Method and Kit of Detecting the Absence of Micro-Organisms

Abstract

Methods of detecting the absence or presence of a micro-organism in a sample comprising: contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, incubating the thus contacted sample under conditions suitable for nucleic acid modifying activity; and specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism. Corresponding kits are also provided.

1 - 59 . (canceled) 
     
     
         60 . A kit comprising at least one nuclease resistant nucleic acid molecule comprising a plurality of nuclease resistant nucleotides, wherein the at least one nuclease resistant nucleic acid molecule can be extended in the presence of polymerase activity or ligated in the presence of ligase activity of a micro-organism in a sample, wherein the at least one nuclease resistant nucleic acid molecule is at least partially double stranded and comprises uracil residues in the complementary strand. 
     
     
         61 . The kit of  claim 60  wherein the nuclease resistant nucleic acid molecule comprises synthetic nucleotides, methylated nucleotides or nucleotides protected of the 3′ and/or 5′ ends. 
     
     
         62 . The kit of  claim 61  wherein the synthetic nucleotides comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides. 
     
     
         63 . The kit of  claim 60  wherein the complementary strand of the nucleic acid molecule comprises a modification at the 3′ end to prevent extension. 
     
     
         64 . The kit of  claim 63  wherein the modification at the 3′ end comprises incorporation of a non-extendible nucleotide. 
     
     
         65 . The kit of  claim 64  wherein the non-extendible nucleotide is a dideoxy nucleotide triphosphate (ddNTP), optionally wherein the ddNTP is dideoxyCytidine. 
     
     
         66 . The kit of  claim 60  further comprising at least one internal positive control (IPC) nucleic acid molecule which comprises identical primer binding sites to the nuclease resistant nucleic acid molecule such that there is competition for primer binding in a nucleic acid amplification reaction containing both the nucleic acid molecule and the IPC. 
     
     
         67 . The kit of  claim 66  wherein the IPC nucleic acid molecule is modified so as to protect it from nuclease activity. 
     
     
         68 . The kit of  claim 67  wherein the modification of the IPC nucleic acid molecule is selected from incorporation of synthetic nucleotides, incorporation of methylation and protection of the 3′ and/or 5′ ends. 
     
     
         69 . The kit of  claim 68  wherein the synthetic nucleotides comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides. 
     
     
         70 . The kit of  claim 60  further comprising a nucleic acid probe which binds to a target probe sequence within the at least one nuclease resistant nucleic acid molecule. 
     
     
         71 . The kit of  claim 70  wherein the nucleic acid probe is labelled. 
     
     
         72 . The kit of  claim 66  further comprising a further nucleic acid probe which binds to a target probe sequence within the IPC nucleic acid molecule. 
     
     
         73 . The kit of  claim 72  wherein the further nucleic acid probe is labelled. 
     
     
         74 . The kit of  claim 60  further comprising a high pH reagent. 
     
     
         75 . The kit of  claim 74  wherein the high pH reagent comprises NaOH or Na 2 CO 3 . 
     
     
         76 . The kit of  claim 74  wherein the concentration of the high pH reagent is around 5 mM or greater. 
     
     
         77 . The kit of  claim 60  further comprising a pH lowering agent. 
     
     
         78 . The kit of  claim 77  wherein the pH lowering reagent comprises a buffer or an acid. 
     
     
         79 . The kit of  claim 78  wherein the buffer comprises a Tris-HCl buffer (pH 7.2 or 8).