Methods of performing nucleic acid stabilization and separation

Abstract

Methods are provided for the stabilization and separation of nucleic acids from a sample via contact of the sample with a lysis and stabilization reagent that includes a cationic detergent. The cationic detergent lyses cells in the sample and stabilizes the released nucleic acids via the formation of nucleic acid-surfactant (NAS) complexes. The NAS complexes are centrifugally precipitated, washed, the resuspended in an aqueous resuspension liquid, forming a NAS complex suspension. The suspension is thermally processed to disintegrate the NAS complexes, thereby releasing the nucleic acids and forming a nucleic acid solution. In some example embodiments, the aqueous resuspension liquid is selected to be suitable for performing molecular amplification assays, such that the nucleic acid solution may be employed for performing a molecular amplification assay in the absence of further nucleic acid extraction. Examples are provided whereby the present methods are adapted for performing transcriptomic biomarker assays.

1 . A method of performing a nucleic acid assay, the method comprising:
 a) obtaining a sample that has been stabilized by the use of a cationic detergent, the cationic detergent having formed complexes with nucleic acids residing within the sample;   b) separating at least a portion of the complexes from the sample and obtaining an aqueous suspension comprising the separated complexes;   c) heating the aqueous suspension to thermally dissociate the separated complexes, thereby obtaining a nucleic acid solution comprising the nucleic acids released from the separated complexes; and   d) in absence of an intervening nucleic acid extractions step, performing the nucleic acid assay to detect at least one nucleic acid present in the nucleic acid solution.   
     
     
         2 . The method according to  claim 1  wherein the aqueous suspension is heated above at least 50 degrees Celsius. 
     
     
         3 . The method according to  claim 1  wherein the aqueous suspension is heated above at least 70 degrees Celsius. 
     
     
         4 . The method according to  claim 1  wherein the aqueous suspension is heated above at least 80 degrees Celsius. 
     
     
         5 . The method according to  claim 1  wherein the aqueous suspension is heated for at least one minute. 
     
     
         6 . The method according to  claim 1  wherein the aqueous suspension is heated by Joule heating through an electric current applied thereto. 
     
     
         7 . The method according to  claim 1  wherein the sample comprises whole blood. 
     
     
         8 . The method according to  claim 1  wherein the sample contains less than 10% by weight of Tetradecyltrimethylammonium oxalate in an acidic solution (pH 3.7) of 2-4% Tartaric acid. 
     
     
         9 . The method according to  claim 1  wherein the complexes are separated centrifugally by:
 centrifuging the sample and precipitating the complexes, thereby obtaining a precipitated complexes; 
 centrifugally washing the precipitated complexes one or more times. 
 
     
     
         10 . The method according to  claim 1  wherein the complexes are separated by filtration. 
     
     
         11 . The method according to  claim 1  wherein the assay is a biomarker assay performed by contacting at least a portion of the nucleic acid solution with assay reagents comprising a primer set configured to detect a biomarker. 
     
     
         12 . The method according to  claim 11  wherein the biomarker is a messenger RNA biomarker. 
     
     
         13 . The method according to  claim 12  wherein the messenger RNA biomarker is associated with a genomic region comprising at least a portion of an intron. 
     
     
         14 . The method according to  claim 11  wherein the biomarker is associated with the response of a host to an infection. 
     
     
         15 . The method according to  claim 14  wherein the nucleic acid assay reagents are first assay reagents, and the primer set is a first primer set, the method further comprising:
 performing a housekeeping gene assay by contacting at least a portion of the nucleic acid solution with second assay reagents comprising a second primer set configured to detect housekeeping messenger RNA associated with a housekeeping gene; 
 wherein the biomarker assay generates a biomarker reverse transcription assay result quantifying an amount of the biomarker present in the nucleic acid solution, and the housekeeping gene assay generates a housekeeping assay result quantifying an amount of the housekeeping messenger RNA present in the nucleic acid solution. 
 
     
     
         16 . The method according to  claim 15  further calculating a comparative measure comparing the biomarker reverse transcription assay result to the housekeeping gene reverse transcription assay result. 
     
     
         17 . The method according to  claim 16  further comprising performing steps a) to d) one or more additional times on samples obtained at different time points; and
 processing the comparative measures associated with the different time points to infer a time-dependent host response. 
 
     
     
         18 . The method according to  claim 14  wherein the primer set is a first primer set, and wherein the assay reagents further comprise a second primer set configured to detect genomic DNA associated with a selected gene, wherein the assay generates a biomarker reverse transcription assay result quantifying an amount of the biomarker present in the nucleic acid solution and a genomic DNA assay result quantifying an amount of the genomic DNA present in the nucleic acid solution, and wherein signals respectively associated with products amplified by the first primer set and the second primer set are distinguished via melt curve analysis. 
     
     
         19 . The method according to  claim 18  wherein at least a portion of the selected gene comprises at least a portion of an intron. 
     
     
         20 . The method according to  claim 18  further calculating a comparative measure comparing the biomarker reverse transcription assay result to the genomic DNA assay result. 
     
     
         21 . The method according to  claim 18  further comprising performing steps a) to d) one or more additional times on samples obtained at different time points; and
 processing comparative measures associated with the different time points to infer a time-dependent host response. 
 
     
     
         22 . The method according to  claim 1  wherein steps b) to d) are performed in a disposable cartridge under the control of an automated instrument.