阅读说明：本技术 检测位于生物全血中血细胞外侧的至少一种核酸的体外方法以及用于该目的的装置和试剂盒 (In vitro method for detecting at least one nucleic acid located outside blood cells in biological whole blood, and device and kit for this purpose ) 是由 拉尔夫·希默尔赖希 伯里克利·西蒙 于 2019-12-18 设计创作，主要内容包括：本发明涉及一种用于检测位于生物全血中血细胞外侧的至少一种核酸(优选DNA和/或RNA,特别优选DNA)的体外方法、装置和试剂盒。根据本发明的方法的优点在于,与现有技术中的已知方法相比,实施该方法需要更少的时间和装置工作,并且使污染的风险和待扩增的无细胞核酸(例如,无细胞DNA)损失的风险最小化。因此,该方法比已知的无细胞核酸(例如,无细胞DNA)检测方法更经济且具有更高的检测精度。此外,与基于全血稀释的现有技术的检测方法相比,检测灵敏度首先得到提高。此外,提出使用提供的装置和提供的试剂盒。(The present invention relates to an in vitro method, device and kit for detecting at least one nucleic acid, preferably DNA and/or RNA, particularly preferably DNA, located outside blood cells in biological whole blood. The advantage of the method according to the invention is that it requires less time and equipment work to carry out the method compared to the methods known in the prior art and minimizes the risk of contamination and the risk of loss of cell-free nucleic acid (e.g. cell-free DNA) to be amplified. Thus, the method is more economical and has higher detection accuracy than known cell-free nucleic acid (e.g., cell-free DNA) detection methods. Furthermore, the detection sensitivity is first improved compared to the prior art detection methods based on whole blood dilution. Furthermore, it is proposed to use the provided device and the provided kit.)

1. An in vitro method for detecting at least one nucleic acid located outside blood cells in biological whole blood, comprising the steps of:

a) providing a reaction mixture adapted to perform an isothermal amplification reaction with at least one primer pair, wherein the reaction mixture comprises at least one primer pair adapted to amplify at least one nucleic acid present in whole blood; or adding at least one such primer pair to the reaction mixture;

b) adding biological whole blood to the reaction mixture;

c) carrying out isothermal amplification reaction at the temperature of less than or equal to 45 ℃; and

d) detecting the DNA by means of at least one detection reagent, wherein the detection takes place during or after step c),

characterized in that said reaction mixture is adapted to prevent lysis of blood cells contained in said biological whole blood.

2. The method of claim 1, wherein the reaction mixture is

i) Forming a liquid mixture with the whole blood, the liquid mixture being substantially isotonic with blood cells; and/or

ii) forming a liquid mixture with the whole blood, which liquid mixture has an osmotic pressure which substantially corresponds to the osmotic pressure of the blood cells, preferably an osmotic pressure of from 230 to 350, preferably from 260 to 320, particularly preferably from 270 to 310, in particular from 280 to 300; and/or

iii) does not comprise any surfactant suitable for performing lysis of blood cells, preferably does not comprise a substance or mixture of substances suitable for performing lysis of blood cells.

3. The method according to any one of the preceding claims, wherein the detection reagent is not adapted to pass through the cell membrane of blood cells contained in the whole blood.

4. The method according to any one of the preceding claims, wherein the detection reagent is selected from the group consisting of fluorescent probes, DNA-binding dye molecules, reagents for detecting pyrophosphate and combinations thereof, wherein preferably

i) The fluorescent probe comprises or consists of an oligonucleotide comprising or consisting of: a quencher, a fluorophore and a DNA sequence present between the quencher and the fluorophore, wherein the DNA sequence is suitable for cleavage by an endonuclease or an exonuclease, preferably the DNA sequence is a DHF or AP DNA sequence; and/or

ii) the dye molecule that binds DNA is selected from the group consisting of EVAGreen, SYBR Green, Syto dye, TOPO dye, and combinations thereof; and/or

iii) the reagent for detecting pyrophosphate is a magnesium salt.

5. The method according to any of the preceding claims, characterized in that the detection of the DNA is performed by a detection unit, wherein the detection unit is preferably selected from the group consisting of an absorption measurement device, a fluorescence measurement device, a turbidimeter, a camera, a cell phone and combinations thereof.

6. Method according to any of the preceding claims, characterized in that the amount of amplified DNA is quantified by an evaluation unit, wherein the evaluation unit is preferably in communication connection with a detection unit.

7. The method according to any of the preceding claims, wherein at least one primer of a primer pair, optionally both primers of a primer pair, is adapted to bind to a repetitive DNA sequence in the biological DNA, preferably to a repetitive sequence selected from the group consisting of a LINE sequence, a SINE sequence and an ALU sequence.

8. Method according to any of the preceding claims, characterized in that at least one primer of a primer pair, optionally both primers of a primer pair, is adapted to bind to a DNA sequence in the DNA of an organism which is present in the blood circulation at, during or after the onset of a disease and/or at, during or after an excessive muscle strain in an organism.

9. Method according to any of the preceding claims, characterized in that in case DNA is amplified during or after step d) it is concluded that there is a disease and/or excessive muscle strain, wherein the disease is in particular selected from the group consisting of inflammation, cancer, autoimmune diseases, coronary arteries, stroke, sepsis, chromosomal aberrations, changes in gene copy number, gene mutations and combinations thereof.

10. The method of any one of the preceding claims, wherein the reaction mixture comprises a plurality of primer pairs, optionally at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 primer pairs, each adapted to amplify at least one nucleic acid present in the whole blood; or a plurality of such primer pairs are added to the reaction mixture, wherein each primer pair is preferably adapted for amplifying a different nucleic acid, preferably a nucleic acid present in whole blood of an organism that is overstrained and/or suffers from a disease selected from the group consisting of: inflammation, cancer, autoimmune disease, coronary artery, stroke, sepsis, chromosomal aberration, changes in gene copy number, gene mutations, and combinations thereof.

11. The method of any preceding claim, wherein the reaction mixture and the detection reagent, optionally the reaction mixture, the primer pair and the detection reagent, are comprised in a lateral flow strip.

12. A device and/or kit suitable for the in vitro detection of at least one nucleic acid present outside blood cells in biological whole blood, comprising

a) A reaction mixture, together with at least one primer pair, adapted to perform an isothermal amplification reaction at a temperature of 45 ℃ or less;

b) at least one primer pair suitable for amplifying at least one nucleic acid present in whole blood, wherein the at least one primer pair is optionally comprised in the reaction mixture;

c) at least one detection reagent for detecting DNA, wherein the at least one detection reagent is optionally contained in the reaction mixture,

characterized in that the reaction mixture is adapted to prevent lysis of blood cells contained in biological whole blood.

13. The device and/or kit of claim 12, wherein the reaction mixture is

i) Adapted to form a liquid mixture with the whole blood that is substantially isotonic with blood cells; and/or

ii) is suitable for forming a liquid mixture with the whole blood, which liquid mixture has an osmotic pressure which substantially corresponds to the osmotic pressure of the blood cells, preferably an osmotic pressure of 230 to 350, preferably 260 to 320, particularly preferably 270 to 310, in particular 280 to 300; and/or

iii) does not comprise any surfactant suitable for performing lysis of blood cells, preferably does not comprise a substance or mixture of substances suitable for performing lysis of blood cells.

14. The device and/or kit of any of claims 12 or 13, wherein the detection reagent is not adapted to pass through the cell membrane of the blood cells contained in the whole blood.

15. The device and/or kit of any one of claims 12 to 14, wherein the detection reagent is selected from the group consisting of a fluorescent probe, a DNA-binding dye molecule, a reagent for detecting pyrophosphate, and combinations thereof, wherein preferably

i) The fluorescent probe comprises or consists of an oligonucleotide comprising or consisting of: a quencher, a fluorophore and a DNA sequence present between the quencher and the fluorophore, wherein the DNA sequence is suitable for cleavage by an endonuclease or an exonuclease, preferably the DNA sequence is a DHF or AP DNA sequence; and/or

ii) the dye molecule that binds DNA is selected from the group consisting of EVAGreen, SYBR Green, Syto dye, TOPO dye, and combinations thereof; and/or

iii) the reagent for detecting pyrophosphate is a magnesium salt.

16. The device and/or kit according to any one of claims 12 to 15, characterized in that the device and/or kit comprises a detection unit for detecting DNA, wherein the detection unit is preferably selected from the group consisting of an absorption measurement device, a fluorescence measurement device, a turbidimeter, a camera, a cell phone, and combinations thereof.

17. The device and/or kit according to any one of claims 12 to 16, characterized in that the device and/or kit comprises an evaluation unit for quantifying the amount of amplified DNA, wherein the evaluation unit is preferably in communicative connection with a detection unit.

18. The device and/or kit according to any one of claims 12 to 17, characterized in that at least one primer of the primer pair, optionally both primers of the primer pair, is adapted to bind to a repetitive DNA sequence in biological DNA, preferably to a repetitive sequence selected from the group consisting of a LINE sequence, a SINE sequence and an ALU sequence.

19. A device and/or kit according to any one of claims 12 to 18, characterised in that at least one primer of the primer pair, optionally both primers of the primer pair, is adapted to bind to a DNA sequence in the DNA of an organism which is present in the blood circulation at, during or after the onset of a disease and/or at, during or after excessive muscle strain in an organism.

20. Device and/or kit according to any of claims 12 to 19, characterized in that in case of detection of DNA by the detection reagent the device and/or kit is configured to show the presence of a disease and/or an excessive muscle strain, optionally through the borders of a device that is transparent to visible light, wherein the disease is in particular selected from the group consisting of inflammation, cancer, autoimmune diseases, coronary arteries, stroke, sepsis, chromosomal aberrations, changes in gene copy number, gene mutations and combinations thereof.

21. The device and/or kit according to any one of claims 12 to 20, characterized in that the device and/or kit comprises a plurality of primer pairs, optionally at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 primer pairs, wherein the primer pairs are each adapted to amplify at least one nucleic acid present in the whole blood, wherein each primer pair is preferably adapted to amplify a different nucleic acid, preferably a nucleic acid present in biological whole blood, which biological excessive muscle strain and/or suffers from a disease selected from the group consisting of: inflammation, cancer, autoimmune disease, coronary artery, stroke, sepsis, chromosomal aberration, changes in gene copy number, gene mutations, and combinations thereof.

22. The device of any one of claims 12 to 21, wherein the device is a lateral flow strip.

23. The kit of any one of claims 12 to 21, wherein the kit has a lateral flow strip, wherein the lateral flow strip is a strip of lateral flow strip

i) Comprising the reaction mixture; or

ii) comprises the reaction mixture and the detection reagent.

24. Use of a device according to any one of claims 12 to 22 or a kit according to any one of claims 12 to 21 or 23 for the direct, semi-quantitative or quantitative detection of at least one nucleic acid in whole blood, preferably for the detection of at least one nucleic acid present outside blood cells in whole blood of an organism suffering from a disease and/or excessive muscle strain.