derived from genomic DNA, RNA, miRNA, mRNA, cell-free DNA, circulating DNA or cDNA of a target microorganism. 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 29 , wherein said forming includes forming in parallel between 10 and 10,000 different amplification products. 
     
     
         38 . The method of  claim 29 , wherein at least two of said amplification reactions each contains a pair of amplification primers configured to amplify a different corresponding target nucleic acid sequence. 
     
     
         39 .- 41 . (canceled) 
     
     
         42 . The method of  claim 29 , wherein said forming includes forming one or more amplification products containing a nucleic acid sequence that is complementary or identical to a portion of a gene listed in Table 2. 
     
     
         43 . The method of  claim 29 , wherein each of said plurality of amplification reactions contains a set of amplification primers configured to produce an amplification product that is between 50 to 300 nucleotides long. 
     
     
         44 .- 46 . (canceled) 
     
     
         47 . The method of  claim 29 , wherein one or more of said amplification reactions further contains a detectably labeled probe that includes a sequence that is identical or complementary to a portion of the amplification product corresponding to said target nucleic acid sequence. 
     
     
         48 . The method of  claim 47 , wherein said detectably labeled probe of at least one amplification reaction is configured to undergo cleavage by a polymerase in a 5′ nuclease assay. 
     
     
         49 . (canceled) 
     
     
         50 . (canceled) 
     
     
         51 . The method of claim of  claim 29 , wherein at least one of said amplification reactions occurs at an individual reaction site present within or upon a support, said support containing one or more individual reaction sites. 
     
     
         52 . (canceled) 
     
     
         53 . The method of  claim 51 , wherein said individual reaction site includes one or more of said amplification primers, and said amplifying further includes distributing a portion of the nucleic acid sample to said individual reaction site. 
     
     
         54 . (canceled) 
     
     
         55 . (canceled) 
     
     
         56 . The method of  claim 29 , wherein said nucleic acid sample is prepared from a vaginal swab specimen. 
     
     
         57 . The method of  claim 29 , comprising preparing said nucleic acid sample from a vaginal swab specimen prior to said distributing. 
     
     
         58 .- 88 . (canceled) 
     
     
         89 . A composition for determining the presence or absence of at least one target nucleic acid from one or more of the microorganisms listed in Table 1 in a biological sample, said composition comprising:
 (a) at least one amplification primer pair, wherein each of said primers of said pair comprises a target hybridization region that is configured to specifically hybridize to all or a portion of a region of said target nucleic acid and wherein under suitable conditions said primer pair generates an amplicon which comprises a sequence listed in Table 4 (i.e., SEQ ID NOs:1 through 34); and   (b) at least one detection probe configured to specifically hybridize to all or a portion of a region of said amplicon produced by said primer pair.   
     
     
         90 . The composition of  claim 89 , wherein said amplicon is between 50 to 300 nucleotides long. 
     
     
         91 .- 105 . (canceled) 
     
     
         106 . A method of profiling gene expression in a human subject, said method comprising determining, for a set of genes, a level of RNA or DNA encoded by said genes in a biological sample of said subject, wherein said set comprises the genes identified as those listed in Table 2 or any combination thereof. 
     
     
         107 . The method of  claim 106 , wherein said method comprises amplification and detection of said set of genes using a set of assays selected from those listed in Table 3. 
     
     
         108 . (canceled) 
     
     
         109 . The method of  claim 106 , wherein said method comprises generation of at least one amplification product corresponding to said RNA or DNA encoded by said genes, characterized in that said amplification product comprises a sequence selected from those listed in Table 3. 
     
     
         110 . The method of  claim 106 , wherein said set of genes comprises 2 to 34 different genes.