Blood sample miRNA releasing agent and application thereof

Abstract

The invention discloses a blood sample miRNA releasing agent which comprises at least one of a reducing agent with the concentration of 0.01%-5%, a surfactant with the concentration of 0.01%-5% and a chelating agent with the concentration of 1 mM-100 mM, and the reducing agent is thioglycerol; the surfactant is Tween-20, and the surfactant is sodium dodecyl benzene sulfonate; the chelating agent is EDTA (Ethylene Diamine Tetraacetic Acid), and the working temperature of the blood sample miRNA releasing agent is not lower than 80 DEG C; the invention further discloses application of the releasing agent in direct PCR amplification detection of sample miRNA. The miRNA releasing agent for the blood sample is adopted for sample dilution, and miRNA can be directly detected through the steps of releasing nucleic acid through high-temperature incubation, removing influence through protein degradation and the like, and the CT value is small. And the blank of direct PCR detection of the miRNA of the blood sample is filled.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a miRNA releasing agent for blood samples and its application. Background Art

[0002] In the field of molecular diagnosis, the detection of microRNA (miRNA) in blood samples is an important biomarker analysis method. The conventional blood miRNA detection process includes nucleic acid extraction, purification, and PCR detection steps. Components such as salts and proteins present in blood samples often interfere with the PCR reaction, making it difficult to directly perform PCR detection on blood sample miRNAs. Existing technologies usually adopt nucleic acid extraction and purification techniques to release and purify nucleic acids in samples and eliminate the interference of salts, proteins, etc. in the samples, such as the phenol-chloroform method, silica membrane column method, and silica magnetic bead method. Although these methods can improve the accuracy of detection, they have cumbersome operation steps, take a long time, and are costly.

[0003] Currently, there is no existing technology that can directly perform PCR detection on blood samples without miRNA extraction and purification. In addition, existing direct amplification diluents for blood DNA samples and virus samples are not applicable in the direct detection of miRNAs. For example, guanidine isothiocyanate used to inhibit nuclease activity does not show any help in the direct amplification of miRNAs. Summary of the Invention

[0004] In order to solve at least one of the above problems, the present invention provides a miRNA releasing agent for blood samples and its application. In serum / plasma samples, by adding a nucleic acid releasing agent and incubating at a high temperature, direct PCR amplification of blood sample miRNAs can be achieved.

[0005] To achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a miRNA releasing agent for blood samples, including at least one of a reducing agent with a concentration of 0.01% - 5%, a surfactant with a concentration of 0.01% - 5%, and a chelating agent with a concentration of 1 mM - 100 mM. The reducing agent is thioglycerol; the surfactant is Tween-20; the chelating agent is EDTA, and the working temperature of the miRNA releasing agent for blood samples is not lower than 80°C.

[0006] In some embodiments of the present invention, preferably, the reducing agent is 1% thioglycerol, the surfactant is 1% Tween-20, and the chelating agent is 10 mM EDTA.

[0007] In some embodiments of the present invention, the reducing agent can also be DTT, b-mercaptoethanol, or tris(2-carboxyethyl)phosphine.

[0008] In some embodiments of the present invention, the surfactant may also be Tween-80, Triton X-100, NP-40.

[0009] In some embodiments of the present invention, the chelating agent may also be sodium citrate, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, hydroxyethylethylenediaminetriacetic acid.

[0010] The second aspect of the present invention provides the use of the blood sample miRNA releasing agent described in the first aspect in the direct PCR amplification detection of sample miRNA.

[0011] In some embodiments of the present invention, the following steps are included: (1) Sample dilution: Dilute the sample 5 - 150 times with the blood sample miRNA releasing agent described in the first aspect; (2) High-temperature incubation: Incubate the diluted mixture of the sample at a temperature above 80°C. (3) Protein degradation: Add proteinase K to the mixture completed in step (2) for incubation and inactivation. (4) Detection: Directly perform PCR detection.

[0012] In some embodiments of the present invention, the temperature range for high-temperature incubation in step (2) is 80 - 100°C.

[0013] In some embodiments of the present invention, the time for high-temperature incubation in step (2) is 5 - 60 min.

[0014] In some embodiments of the present invention, the incubation conditions for protein degradation in step (3) are 50 - 60°C for 10 - 15 min.

[0015] In some embodiments of the present invention, the inactivation conditions in step (2) are inactivation at 90 - 100°C for 15 - 20 min.

[0016] The present invention also provides the use of the blood sample miRNA releasing agent described in the first aspect in the preparation of a miRNA detection kit.

[0017] The present invention also provides a miRNA detection kit, which contains the blood sample miRNA releasing agent described in the first aspect and also contains proteinase K.

[0018] The present invention also provides a method for directly PCR amplifying and detecting sample miRNA using the miRNA detection kit, and the method is the same as the method described in the second aspect.

[0019] In some embodiments of the present invention, the blood sample is a plasma or serum sample.

[0020] Advantages of the present invention Compared with the prior art, the present invention has the following advantages: The present invention provides a miRNA releasing agent for blood samples, which comprises chelating agents, surfactants and reducing agents represented by EDTA, Tween-20 and thioglycerol. After the sample added with the nucleic acid releasing agent is incubated at a temperature of 80 °C or above, miRNA is released and can be detected by direct PCR. The introduction of the reducing agent significantly advances the CT value of the direct detection of miRNA. In the direct PCR amplification detection of miRNA in the sample using the miRNA releasing agent for blood samples, by steps such as sample dilution, high-temperature incubation of the nucleic acid releasing agent to release nucleic acids, and removal of interference by protein degradation, the CT value of the direct detection of miRNA is further advanced. It fills the blank of direct PCR detection of miRNA in blood samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shows the CT values of RT-PCR detection of each scheme in Example 1 of the present invention; Figure 2 Shows the CT values of RT-PCR detection of each scheme in Example 2 of the present invention; Figure 3 Shows the CT values of RT-PCR detection of each scheme in Example 3 of the present invention; Figure 4 Shows the CT values of RT-PCR detection of the nucleic acid releasing agent scheme that can be directly amplified in Example 4 of the present invention; Figure 5 Shows the CT values of RT-PCR detection of the nucleic acid releasing agent scheme that cannot be directly amplified in Example 4 of the present invention; Figure 6 Shows the CT values of RT-PCR detection of each scheme in Example 5 of the present invention; Figure 7 Shows the CT values of RT-PCR detection of each scheme in Example 6 of the present invention; Among them, target 1 is hsa-miR-615-5p; target 2 is hsa-miR-542-3p. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following examples are used herein to demonstrate the preferred embodiments of the present invention. Those skilled in the art will understand that the technologies disclosed in the following examples represent the technologies discovered by the inventors that can be used to implement the present invention, and thus can be regarded as the preferred solutions for implementing the present invention. However, those skilled in the art should understand from this specification that the specific embodiments disclosed here can be modified in many ways and still obtain the same or similar results without departing from the spirit or scope of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0024] The present invention provides a miRNA releasing agent for blood samples, comprising at least one of a reducing agent, a surfactant, and a chelating agent. The concentration of the reducing agent is 0.01% - 5%, and the reducing agent is one of DTT, thioglycerol, β-mercaptoethanol, and tris(2-carboxyethyl)phosphine; the concentration of the surfactant is 0.01% - 5%, and the surfactant is one of Tween-20, Tween-80, Triton X-100, and NP-40; the concentration of the chelating agent is 1 mM - 100 mM, and the chelating agent is one of EDTA, sodium citrate, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, and hydroxyethylethylenediaminetriacetic acid. The working temperature of the miRNA releasing agent for blood samples is not lower than 80°C.

[0025] Preferably, the miRNA releasing agent for blood samples is a mixture of thioglycerol, Tween-20, and EDTA, and its optimal concentration is 1% thioglycerol, 1% Tween-20, and 10 mM EDTA.

[0026] The application of the miRNA releasing agent for blood samples described above in the direct PCR amplification detection of sample miRNA includes the following steps: (1) Sample dilution: Dilute the sample 5 - 150 times using the miRNA releasing agent for blood samples described in the first aspect; (2) High-temperature incubation: Incubate the diluted mixture of the sample at a temperature above 80°C for 5 - 60 min; (3) Protein degradation: Add proteinase K to the mixture completed in step (2) and incubate at 50 - 60°C, and inactivate at 90 - 100°C for 15 - 20 min; (4) Detection: Directly perform PCR detection.

[0027] The application of the miRNA releasing agent for blood samples in the preparation of a miRNA detection kit.

[0028] A miRNA detection kit, which contains the miRNA releasing agent for blood samples described in the first aspect and also contains proteinase K.

[0029] A method for directly detecting miRNA in a sample by using an miRNA detection kit, which is the same as the method described in the second aspect. Further, the blood sample is a plasma or serum sample.

[0030] The technical solutions of the present application will be further described in detail below in conjunction with some specific embodiments.

[0031] Example 1 Exploration of the types of nucleic acid release agents 1. Select nuclease-free water (NFW), SDS, EDTA, thioglycerol, Tween-20, Triton, Tris-HCI (PH=8), TE buffer, guanidine isothiocyanate, etc. as nucleic acid release agents for exploration experiments.

[0032] Prepare samples: Take 10 μL of platelet-poor plasma samples and add 90 μL of the above nucleic acid release agents respectively, mix well by shaking, centrifuge, and let stand at room temperature for 10 min to obtain experimental samples.

[0033] 2. Take 5 μL of the sample for RT-PCR detection. The sample addition system is as follows:

[0034] The reaction program is as follows:

[0035] The detection results of RT-PCR are as Figure 1 shown.

[0036] The results show that none of the nucleic acid release agents with the above formulations can achieve the purpose of direct amplification, indicating that the nucleic acid release agents with the above components are not suitable for the direct detection of miRNA or the treatment conditions of the nucleic acid release agents are inappropriate, and the nucleic acid release agents fail to achieve the desired effect.

[0037] Example 2 Exploration of temperature incubation 1. Dilute the plasma sample 10-fold with 5 mM EDTA as the nucleic acid release agent. 2. Set the incubation time to 30 min, and incubate the diluted sample at temperature gradients of 37°C, 60°C, 80°C, and 95°C respectively to achieve the release of miRNA. 3. Incubate the sample under the above temperature conditions. The reaction system and reaction program are the same as in Example 1. After incubation at temperature gradients, the detection results of RT-PCR are as Figure 2 shown.

[0038] The results showed that when the plasma sample was diluted 10-fold with 5 mM EDTA as the nucleic acid releasing agent, it was found that the sample could be detected when incubated at temperatures above 80°C. After incubation at 95°C, the CT value of the direct PCR detection of miRNA in the sample was the lowest and the effect was the best.

[0039] Example 3 Exploration of Incubation Time 1. Dilute the plasma sample 10-fold with 5 mM EDTA as the nucleic acid releasing agent; 2. Set the incubation temperature at 95°C and release the miRNA in the sample with time gradients of 1 min, 5 min, 10 min, 20 min, 30 min, and 60 min; 3. Incubate the sample at the above temperature conditions for different durations. The reaction system and reaction procedure are the same as those shown in the examples. After incubation with different incubation time gradients, the detection results of RT-PCR are as Figure 3 shown.

[0040] The results showed that when the plasma sample was diluted 10-fold with 5 mM EDTA as the nucleic acid releasing agent, it was found that the sample could be detected when incubated at 95°C for more than 5 min. Incubation for 10 - 30 min had a lower CT value and better effect on the direct PCR detection of miRNA in the sample.

[0041] Example 4 Exploration of Nucleic Acid Releasing Agent Concentration Dilute the plasma sample 10-fold with different types of nucleic acid releasing agents at different concentrations or pH conditions, and test after incubation at 95°C for 10 min. Different test schemes are shown in Table 1 below: Table 1 Test Schemes of Nucleic Acid Releasing Agents

[0042] The experimental results are as follows Figure 4 and Figure 5 shown.

[0043] The results showed that when the concentration of EDTA was 10 mM, the concentration of thioglycerol was 1%, the concentration of Tween-20 was 1%, and the concentration of Triton was 1%, the CT value of the direct PCR detection of miRNA in the sample was the lowest and the effect was the best. SDS, guanidine isothiocyanate, and Tris-HCl failed to achieve the purpose of direct amplification.

[0044] Example 5 Exploration of the Combined Effect of Nucleic Acid Releasing Agents On the basis of Example 4, we used the nucleic acid releasing agents with better effects by adding single components for combined testing. Different test schemes are shown in Table 2 below: Table 2 Combined Test Schemes of Nucleic Acid Releasing Agents

[0045] The experimental results are as follows Figure 6 as shown below

[0046] The results show that the introduction of the reducing agent thioglycerol significantly advances the CT value of miRNA. The multi-component nucleic acid release agent can play a better role than the single sample treatment solution, and the reducing agent thioglycerol plays a relatively important role in the nucleic acid release agent.

[0047] The nucleic acid release agent of the three components of EDTA, Tween-20 and thioglycerol has the lowest CT value and the best effect on the direct PCR detection of miRNA in the sample.

[0048] Investigation on eliminating the influence of protein in the direct PCR detection of miRNA in Example 6 samples In order to eliminate the influence of protein in the direct PCR detection of miRNA in the sample, we added proteinase K to the sample after high-temperature incubation and release of the nucleic acid release agent containing 10 mM EDTA, 1% Tween-20 and 1% thioglycerol to eliminate the influence of protein.

[0049] Add 3 μL of proteinase K to 100 μL of the sample mixture and incubate at 55 °C for 10 min, and inactivate at 95 °C for 15 min to avoid the influence of proteinase K on the PCR enzyme. At the same time, set a group without adding proteinase K as a control.

[0050] The detection results are as Figure 7 shown below

[0051] The results show that after the introduction of proteinase K digestion, the CT value of the target increases by 0.5, the overall volatility decreases, and the stability is better, indicating that the introduction of proteinase K helps the direct PCR detection of miRNA in blood samples.

[0052] In summary: Using 10 mM EDTA, 1% Tween and 1% thioglycerol as the nucleic acid release agent, diluting the plasma sample 10-fold, incubating at 95 °C for 10 min, adding PK and digesting at 55 °C for 10 min, and inactivating at 95 °C for 15 min to achieve the direct PCR detection of miRNA in blood samples.

[0053] In the above examples, the reducing agent thioglycerol can also be replaced by DTT, β-mercaptoethanol, tris(2-carboxyethyl)phosphate, the surfactant Tween-20 can also be replaced by Tween-80, TritonX-100, NP-40; the chelating agent EDTA can also be replaced by sodium citrate, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, hydroxyethylethylenediaminetriacetic acid, and the purpose of direct PCR detection of miRNA in blood samples can also be achieved.

[0054] When different concentrations of each reagent were replaced, the CT values of direct PCR detection of miRNA in blood samples are shown in Table 3.

[0055] Table 3 CT value results of direct PCR detection of samples with different concentrations of reagents replaced

[0056] From the above results, it can be seen that after replacement, the purpose of direct PCR detection of miRNA in blood samples can basically be achieved.

[0057] All the documents mentioned in the present invention are cited in this application for reference as if each document was cited separately for reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by this application.

Claims

1. Blood sample miRNA releasing agent, characterized in that: It includes at least one of a reducing agent with a concentration of 0.01% - 5%, a surfactant with a concentration of 0.01% - 5%, and a chelating agent with a concentration of 1 mM - 100 mM. The reducing agent is thioglycerol; the surfactant is Tween-20; the chelating agent is EDTA, and the working temperature of the miRNA releasing agent for blood samples is not lower than 80°C.

2. The miRNA releasing agent for blood samples according to claim 1, wherein: The reducing agent can also be DTT, β-mercaptoethanol, tris(2-carboxyethyl)phosphine.

3. The miRNA releasing agent for blood samples according to claim 1, wherein: The surfactant can also be Tween-80, TritonX-100, NP-40.

4. The miRNA releasing agent for blood samples according to claim 1, wherein: The chelating agent can also be sodium citrate, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, hydroxyethylethylenediaminetriacetic acid.

5. Use of the miRNA releasing agent for blood samples according to any one of claims 1 - 4 in the direct PCR amplification detection of sample miRNA.

6. The application according to claim 5, characterized in that, It includes the following steps: (1) Sample dilution: Dilute the sample 5 - 150 times using the miRNA releasing agent for blood samples according to any one of claims 1 - 4; (2) High-temperature incubation: Incubate the diluted mixture of the sample at a temperature above 80°C; (3) Protein degradation: Add proteinase K to the mixture after completing step (2) and incubate it and then inactivate it; (4) Detection: Directly perform PCR detection.

7. The application according to claim 6, characterized in that: The incubation temperature range in step (2) is 80 - 100°C.

8. The application according to claim 6, wherein: The incubation time in step (2) is 5 - 60 min.

9. The application according to claim 6, wherein: The protein degradation incubation conditions in step (3) are 50 - 60°C for 10 - 15 min.

10. The application according to claim 6, characterized in that: The inactivation conditions in step (2) are inactivation at 90 - 100°C for 15 - 20 min.

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