Enhanced methods of ribonucleic acid hybridization

Abstract

This disclosure relates to a method for increasing the hybridization efficiency of a probe and a target RNA in a sample, for example to identify a particular RNA present in the sample. The method includes heating a lysate sample comprising at least one target RNA, such as a tRNA, mRNA or rRNA, at a temperature of about 95° C. for a time sufficient to interfere with secondary structure of the RNA, wherein the time is short enough, such that the RNA in the cell lysate sample are not significantly degraded, and wherein the lysate comprises a cell lysis buffer comprising a chemical denaturant. To detect a target RNA in the lysate, the lysate is contacted with at least one detectable probe, such as a labeled probe, designed to specifically hybridize to the target RNA in the lysate.

We claim: 
     
         1 . A method of identifying a pathogen in a crude sample, the method comprising:
 providing a crude sample lysate from a subject suspected of having an infection with one or more pathogenic organisms;   contacting the sample lysate with one or more detectable nucleic acids capable of hybridizing to one or more target nucleic acids in the sample lysate, wherein the contacting takes place under conditions that allow hybridization of the one or more detectable nucleic acids to the one or more target nucleic acids and wherein hybridization takes place for one hour or less; and   detecting the one or more target nucleic acids, thereby identifying the pathogen in the sample lysate.   
     
     
         2 . The method of  claim 1 , wherein the one or more target nucleic acids are target RNA. 
     
     
         3 . The method of  claim 1 , wherein the pathogen is an infectious disease pathogen. 
     
     
         4 . The method of  claim 1 , wherein the pathogen is a virus. 
     
     
         5 . The method of  claim 1 , wherein the subject is human. 
     
     
         6 . The method of  claim 1 , further comprising two or more detectable nucleic acids with non-overlapping sequences. 
     
     
         7 . The method of  claim 1 , wherein the one or more target nucleic acids have a sequence that is conserved among strains of the same species of a pathogen. 
     
     
         8 . The method of  claim 1 , wherein the method comprises use of a microfluidic device or a microarray. 
     
     
         9 . The method of  claim 1 , wherein two or more detectable nucleic acids are used that bind specifically to a target nucleic acid that identifies the pathogen. 
     
     
         10 . The method of  claim 1 , further comprising contacting the sample lysate with a fluorescently labeled reporter nucleic acid that uniquely identifies a given RNA molecule. 
     
     
         11 . The method of  claim 1 , wherein the one or more detectable nucleic acids are detectably labeled with an isotopic or non-isotopic label. 
     
     
         12 . The method of  claim 11 , wherein the non-isotopic label comprises enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. 
     
     
         13 . A kit comprising a plurality of detectable nucleic acids for use in the method of  claim 1 , and instructions for its use. 
     
     
         14 . A kit comprising a plurality of detectable nucleic acids for use in the method of  claim 13 , and instructions for its use; wherein the reporter probes comprise a fluorescent tag.