Methods and materials for detecting viral or microbial infections

Abstract

This document provides methods and materials for detecting target nucleic acid. For example, methods and materials for detecting the presence or absence of target nucleic acid, methods and materials for detecting the amount of target nucleic acid present within a sample, kits for detecting the presence or absence of target nucleic acid, kits for detecting the amount of target nucleic acid present within a sample, and methods for making such kits are provided.

What is claimed is: 
     
         1 . A method for assessing a mammal for an infection, said method comprising:
 (a) contacting a sample from said mammal with a probe nucleic acid comprising an amplifying restriction endonuclease and a nucleotide sequence complementary to a sequence of a target nucleic acid present within a microorganism or virus under conditions wherein, if said target nucleic acid is present in said sample, at least a portion of said target nucleic acid hybridizes to at least a portion of said probe nucleic acid to form a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site,   (b) contacting said double-stranded portion of nucleic acid with a recognition restriction endonuclease having the ability to cut said double-stranded portion of nucleic acid at said restriction endonuclease cut site under conditions wherein said recognition restriction endonuclease cleaves said double-stranded portion of nucleic acid at said restriction endonuclease cut site, thereby separating a portion of said probe nucleic acid comprising said amplifying restriction endonuclease from at least another portion of said probe nucleic acid,   (c) contacting said portion of said probe nucleic acid comprising said amplifying restriction endonuclease with a reporter nucleic acid comprising a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of said amplifying restriction endonuclease under conditions wherein said amplifying restriction endonuclease cleaves said reporter nucleic acid at said restriction endonuclease cut site of said amplifying restriction endonuclease, thereby separating a portion of said reporter nucleic acid from at least another portion of said reporter nucleic acid, and   (d) determining the presence or absence of said portion of said reporter nucleic acid, wherein the presence of said portion of said reporter nucleic acid indicates that said sample contains said target nucleic acid, thereby indicating that said mammal is infected with said microorganism or virus, and wherein the absence of said portion of said reporter nucleic acid indicates that said sample does not contain said target nucleic acid, thereby indicating that said mammal is not infected with said microorganism or virus.   
     
     
         2 . The method of  claim 1 , wherein said mammal is a human. 
     
     
         3 . The method of  claim 1 , wherein said mammal is a farm animal selected from the group consisting of bovine, porcine, and equine species. 
     
     
         4 . The method of  claim 1 , wherein said mammal is a dog or cat. 
     
     
         5 . The method of  claim 1 , wherein said infection is a microbial infection, and wherein said target nucleic acid is present within a microorganism. 
     
     
         6 . The method of  claim 1 , wherein said infection is a viral infection, and wherein said target nucleic acid is present within a virus. 
     
     
         7 . The method of  claim 1 , wherein said sample comprises a nasal or throat swab sample. 
     
     
         8 . The method of  claim 1 , wherein said sample is selected from the group consisting of nasal samples, throat samples, sputum samples, bronchial lavage samples, tissue samples, cellular samples, and blood samples. 
     
     
         9 . The method of  claim 1 , wherein, prior to step (a), said sample was cultured to enrich the population of microorganisms or viruses, if present, within said sample. 
     
     
         10 . The method of  claim 9 , wherein said sample was cultured for at least 30 minutes in the presence of enrichment medium. 
     
     
         11 . The method of  claim 1 , wherein, prior to step (a), said sample was processed to remove non-nucleic acid material from said sample, thereby increasing the concentration of nucleic acid, if present, within said sample. 
     
     
         12 . The method of  claim 11 , wherein said sample was subjected to a nucleic acid extraction technique. 
     
     
         13 . The method of  claim 1 , wherein, prior to step (a), said sample was subjected to a nucleic acid amplification technique to increase the concentration of microbial or viral nucleic acid, if present, within said sample. 
     
     
         14 . The method of  claim 13 , wherein said sample was subjected to a PCR-based technique designed to amplify said target nucleic acid. 
     
     
         15 . The method of  claim 1 , wherein, prior to step (a), said method comprises (i) culturing said sample to enrich the population of microorganisms or viruses, if present, within said sample and removing non-nucleic acid material from said sample, thereby increasing the concentration of nucleic acid, if present, within said sample or (ii) culturing said sample to enrich the population of microorganisms or viruses, if present, within said sample, removing non-nucleic acid material from said sample, thereby increasing the concentration of nucleic acid, if present, within said sample, and performing a nucleic acid amplification technique to increase the concentration of microbial or viral nucleic acid, if present, within said sample. 
     
     
         16 . The method of  claim 1 , wherein said probe nucleic acid is single-stranded probe nucleic acid. 
     
     
         17 . The method of  claim 1 , wherein said determining step comprises determining the amount of said target nucleic acid present within said sample. 
     
     
         18 . A method for assessing a mammal for an infection, said method comprising:
 (a) contacting a sample from said mammal with a probe nucleic acid comprising an initial amplifying restriction endonuclease and a nucleotide sequence complementary to a sequence of a target nucleic acid present within a microorganism or virus under conditions wherein, if said target nucleic acid is present in said sample, at least a portion of said target nucleic acid hybridizes to at least a portion of said probe nucleic acid to form a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site,   (b) contacting said double-stranded portion of nucleic acid with a recognition restriction endonuclease having the ability to cut said double-stranded portion of nucleic acid at said restriction endonuclease cut site under conditions wherein said recognition restriction endonuclease cleaves said double-stranded portion of nucleic acid at said restriction endonuclease cut site, thereby separating a portion of said probe nucleic acid comprising said initial amplifying restriction endonuclease from at least another portion of said probe nucleic acid,   (c) contacting said portion of said probe nucleic acid comprising said initial amplifying restriction endonuclease with a first nucleic acid comprising a secondary amplifying restriction endonuclease and a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of said initial amplifying restriction endonuclease under conditions wherein said initial amplifying restriction endonuclease cleaves said first nucleic acid at said restriction endonuclease cut site of said initial amplifying restriction endonuclease, thereby separating a portion of said first nucleic acid comprising said secondary amplifying restriction endonuclease from at least another portion of said first nucleic acid,   (d) contacting said portion of said first nucleic acid comprising said secondary amplifying restriction endonuclease with a second nucleic acid comprising said initial amplifying restriction endonuclease and a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of said secondary amplifying restriction endonuclease under conditions wherein said secondary amplifying restriction endonuclease cleaves said second nucleic acid at said restriction endonuclease cut site of said secondary amplifying restriction endonuclease, thereby separating a portion of said second nucleic acid comprising said initial amplifying restriction endonuclease from at least another portion of said second nucleic acid,   (e) contacting said portion of said second nucleic acid comprising said initial amplifying restriction endonuclease with a reporter nucleic acid comprising a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of said initial amplifying restriction endonuclease under conditions wherein said initial amplifying restriction endonuclease cleaves said reporter nucleic acid at said restriction endonuclease cut site of said initial amplifying restriction endonuclease, thereby separating a portion of said reporter nucleic acid from at least another portion of said reporter nucleic acid, and   (f) determining the presence or absence of said portion of said reporter nucleic acid, wherein the presence of said portion of said reporter nucleic acid indicates that said sample contains said target nucleic acid, thereby indicating that said mammal is infected with said microorganism or virus, and wherein the absence of said portion of said reporter nucleic acid indicates that said sample does not contain said target nucleic acid, thereby indicating that said mammal is not infected with said microorganism or virus.   
     
     
         19 . A method for assessing a mammal for an infection, said method comprising:
 (a) contacting a sample from said mammal with a probe nucleic acid comprising an initial amplifying restriction endonuclease and a nucleotide sequence complementary to a sequence of a target nucleic acid present within a microorganism or virus under conditions wherein, if said target nucleic acid is present in said sample, at least a portion of said target nucleic acid hybridizes to at least a portion of said probe nucleic acid to form a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site,   (b) contacting said double-stranded portion of nucleic acid with a recognition restriction endonuclease having the ability to cut said double-stranded portion of nucleic acid at said restriction endonuclease cut site under conditions wherein said recognition restriction endonuclease cleaves said double-stranded portion of nucleic acid at said restriction endonuclease cut site, thereby separating a portion of said probe nucleic acid comprising said initial amplifying restriction endonuclease from at least another portion of said probe nucleic acid,   (c) contacting said portion of said probe nucleic acid comprising said initial amplifying restriction endonuclease with a first reporter nucleic acid comprising a secondary amplifying restriction endonuclease and a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of said initial amplifying restriction endonuclease under conditions wherein said initial amplifying restriction endonuclease cleaves said first reporter nucleic acid at said restriction endonuclease cut site of said initial amplifying restriction endonuclease, thereby separating a portion of said first nucleic acid comprising said secondary amplifying restriction endonuclease from at least another portion of said first nucleic acid,   (d) contacting said portion of said first reporter nucleic acid comprising said secondary amplifying restriction endonuclease with a second reporter nucleic acid comprising said initial amplifying restriction endonuclease and a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of said secondary amplifying restriction endonuclease under conditions wherein said initial amplifying restriction endonuclease cleaves said second nucleic acid at said restriction endonuclease cut site of said secondary amplifying restriction endonuclease, thereby separating a portion of said second nucleic acid comprising said initial amplifying restriction endonuclease from at least another portion of said second nucleic acid, and   (e) determining the presence or absence of said portion of said first reporter nucleic acid, said second reporter nucleic acid, or both said first reporter nucleic acid and said second reporter nucleic acid, wherein said presence indicates that said sample contains said target nucleic acid, thereby indicating that said mammal is infected with said microorganism or virus, and wherein said absence indicates that said sample does not contain said target nucleic acid, thereby indicating that said mammal is not infected with said microorganism or virus.   
     
     
         20 . A kit for assessing a mammal for an infection, said kit comprises a probe nucleic acid comprising an amplifying restriction endonuclease and a nucleotide sequence complementary to a sequence of a target nucleic acid present in a microorganism or virus, wherein at least a portion of said target nucleic acid is capable of hybridizing to at least a portion of said probe nucleic acid to form a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site.