Methods for analyzing a target nucleic acid from red blood cells

Abstract

The invention provides a lysis reagent for lysing red blood cells, thereby releasing a target, such as RNA from a parasitic organism, in a form suitable for analysis. The reagent includes at least ammonium chloride and an anionic detergent, and may include an anti-coagulant. The reagent serves to lyse red blood cells, protect the released target from degradation in the lysate, and is compatible with subsequent steps for analysis of the target such as target capture, amplification, detection, or sequencing.

What is claimed is: 
     
         1 . A method of analyzing a target nucleic acid from red blood cells comprising:
 (a) contacting red blood cells with a reagent comprising ammonium chloride at a concentration of 200-300 mM and lithium lauryl sulfate (LLS) at a concentration of 4% to 15% (w/v), the reagent being effective to lyse the red blood cells and inhibit degradation of a target nucleic acid released from the red blood cells, wherein the target nucleic acid is from a pathogenic organism of the genus  Plasmodium ; and   (b) analyzing the target nucleic acid released from the red blood cells, wherein said analyzing comprises performing an amplification reaction to amplify the target nucleic acid.   
     
     
         2 . The method of  claim 1 , wherein the target nucleic acid is RNA. 
     
     
         3 . The method of  claim 1 , wherein analyzing the target nucleic further comprises contacting the released target nucleic acid with a capture probe and an immobilized probe prior to performing the amplification reaction, the capture probe having a first segment complementary to the target nucleic acid, and a second segment complementary to the immobilized probe, wherein the target nucleic acid binds to the capture probe, and wherein the bound capture probe binds to the immobilized probe. 
     
     
         4 . The method of  claim 3 , wherein the target nucleic acid is RNA and the amplification reaction is a transcription mediated amplification reaction, and wherein analyzing the target RNA further comprises detecting the resulting amplification product with a detection probe. 
     
     
         5 . The method of  claim 1 , wherein the pathogenic organism is of the species  Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax , or  Plasmodium knowlesi.    
     
     
         6 . The method of  claim 1 , wherein the concentration of lithium lauryl sulfate (LLS) is 5% to 8% (w/v). 
     
     
         7 . The method of  claim 1 , wherein the reagent further comprises an anti-coagulant. 
     
     
         8 . The method of  claim 6 , wherein the reagent further comprises sodium bicarbonate buffer. 
     
     
         9 . The method of  claim 8 , wherein the pH of the buffer is 7-8. 
     
     
         10 . The method of  claim 7 , wherein the anti-coagulant is EDTA, heparin, or citrate. 
     
     
         11 . The method of  claim 6 , wherein the concentration of ammonium chloride is 250 mM. 
     
     
         12 . The method of  claim 7 , wherein the concentration of LLS is 5% (w/v). 
     
     
         13 . The method of  claim 8 , wherein the concentration of sodium bicarbonate is 10-20 mM. 
     
     
         14 . The method of  claim 1 , wherein the reagent is admixed with whole blood at step (a) in a ratio of 3:1 (v/v). 
     
     
         15 . A method of analyzing a target nucleic acid from red blood cells comprising:
 (a) contacting red blood cells with a reagent comprising ammonium chloride at a concentration of 100-500 mM, lithium lauryl sulfate (LLS) at a concentration of 4% to 15% (w/v), and sodium bicarbonate buffer at a concentration of 10-20 mM and at pH 7-8, the reagent being effective to lyse the red blood cells and inhibit degradation of a target nucleic acid released from the red blood cells, wherein the target nucleic acid is from a pathogenic organism of the genus  Plasmodium ; and   (b) analyzing the target nucleic released from the red blood cells, wherein said analyzing comprises performing an amplification reaction to amplify the target nucleic acid.   
     
     
         16 . The method of  claim 15 , wherein the reagent further comprises an anti-coagulant. 
     
     
         17 . The method of  claim 15 , wherein analyzing the target nucleic acid further comprises contacting the released target nucleic acid with a capture probe and an immobilized probe prior to performing the amplification reaction, the capture probe having a first segment complementary to the target nucleic acid, and a second segment complementary to the immobilized probe, wherein the target nucleic acid binds to the capture probe, and wherein the bound capture probe binds to the immobilized probe. 
     
     
         18 . The method of  claim 15 , wherein the concentration of lithium lauryl sulfate (LLS) is 5% to 8% (w/v). 
     
     
         19 . A method for isolating a target nucleic acid from red blood cells, the method comprising:
 (a) mixing a sample containing red blood cells with a reagent comprising ammonium chloride at a concentration of 200-300 mM, lithium lauryl sulfate (LLS) at a concentration of 4% to 15% (w/v), and an anti-coagulant;   (b) incubating the mixture of step (a), wherein the red blood cells are lysed and a target nucleic acid in the red blood cells is released, wherein the target nucleic acid is from a pathogenic organism of the genus  Plasmodium;      (c) adding a target capture oligomer and an immobilized probe to the mixture, wherein the target capture oligomer comprises (i) a target hybridizing sequence that hybridizes with the target nucleic acid and (ii) a segment for attaching the target capture oligomer to the immobilized probe, thereby forming a capture hybrid complex comprising the target capture oligomer and the target nucleic acid; and   (d) separating the capture hybrid complex away from other components of the sample, thereby isolating the target nucleic acid from the red blood cells.   
     
     
         20 . The method of  claim 19 , wherein the target nucleic acid is RNA.