Crude biological derivatives competent for nucleic acid detection
View Patent ↗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. A method comprising:
a) mixing at least one biological unit containing RNA, a non-ionic detergent, and a buffer having a pH below pH 3.5 to prepare a low pH lysate; and
b) mixing at least a portion of the low pH lysate directly with a composition comprising an enzyme that uses RNA as a substrate to form a reaction mixture;
wherein the RNA is not isolated from the lysate prior to b), and
wherein the lysate is not incubated with a protease prior to b); and
wherein the method further comprises a step of detecting one or more protein(s) in the low pH lysate.
2. The method of claim 1 , wherein the enzyme is in a buffer that adjusts the pH of the reaction mixture to a level suitable for enzyme function upon mixing.
3. The method of claim 1 , wherein the buffer comprises an acid having a pKa of <3.
4. The method of claim 3 , wherein the acid is arginine, glycine, or chloroacetic acid.
5. The method of claim 1 , wherein the enzyme that uses RNA as a substrate comprises reverse transcriptase.
6. The method of claim 5 , further comprising amplifying at least one cDNA product of the reverse transcription reaction.
7. The method of claim 5 , further comprising determining the presence of and/or quantity of an RNA in the biological unit.
8. The method of claim 5 , further comprising admixing an RNA control with the reaction mixture or the portion of the lysate prior to reverse transcription.
9. The method of claim 1 , wherein the protein is detected in an antibody-based assay.
10. The method of claim 9 , wherein the antibody-based assay comprises immunoblotting, ELISA, or immunoprecipitation.