US2021130873A1PendingUtilityA1

Method and Kit of Detecting the Absence of Micro-Organisms

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Assignee: MOMENTUM BIOSCIENCE LTDPriority: Jul 10, 2014Filed: Aug 25, 2020Published: May 6, 2021
Est. expiryJul 10, 2034(~8 yrs left)· nominal 20-yr term from priority
Inventors:Matthew A. CrowHelen V. BennettDaniel S. WrattingWilliam H. Mullen
G01N 2333/91245C12Q 2521/101C12Q 1/04C12Q 1/689C12Q 2525/113C12Q 1/6806C12Q 2525/125C12Q 1/6848C12Q 2525/101C12Q 2535/113C12Q 2545/107C12Q 1/44C12Q 2533/101C12Q 1/06
62
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Claims

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.

Claims

exact text as granted — not AI-modified
1 - 59 . (canceled) 
     
     
         60 . A method of detecting the presence of a micro-organism in a sample, the method comprising:
 (a) contacting the sample with a nuclease resistant nucleic acid molecule comprising a plurality of nuclease resistant nucleotides which is either extended by polymerase activity or is ligated by ligase activity of the micro-organism in the sample,   (b) incubating the thus contacted sample under conditions suitable for polymerase activity or ligase activity; and   (c) detecting in the sample a nucleic acid molecule that has been extended by the polymerase activity or ligated by the ligase activity of the micro-organism as compared to a negative control, thereby indicating the presence of the micro-organism in the sample.   
     
     
         61 . The method of  claim 60  wherein the nuclease resistant nucleic acid molecule comprises methylated nucleotides, nucleotides protected at the 3′ and/or 5′ ends or synthetic nucleotides. 
     
     
         62 . The method of  claim 61  wherein the synthetic nucleotides comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides. 
     
     
         63 . The method of  claim 60  wherein the action of the polymerase activity or ligase activity on the nuclease resistant nucleic acid molecule produces an extended nucleic acid molecule. 
     
     
         64 . The method of  claim 60  wherein step (a) comprises contacting the sample with a nucleic acid molecule which is either extended by polymerase activity or is ligated by ligase activity of the micro-organism in the sample together with an internal positive control (IPC) nucleic acid molecule, wherein:
 (a) the IPC nucleic acid molecule is susceptible to nuclease activity and is used to identify contaminating nuclease activity in the sample; or 
 (b) the IPC nucleic acid molecule is nuclease resistant and comprises a plurality of nucleotides that are resistant to nuclease activity. 
 
     
     
         65 . The method of  claim 63  wherein a nucleic acid probe is added in step (c) which binds to a target probe sequence within the nucleic acid molecule, optionally wherein the nucleic acid probe is labelled. 
     
     
         66 . The method of  claim 64  wherein a further nucleic acid probe is added in step (c) which binds to a target probe sequence within the IPC nucleic acid molecule, optionally wherein the further nucleic acid probe is labelled. 
     
     
         67 . The method of  claim 65  wherein:
 (a) the nucleic acid probe does not bind to the IPC nucleic acid molecule and the further nucleic acid probe does not bind to the nucleic acid molecule; or 
 (b) the nucleic acid probe and further nucleic acid probe are differently labelled. 
 
     
     
         68 . The method of  claim 60  wherein the nucleic acid molecule is at least partially double stranded and comprises uracil residues in the complementary strand and step (c) comprises adding Uracil DNA Glycosylase (UDG) to the sample in order to degrade the uracil residues in the complementary strand. 
     
     
         69 . The method of  claim 68  wherein the complementary strand of the nucleic acid molecule comprises a modification at the 3′ end to prevent extension. 
     
     
         70 . The method of  claim 69  wherein the modification at the 3′ end comprises incorporation of a non-extendible nucleotide. 
     
     
         71 . The method of  claim 70  wherein the non-extendible nucleotide is a dideoxy nucleotide triphosphate (ddNTP), optionally wherein the ddNTP is dideoxyCytidine. 
     
     
         72 . The method of  claim 60  wherein step (c) comprises a nucleic acid amplification step. 
     
     
         73 . A method of detecting the absence of a micro-organism in a sample, the method comprising:
 (a) contacting the sample with a nuclease resistant nucleic acid molecule comprising a plurality of nuclease resistant nucleotides which is either extended by polymerase activity or is ligated by ligase activity of the micro-organism in the sample,   (b) incubating the thus contacted sample under conditions suitable for polymerase activity or ligase activity; and   (c) detecting in the sample a lack of a nucleic acid molecule that has been extended by the polymerase activity or ligated by the ligase activity of the micro-organism as compared to a positive control, thereby indicating the absence of the micro-organism in the sample.   
     
     
         74 . The method of  claim 73  wherein the nuclease resistant nucleic acid molecule comprises methylated nucleotides, nucleotides protected at the 3′ and/or 5′ ends or synthetic nucleotides. 
     
     
         75 . The method of  claim 74  wherein the synthetic nucleotides comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides. 
     
     
         76 . The method of  claim 73  wherein the positive control is an internal positive control (IPC). 
     
     
         77 . The method of  claim 73  wherein the nucleic acid molecule is at least partially double stranded and comprises uracil residues in the complementary strand and step (c) comprises adding Uracil DNA Glycosylase (UDG) to the sample in order to degrade the uracil residues in the complementary strand. 
     
     
         78 . The method of  claim 77  wherein the complementary strand of the nucleic acid molecule comprises a modification at the 3′ end to prevent extension. 
     
     
         79 . The method of  claim 73  wherein step (c) comprises a nucleic acid amplification step. 
     
     
         80 . 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 the micro-organism in the 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. 
     
     
         81 . The kit of  claim 80  wherein the nuclease resistant nucleic acid molecule comprises synthetic nucleotides, methylated nucleotides or nucleotides protected of the 3′ and/or 5′ ends. 
     
     
         82 . The kit of  claim 81  wherein the synthetic nucleotides comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides. 
     
     
         83 . The kit of  claim 80  wherein the complementary strand of the nucleic acid molecule comprises a modification at the 3′ end to prevent extension. 
     
     
         84 . The kit of  claim 83  wherein the modification at the 3′ end comprises incorporation of a non-extendible nucleotide. 
     
     
         85 . The kit of  claim 84  wherein the non-extendible nucleotide is a dideoxy nucleotide triphosphate (ddNTP), optionally wherein the ddNTP is dideoxyCytidine. 
     
     
         86 . The kit of  claim 80  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. 
     
     
         87 . The kit of  claim 86  wherein the IPC nucleic acid molecule is modified so as to protect it from nuclease activity. 
     
     
         88 . The kit of  claim 87  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. 
     
     
         89 . The kit of  claim 88  wherein the synthetic nucleotides comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides.

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