Method, automated system, and cartridge for extracting cell-free nucleic acids from blood samples

Abstract

This invention provides a method for extracting cell-free nucleic acid fragments from blood samples to facilitate cancer diagnosis, prognosis, and monitoring, as well as prenatal screening. [Solution] The present invention provides a cartridge comprising a first compartment for filtering plasma from a blood sample, preferably also for fixing and rinsing cells to improve yield, and a second compartment for separating nucleic acids, wherein the first compartment comprises a hollow fiber membrane and the second compartment comprises a substance for binding nucleic acids or an electrophoretic gel. The present invention also provides an automated system comprising a device having a docking portion adapted to receive the cartridge, wherein the device comprises means adapted to operate the plasma filtration process in the cartridge and means adapted to operate the separation of nucleic acids in the cartridge.

Claims

1. A method for extracting nucleic acid fragments from a blood sample, a) A step of providing a blood sample collected from an individual, b) Preferably a step of stabilizing the blood cells in the sample with a fixation reagent, c) To separate plasma from the blood sample, a hollow fiber filter is used. The steps include filtering the blood sample and d) Preferably, the fraction containing blood cells obtained from step c) is washed with a washing solution. The steps include: collecting the bloat, e) To purify the nucleic acid fragments present in the plasma sample, the plasma sample obtained in step b) The material, and preferably the washout recovered in step d), is subjected to nucleic acid-specific bonding. Contact with the composite material, or instead with the plasma sample, and preferably in step d) The step of subjecting the recovered washout to electrophoresis in a separation medium, wherein the separation The medium is configured such that nucleic acid fragments are mobile within the medium and separable within the medium based on their size. The steps are prepared as follows: f) Recovering nucleic acid fragments bound to the binding substance or that have moved through the separation medium. Step and, g) A step of mixing the nucleic acid fragment recovered in step f) with a preservative or stabilizer. , including, Steps b) to g) are for filtering plasma from a blood sample in an automated system. The first section and the plasma sample obtained in step c) are brought into contact with a nucleic acid-specific binding substance. This is done on a cartridge that includes a second section, or alternatively, the second section is electric A method including means for performing pneumophoresis.

2. In step e), the resulting mixture of the nucleic acid fragment and the preservative or stabilizer The method according to claim 1, wherein the contents are stored in a removable container.

3. The method according to claim 2, wherein the mixture comprises nucleic acid fragments bound to the binding substance.

4. The length of the nucleic acid fragment is less than 400 base pairs, as per any one of claims 1 to 3. Law.

5. The blood sample obtained in step a) is mixed with an anticoagulant before being subjected to the filtration step b). The method according to any one of claims 1 to 4, which is combined.

6. The method according to any one of claims 1 to 5, wherein the hollow fiber filter is a hollow fiber membrane.

7. In step c), the plasma sample comes into contact with a nucleic acid-specific binding agent, claims 1 to 6 The method described in any one of the following items.

8. The method according to claim 7, wherein the binding material includes magnetic beads.

9. The steps include contacting the nucleic acid fragment bound to the binding substance with a nucleic acid binding dye, and the recovered The method according to any one of claims 1 to 8, further comprising the step of measuring the amount of nucleic acid fragments. Law.

10. The separation medium in step c) is a size-selection medium for nucleic acid fragments, as described in claim 1. The method.

11. The method according to claim 10, wherein the separation medium is an electrophoretic gel such as an agarose gel.

12. Steps b) to e) above are performed in an automated system to filter plasma from a blood sample. The process is performed on a cartridge that includes a first compartment for the eye and a second compartment for performing electrophoresis. The method according to claim 10 or 11, wherein the second compartment contains an electrophoresis gel.

13. A first compartment for filtering plasma from a blood sample, and a plasma sample that binds to nucleic acids with a specific binding agent. A cartridge comprising a second compartment for contact, wherein the first compartment is a hollow fiber filtration The cartridge includes a second compartment which includes a chamber for nucleic acid purification, and the cartridge includes the The cartridge contains a nucleic acid-specific binding substance, and the binding substance is preferably A cartridge containing magnetic beads.

14. The cartridge according to claim 13, wherein the first compartment includes means for receiving a blood sample.

15. The first compartment is used to mix the blood sample with the cell fixation reagent sealed in the first compartment. The first compartment includes a step, and preferably means for washing cells filtered from the blood sample. The cleaning buffer is preferably sealed in the cartridge, as described in claim 14. cartridge.

16. The first compartment contains the plasma sample separated from the blood sample in a hollow fiber membrane. Any of claims 13 to 15, comprising means for mixing with an enzyme or chemical reagent enclosed in one compartment. The cartridge described in item 1.

17. The first compartment receives the separated plasma mixed with the enzyme or chemical reagent from the first compartment. A cartridge according to any one of claims 13 to 16, including means for transporting to the second compartment. Ji.

18. The preservative or stabilizer sealed in the cartridge, and the preservative or stabilizer in front A means for mixing a nucleic acid fragment bound to a binding substance with the nucleic acid fragment and the preservative or stabilizer. A removable container for storing a mixture of chemical agents, as described in any one of claims 13 to 17. The included cartridge.

19. A cartridge according to any one of claims 13 to 18, which contains a nucleic acid-binding dye. 。

20. The second compartment is used to measure the amount of isolated nucleic acid bound to the dye by optical means. The ca according to claim 19, comprising a transparent window positioned to guide light rays into the sample or mixture. Tridge.

21. A dock adapted to receive the cartridge described in any one of claims 13 to 20 An automated system for extracting nucleic acid fragments from blood samples, including a device having an extraction site. The apparatus is adapted to operate the plasma filtration process in the cartridge. The means described above, and the nucleic acid purification process in the cartridge using a nucleic acid-specific binding substance An automated system including means adapted to perform the task.

22. The first compartment is for filtering plasma from a blood sample, and the second compartment is for performing gel electrophoresis. A cartridge comprising a first compartment containing a hollow fiber membrane and a second compartment containing electrophoresis A cartridge containing a gel for use.

23. The electrophoresis gel in the second compartment is an agarose gel, as described in claim 22. Tridge.

24. The cartridge according to claim 22 or 23, wherein the first compartment includes means for receiving a blood sample. Jig.

25. The first compartment is used to mix the blood sample with the cell fixation reagent sealed in the first compartment. The cartridge according to claim 24, including a step.

26. The first compartment contains the plasma sample separated from the blood sample in the hollow fiber membrane. Any of claims 22 to 25, comprising means for mixing with an enzyme or chemical reagent enclosed in one compartment. The cartridge described in item 1.

27. The first compartment receives the separated plasma mixed with the enzyme or chemical reagent from the first compartment. A cartridge according to any one of claims 22 to 26, including means for transporting to the second compartment. Ji.

28. The cartridge contains a preservative or stabilizer, and the preservative or stabilizer is contained within it. Means for mixing with acid fragments, and for preserving the mixture of nucleic acid fragments and the preservative or stabilizer. A cartridge according to one of claims 22 to 27, comprising a detachable container for that purpose.

29. A cartridge according to any one of claims 22 to 28, which contains a nucleic acid-binding dye. 。

30. docking section adapted to receive the cartridge described in one of claims 22 to 29 An automated system for extracting nucleic acid fragments from a blood sample, comprising a device having a position, The apparatus is equipped with a hand adapted to operate the plasma filtration process in the cartridge. The cartridge includes a step and means adapted for operating gel electrophoresis. , automation system.

31. A method for extracting protein biomarkers from blood samples, a) A step of providing a blood sample collected from an individual, b) Preferably, the step of stabilizing the blood cells in the sample with a fixation reagent, c) To separate plasma from the blood sample, a hollow fiber filter is used. The steps include filtering the blood sample and d) Preferably, the separated cell fraction is washed with a washing reagent and the washout is collected. The steps, e) To always purify the biomarkers present in the plasma sample, obtain the biomarkers obtained in step c) The plasma sample, and preferably the washout recovered in step d), is treated with protein The steps include contacting the biomarker with a binding substance specific to that biomarker, f) A step of recovering the protein bound to the binding substance, g) A step in which the protein recovered in step f) is mixed with a preservative or stabilizer. and, Includes, Steps b) to g) are for filtering plasma from a blood sample in an automated system. Section 1 and the plasma sample obtained in step c) specifically for the protein biomarker A method performed on a cartridge including a second compartment for contacting a binding substance.

32. In step e), the obtained mixture of the protein and the preservative or stabilizer The method according to claim 31, wherein the product is stored in a removable container.

33. The method according to claim 32, wherein the mixture comprises a protein bound to the binding substance.

34. The blood sample obtained in step a) is mixed with an anticoagulant before being subjected to the filtration step b). The method according to any one of claims 31 to 33, which is combined.

35. The hollow fiber filter is a hollow fiber membrane, as described in any one of claims 31 to 34. Law.

36. In step c), the plasma sample is a binding substance specific to the protein biomarker. The method according to any one of claims 1 to 35, wherein contact is made with

37. The method according to claim 36, wherein the binding material includes magnetic beads.

38. The magnetic beads are coated with antibodies specific to the protein biomarker. The method according to claim 36.

39. The first compartment is for filtering plasma from a blood sample, and the plasma sample is used as a protein biomarker. A cartridge comprising a second section for contacting a specific binding substance, the aforementioned One compartment contains a hollow fiber filter, and the second compartment contains a chamber for protein purification. The cartridge contains the protein biomarker sealed inside the cartridge. - Contains a specific binding substance, the binding substance preferably contains magnetic beads, cartridge Ji.

40. The magnetic beads are coated with antibodies specific to the protein biomarker. The cartridge according to claim 39.

41. The preservative or stabilizer sealed in the cartridge, and the preservative or stabilizer in front A means for mixing a nucleic acid fragment bound to a binding substance with the nucleic acid fragment and the preservative or stabilizer. The car according to claim 39 or 40, comprising a removable container for storing a mixture of chemical agents. Trash.

42. The ca according to claim 40 or 41, comprising a binding substance specific to a protein biomarker. A device including a docking site adapted to receive a tamper, from a blood sample An automated system for extracting nucleic acid fragments, wherein the apparatus is located in the cartridge. Means adapted to operate the plasma filtration process, and the protein biomarker - A specific binding substance is used to perform protein purification in the cartridge. An automated system including means conforming to the requirements.

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