Catalytic nucleic acid probes as microbial indicators

Abstract

The disclosure relates to a method of generating catalytic nucleic acid probes useful for detecting microorganisms such as bacterial pathogens. In one embodiment, the catalytic nucleic acid probes are fluorogenic DNAzymes. The disclosure also relates to catalytic nucleic acid probes and methods of using the probes for detecting microorganisms.

1 - 37 . (canceled) 
     
     
         38 . A method of generating a catalytic nucleic acid probe for detecting a microorganism, the method comprising:
 (a) contacting a plurality of candidate nucleic acid molecules with a microorganism target, wherein each of the candidate nucleic acid molecules comprises a variable region of nucleic acid sequence and a constant region of nucleic acid sequence comprising a detectable substrate, wherein the detectable substrate is detectable only upon cleavage of the detectable substrate, and   (b) detecting the candidate nucleic acid molecule which (i) interacts with the microorganism target and (ii) has catalytic activity to cleave the detectable substrate upon interaction with the target, thereby generating a detectable signal,   wherein the detected candidate nucleic acid molecule is a catalytic nucleic acid probe.   
     
     
         39 . The method of  claim 38 , wherein the method further comprises contacting the plurality of candidate nucleic acid molecules with a negative control target prior to contacting the plurality of candidate nucleic acids with the microorganism target and detecting a candidate nucleic acid which (i) interacts with the microorganism target, (ii) has catalytic activity to cleave the detectable substrate upon interaction with the target, thereby generating a detectable signal and (iii) does not cleave the detectable substrate upon interaction with the negative control target. 
     
     
         40 . The method of  claim 38 , wherein the method further comprises:
 (A) selecting a plurality of candidate nucleic acid molecules which (i) interact with the microorganism target and (ii) have catalytic activity to cleave the detectable substrate upon interaction with the target, thereby generating a detectable signal, wherein the plurality of candidate nucleic acid molecules constitute a pool of candidate nucleic acid molecules,   (B) amplifying the variable region of nucleic acid sequence of each of the candidate nucleic acid molecules in the pool of candidate nucleic acid molecules,   (C) ligating each of the amplified regions to the constant region of nucleic acid sequence,   (D) contacting the ligated molecules with the microorganism target, and   (E) detecting a further plurality of candidate nucleic acid molecules which (i) interact with the microorganism target and (ii) have catalytic activity to cleave the detectable substrate upon interaction with the target, thereby generating a detectable signal, wherein the further plurality of candidate nucleic acid molecules constitute an enriched pool of candidate nucleic acid molecules.   
     
     
         41 . The method of  claim 40 , wherein the method comprises multiple cycles of steps (A) through (E). 
     
     
         42 . The method of  claim 38 , wherein the catalytic nucleic acid probe is a DNAzyme. 
     
     
         43 . The method of  claim 38 , wherein the detectable substrate is a single ribonucleotide linkage embedded in a DNA sequence. 
     
     
         44 . The method of  claim 43 , wherein the ribonucleotide linkage is flanked by a fluorophore modified nucleic acid residue on one side and a quencher modified nucleic acid residue on the other side and the fluorophore is quenched until the ribonucleotide linkage is cleaved, thereby generating a fluorogenic signal. 
     
     
         45 . The method of  claim 38 , wherein the microorganism target is found in the extracellular matrix of the microorganism. 
     
     
         46 . The method of  claim 38 , wherein the microorganism target comprises a protein, a DNA molecule, a RNA molecule, a small molecule, extracellular matrix, intracellular matrix, a cell of the microorganism, or any combination thereof. 
     
     
         47 . The method of  claim 38 , wherein the method further comprises the use of the catalytic nucleic acid probe for detecting the microorganism. 
     
     
         49 . A catalytic nucleic acid probe for detecting a microorganism, wherein the catalytic nucleic acid probe comprises a first nucleic acid region that (i) interacts with a microorganism target and (ii) has catalytic activity to cleave a detectable substrate upon interaction with the target, thereby generating a detectable signal. 
     
     
         50 . The catalytic nucleic acid probe of  claim 49 , wherein the catalytic nucleic acid probe further comprises a second nucleic acid region comprising the detectable substrate. 
     
     
         51 . The catalytic nucleic acid probe of  claim 50 , wherein the detectable substrate is a single ribonucleotide linkage embedded in a DNA sequence and the ribonucleotide linkage is flanked by a fluorophore modified nucleic acid residue on one side and a quencher modified nucleic acid residue on the other side such that the fluorophore is quenched until the ribonucleotide linkage is cleaved, thereby generating a fluorogenic signal. 
     
     
         52 . The catalytic nucleic acid probe of  claim 49 , wherein the catalytic nucleic acid probe comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or residues 1-99 of SEQ ID NO: 15 or a sequence with at least 80%, 90%, 95% or 99% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or residues 1-99 of SEQ 1D NO: 15. 
     
     
         53 . The catalytic nucleic acid probe of  claim 49 , wherein the catalytic nucleic acid probe comprises SEQ ID NO: 5 or a sequence with at least 80%, 90%, 95% or 99% sequence identity to SEQ ID NO: 5. 
     
     
         54 . The catalytic nucleic acid probe of  claim 49 , wherein the nucleic acid probe comprises (a) a sequence with at least 80%, 90%, 95% or 99% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and residues 1-99 of SEQ ID NO: 15 and (b) a sequence with at least 80%, 90%, 95% or 99% sequence identity to SEQ ID NO: 5. 
     
     
         55 . A method of detecting a microorganism in a sample comprising:
 exposing the sample to a catalytic nucleic acid probe, wherein the catalytic nucleic acid probe (i) interacts with a microorganism target and (ii) cleaves a detectable substrate upon interaction of the catalytic nucleic acid probe with the target thereby generating a signal that indicates the presence of the microorganism in the sample.   
     
     
         56 . The method of  claim 55 , wherein the detectable substrate is a single ribonucleotide linkage embedded in a DNA sequence and the ribonucleotide linkage is flanked by a fluorophore modified nucleic acid residue on one side and a quencher modified nucleic acid residue on the other side such that the fluorophore is quenched until the ribonucleotide linkage is cleaved, thereby generating a fluorogenic signal. 
     
     
         57 . The method of  claim 55 , wherein a single colony forming unit of the microorganism is detected. 
     
     
         58 . The method of  claim 55 , wherein the microorganism is a bacterial pathogen.