Red blood cell lysis solution
View Patent ↗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.
1. A method of analyzing a target RNA 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 target RNA released from the red blood cells; and
(b) analyzing the target RNA released from the red blood cells, wherein said analyzing comprises performing an amplification reaction to amplify the target RNA.
2. The method of claim 1 , wherein the target RNA is a pathogen-derived target.
3. The method of claim 1 , wherein analyzing the target RNA further comprises contacting the released target RNA 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 RNA, and a second segment complementary to the immobilized probe, wherein the target RNA binds to the capture probe, and wherein the bound capture probe binds to the immobilized probe.
4. The method of claim 3 , wherein the amplification reaction is a transcription mediated amplification reaction and analyzing the target RNA further comprises detecting the resulting amplification product with a detection probe.
5. The method of claim 1 , wherein the target RNA is 18S rRNA from a pathogenic organism of the genus Babesia.
6. The method of claim 5 , wherein the pathogenic organism is of the species Babesia microti.
7. The method of claim 6 , wherein the limit of detection is at least 2×10 3 copies of Babesia microti 18S rRNA per 1 mL of whole blood.
8. The method of claim 1 , wherein the concentration of lithium lauryl sulfate (LLS) is 5% to 8% (w/v).
9. The method of claim 1 , wherein the reagent further comprises an anti-coagulant.
10. The method of claim 8 , wherein the reagent further comprises sodium bicarbonate buffer.
11. The method of claim 10 , wherein the pH of the buffer is 7-8.
12. The method of claim 9 , wherein the anti-coagulant is EDTA, heparin, or citrate.
13. The method of claim 8 , wherein the concentration of ammonium chloride is 250 mM.
14. The method of claim 9 , wherein the concentration of LLS is 5% (w/v).
15. The method of claim 10 , wherein the concentration of sodium bicarbonate is 10-20 mM.
16. The method of claim 1 , wherein the reagent is admixed with whole blood at step (a) in a ratio of 3:1 (v/v).
17. A method of analyzing a target RNA 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 target RNA released from the red blood cells; and
(b) analyzing the target RNA released from the red blood cells, wherein said analyzing comprises performing an amplification reaction to amplify the target RNA.
18. The method of claim 17 , wherein the reagent further comprises an anti-coagulant.
19. The method of claim 17 , wherein analyzing the target RNA further comprises contacting the released target RNA 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 RNA, and a second segment complementary to the immobilized probe, wherein the target RNA binds to the capture probe, and wherein the bound capture probe binds to the immobilized probe.
20. The method of claim 17 , wherein the concentration of lithium lauryl sulfate (LLS) is 5% to 8% (w/v).