Crude biological derivatives competent for nucleic acid detection

Abstract

The invention relates generally to the fields of making biological unit lysates or admixtures of body fluids and of RNA analysis. More specifically, it relates to direct methods for the detection of a specific sequence of RNA in a biological unit, for example a virus, cell or tissue sample, or a body fluid, for example saliva, sputum, blood plasma, etc. More generally, the invention may be used to enzymatically manipulate and protect the RNA in lysate or bodily fluids for a number of applications.

1 - 66 . (canceled) 
     
     
         67 . A method comprising:
 obtaining at least one biological unit containing RNA or sample comprising RNA not comprised in a biological unit;   obtaining a high pH buffer;   mixing the biological unit and the buffer to prepare a high pH lysate or mixing the sample and the buffer to prepare a high pH admixture; and   mixing at least a portion of the lysate or admixture with a composition comprising an enzyme using RNA as a substrate to form a reaction mixture.   
     
     
         68 . The method of  claim 67 , wherein the lysate or admixture has a pH of from 9 to 14. 
     
     
         69 . The method of  claim 68 , wherein the lysate or admixture has a pH of greater than or equal to 11 and less than 14. 
     
     
         70 . The method of  claim 69 , further comprising mixing at least a portion of the lysate or admixture with a composition comprising reverse transcriptase to form a reverse transcriptase reaction mixture and incubating the reaction mixture under conditions resulting in a reverse transcription reaction. 
     
     
         71 . The method of  claim 70 , wherein the reverse transcriptase is comprised in a reverse transcriptase buffer that adjusts the pH of the reaction mixture to a level suitable for reverse transcriptase function. 
     
     
         72 . The method of  claim 71 , wherein the pH of the reaction mixture is between 7.0 and 9.5. 
     
     
         73 . The method of  claim 72 , wherein the pH of the reaction mixture is between 8.0 and 8.4. 
     
     
         74 . A method comprising:
 obtaining at least one biological unit containing RNA or sample comprising RNA not comprised in a biological unit;   obtaining a buffer;   mixing the biological unit and the buffer to prepare a low pH lysate or mixing the sample and the buffer to prepare an admixture, wherein the buffer precipitates RNA in the lysate or admixture; and   detecting and/or quantifying RNA in the biological unit or sample.   
     
     
         75 . The method of  claim 74 , wherein the buffer is a low pH buffer. 
     
     
         76 . The method of  claim 74 , wherein the buffer is a high pH buffer. 
     
     
         77 . The method of  claim 74 , wherein the buffer comprises a detergent. 
     
     
         78 . The method of  claim 77 , wherein the buffer comprises a non-ionic detergent. 
     
     
         79 . The method of  claim 77 , wherein the detergent comprises an anionic or cationic detergent.