Method for the preparation of purified HCV RNA by exosome separation
View Patent ↗The invention relates to a method for the isolation of hepatitis C virus. The method comprises the separation of particles termed exosomes from the blood plasma of an individual infected with hepatitis C virus (HCV) and the extraction or RNA from these exosome particles.
1. A method for preparation of purified HCV RNA comprising separating exosome particles from the supernatant of a cell culture infected with HCV and extracting RNA from the exosome particles.
2. A method according to claim 1 , wherein the exosome particles are separated from the plasma of an individual infected with hepatitis C virus.
3. A method according to claim 1 , wherein said exosome particles are prepared from the cell culture supernatant by differential centrifugation.
4. A method according to claim 3 , additionally comprising the step of incubating the cell culture supernatant with beads coated with antibody.
5. A method according to claim 1 , wherein said exosome particles are prepared from the cell culture supernatant by incubating the cell culture supernatant with beads coated with antibody.
6. A method according to claim 4 , wherein said antibody is anti-CD81 or anti-CD82 antibody.
7. A method according to claim 4 , wherein said beads are magnetic beads.
8. A method according claim 4 , wherein said beads are polystyrene beads.
9. A method according to claim 1 , wherein said HCV RNA is extracted from the exosome particles using a viral extraction kit.
10. A method according to claim 1 , wherein said exosome particles are enriched in CD81 protein.
11. A method according to claim 1 , wherein said cell culture is a human cell culture.
12. A method of diagnosing an individual as being infected with HCV, comprising obtaining a preparation of cells from the individual, preparing exosome particles from the cellular supernatant and testing the exosome particles for the presence of HCV RNA and proteins.
13. A method according to claim 12 , wherein said preparation of cells is a blood plasma preparation.
14. A method for preparing purified HCV RNA comprising:
(a) providing plasma from a subject infected with HCV;
(b) subjecting the plasma to differential centrifugation to produce an exosome-enriched supernatant;
(c) incubating the supernatant with beads coated with an anti-CD81 or anti-CD82 antibody to isolate exosome particles; and
(d) extracting HCV RNA from the exosome particles.
15. The method of claim 14 , wherein the subject is human.
16. The method of claim 14 , wherein the supernatant is incubated with an anti-CD81 antibody.
17. The method of claim 14 , wherein the supernatant is incubated with an anti-CD82 antibody.
18. The method of claim 14 , wherein the beads are magnetic beads.
19. The method of claim 14 , wherein the beads are polystyrene beads.
20. The method of claim 14 , wherein the HCV RNA is extracted from the exosome particles using a viral extraction kit.
21. A method for preparing purified HCV RNA comprising:
(a) providing plasma from a human infected with HCV;
(b) subjecting the plasma to differential centrifugation to produce an exosome-enriched supernatant;
(c) incubating the supernatant with magnetic beads coated with an anti-CD81 or anti-CD82 antibody to isolate exosome particles; and
(d) extracting HCV RNA from the exosome particles using a viral extraction kit.