CN114350768A

CN114350768A - Blood sample DNA direct amplification reagent and application thereof

Info

Publication number: CN114350768A
Application number: CN202111658527.XA
Authority: CN
Inventors: 邹永龙; 何宗顺; 曲峰; 张佳斌
Original assignee: Suzhou Cretaceous Biotechnology Co ltd
Current assignee: Suzhou Cretaceous Biotechnology Co ltd
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2022-04-15
Anticipated expiration: 2041-12-30
Also published as: CN114350768B

Abstract

The invention discloses a blood sample DNA direct amplification reagent and application thereof. Specifically, the present invention provides a sample-releasing agent comprising Tris, sodium bicarbonate, cyanoguanidine isosulfate, sodium dodecyl diphenyl ether disulfonate, Brij30, and sucrose; containing Tris-HCl, KCl, dNTPs, BSA, MgCl₂Brij52, direct amplification reagents for crude samples of hot-start DNA polymerase and tetramethylammonium chloride and kits comprising the reagents required to formulate them; a method for quantitative and/or qualitative analysis of DNA in a blood sample using said sample releasing agent and/or crude sample direct amplification reagent.

Description

Blood sample DNA direct amplification reagent and application thereof

Technical Field

The invention relates to a reagent for directly amplifying nucleic acid from a crude sample, in particular to a reagent for directly amplifying DNA from a blood sample and application thereof.

Background

Polymerase chain reaction, PCR for short, is an important molecular biology technique. Since the 80 s of the 20 th century, Mullis et al developed PCR technology, it has been widely used in various related fields such as biology, medicine, and forensic medicine. The quality and quantity of the template nucleic acid generally directly determine the success or failure of the PCR reaction. The existing detection method and detection means based on PCR technology need to extract and purify nucleic acid of biological samples before amplification. However, conventional nucleic acid extraction methods such as organic solvent method, silicon column method, etc. generally involve the following technical drawbacks: 1) the operation steps are complicated; 2) the time consumption is long; 3) a large amount of manpower, material resources and financial resources are consumed; 4) relates to the use of toxic and harmful organic reagents. In order to overcome the bottleneck of high-throughput detection of the samples and improve the detection efficiency of the samples, the magnetic bead method which is popular in the market at present solves the problem of high-throughput automation, but needs to rely on an expensive automatic extraction workstation, and the financial burden is heavy.

Many researchers try to omit a DNA extraction step and directly perform a PCR amplification operation on a proper amount of biological samples, namely, a so-called Direct amplification technique (Direct PCR), also called a Direct amplification technique or a hands-free amplification technique, which can directly add original samples without DNA extraction and other pretreatment steps into a PCR reaction system and obtain amplification products, reduce detection steps and reduce detection cost, and this is undoubtedly a great revolutionary progress for rapid and efficient detection of biological samples. The existing commercial PCR reagent can be used for treating the DNA direct amplification of samples such as rat tail, plants and the like. With the continuous development of the technology, the PCR technology as a qualitative technology has not been able to meet the requirements of scientific research and clinic, and the second generation PCR technology (fluorescent quantitative PCR) based on relative quantification has been widely used. Therefore, direct amplification using fluorescent quantitative PCR is a difficult problem that many scientists want to overcome.

The genome DNA sample comes from blood at most, and the blood sample has strong inhibiting effect on the collection of fluorescence, so that the fluorescence quantitative PCR can not be accurately quantified, and the detection result is influenced.

Chinese patent CN101492670A discloses a method for directly amplifying nucleic acid by using whole blood, wherein PCR amplification can be completed by directly adding a blood sample, but the whole blood sample amplification is not performed by deep fluorescence quantitative PCR.

Chinese patent CN 113151583 a discloses a detection kit and a detection method thereof capable of directly amplifying a sample containing SARS CoV-2 virus, which can perform a simple lysis treatment on the sample and then directly perform a quantitative analysis by using a fluorescent quantitative PCR, but the method is only applicable to samples with few inhibitors such as nasopharyngeal swab, and can not perform amplification of DNA in blood samples.

In view of the above, the prior art fails to provide a method or reagent that can perform quantitative analysis of fluorescence quantitative PCR on DNA in blood samples ideally to meet the practical needs.

Disclosure of Invention

The invention aims to provide a reagent for carrying out fluorescence quantitative PCR on DNA in a whole blood sample and an application method thereof. The reagent of the present invention comprises a releasing agent for a blood sample and a reagent for directly amplifying a crude sample of DNA released from blood.

In order to achieve the above object, the present invention provides, in a first aspect, a sample-releasing agent that can rapidly release DNA in blood and remove most of inhibitors, obtaining a crude sample that can be used for direct amplification for qualitative and/or quantitative analysis of blood sample DNA.

Specifically, the sample-releasing agent of the present invention comprises: tris, sodium bicarbonate, guanidine isosulfate, sodium dodecyl diphenyl ether disulfonate, Brij30 (CAS:9002-92-0), sucrose, and water.

Preferably, the sample-releasing agent of the present invention comprises: 1-50mM Tris, 1-50mM sodium bicarbonate, 1-20mM guanidine isothiocyanate, 0.01-0.2% sodium dodecyl diphenyl ether disulfonate, 0.01-2% Brij30, 0.5-10% sucrose and the balance water; more preferably, the pH of the sample-releasing agent is 9.0 to 12.5.

More preferably, the sample-releasing agent comprises: 10-20mM Tris, 5-40mM sodium bicarbonate, 2-10mM guanidine isothiocyanate, 0.02-0.1% sodium dodecyl diphenyl ether disulfonate, 0.02-1% Brij30, 1-5% sucrose and the balance water; more preferably, the pH of the sample-releasing agent is 10.0 to 12.0.

Further preferably, the sample-releasing agent comprises: 20mM Tris, 25mM sodium bicarbonate, 10mM cyanoguanidine isosulfate, 0.05% sodium dodecyl diphenyl oxide disulfonate, 0.5% Brij30, 2% sucrose, and balance water, the pH of the sample release agent being 11.5.

In the sample releasing agent of the present invention, the combination of Tris and sodium bicarbonate provides a stable weakly alkaline environment, which can maintain the pH buffer of cells during the lysis process, and can assist the sodium dodecyl diphenyl oxide disulfonate and Brij30 to rapidly open the cell membrane and release nucleic acid.

In the sample releasing agent, the guanidine isocyanate can inhibit the activity of nuclease released in the cell lysis process, so that the nuclease cannot degrade nucleic acid, and the integrity of the nucleic acid is ensured.

In the sample-releasing agent of the present invention, the sodium dodecyl diphenyl oxide disulfonate is a high-performance anionic surfactant, shows very good stability in a strong alkaline solution, can lyse cells at room temperature, and denatures proteins. Surprisingly, under weakly alkaline conditions, the interaction of sodium dodecyl diphenyl oxide disulfonate with Brij30 rapidly lyses cells, releasing DNA into solution.

In the sample releaser, Brij30 is a nonionic surfactant, can be matched with sodium dodecyl diphenyl oxide disulfonate to rapidly crack cells under weak alkaline to release nucleic acid, and Brij30 is a nonionic surfactant with small influence on PCR and does not influence downstream amplification reaction.

In the sample releasing agent of the present invention, the sucrose may be fixed and denatured at a high temperature (about 90 ℃) of hemoglobin released from blood cells, and heme may not be dispersed into a solution, thereby weakening the collection and analysis of fluorescence.

The present invention also provides a sample release agent kit comprising reagents for formulating a sample release agent according to the first aspect of the present invention. Preferably, the reagent for formulating the sample-releasing agent according to the first aspect of the present invention comprises at least one, at least two, at least three, at least four, at least five, at least six, preferably all of the above-mentioned reagents for formulating the sample-releasing agent, selected from the group consisting of Tris, sodium bicarbonate, cyanoguanidine iso-sulfate, sodium dodecyl diphenyl oxide disulfonate, Brij30 and sucrose, or comprises all of the reagents required for formulating the sample-releasing agent (for example comprising the above-listed reagents and water and a pH adjusting agent for adjusting pH such as sodium hydroxide).

The present invention provides in a second aspect a reagent that can be used for Direct amplification of a crude sample (hereinafter sometimes referred to as a crude sample Direct amplification reagent or a Direct amplification reagent), wherein the crude sample Direct amplification reagent (preferably formulated at a 2-fold working solution concentration prior to use, i.e., such a Direct amplification reagent is herein sometimes referred to as a 2X Direct qPCR Master Mix) comprises the following: Tris-HCl, KCl, dNTPs, BSA, MgCl₂Brij52 (CAS:9004-95-9), hot start DNA polymerase and tetramethylammonium chloride.

Preferably, the crude sample direct amplification reagent comprises: 10-150mM Tris-HCl, 10-150mM KCl, 100-₂0.1-1% Brij52, 1-5U of hot start DNA polymerase and 1-20mM tetramethylammonium chloride.

More preferably, the crude sample direct amplification reagent comprises: 20-100mM Tris-HCl, 25-100mM KCl, 200-₂0.2-0.5% Brij52, 2-5U of hot start DNA polymerase and 1-5mM tetramethylammonium chloride.

Further preferably, the crude sample direct amplification reagent comprises: 60mM Tris-HCl, 75mM KCl, 250. mu.M dNTPs, 250. mu.g/mL BSA, 2.5mM MgCl₂0.5% Brij52, 2U of hot start DNA polymerase and 2mM tetramethylammonium chloride.

In the direct amplification reagent of the present invention, BSA and Brij52 can neutralize impurities such as hemoglobin in a crude sample, and tetramethylammonium chloride can aggregate denatured hemoglobin at a high temperature so that it is not dispersed in an amplification solution, thereby not affecting the acquisition of a fluorescence signal.

Preferably, the promoter DNA polymerase may be Jumbo^TMThe Taq Hot-Start DNA Polymerase is directionally modified and has the characteristics of strong inhibition resistance and high sensitivity.

The present invention also provides a crude sample direct amplification kit comprising reagents for formulating the crude sample direct amplification reagent according to the second aspect of the present invention. Preferably, theThe kit comprises Tris-HCl, KCl, dNTPs, BSA, MgCl₂Brij52, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight of the group consisting of tetramethylammonium chloride, preferably including all of the above listed reagents used to formulate the crude sample direct amplification reagent.

The present invention provides in a third aspect a crude sample direct amplification kit comprising:

(1) reagents for formulating a sample-releasing agent according to the first aspect of the present invention; and/or

(2) Reagents for formulating a crude sample direct amplification reagent according to the second aspect of the present invention.

As the reagent for formulating the sample-releasing agent according to the first aspect of the present invention, it may, for example, be at least one, at least two, at least three, at least four, at least five, at least six, preferably all of the above-mentioned reagents for formulating the sample-releasing agent, selected from the group consisting of Tris, sodium bicarbonate, cyanoguanidine isothiocyanate, sodium dodecyldiphenyloxide disulfonate, Brij30 and sucrose, or all of the reagents required for formulating the sample-releasing agent (for example, including the above-listed reagents and water and a pH adjusting agent such as sodium hydroxide for adjusting pH).

As the reagent for formulating the crude sample direct amplification reagent according to the second aspect of the present invention, it may, for example, be selected from Tris-HCl, KCl, dNTPs, BSA, MgCl₂Brij52, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight of the group consisting of tetramethylammonium chloride, preferably including all of the above listed reagents used to formulate the crude sample direct amplification reagent.

In a fourth aspect, the present invention provides a method for quantitative and/or qualitative analysis of DNA in a blood sample, said method employing a sample-releasing agent according to the first aspect of the invention to prepare a crude sample that can be used for direct amplification; and/or using the crude sample direct amplification reagent according to the second aspect of the invention for amplification to quantify and/or characterize DNA in a blood sample.

Preferably, the method is carried out using a crude sample direct amplification kit according to the third aspect of the invention.

More preferably, the method comprises the steps of:

(1) adding a sample releasing agent into the blood sample and uniformly mixing to obtain a crude sample;

(2) adding a crude sample direct amplification reagent, a primer and a probe of a corresponding detection target into the crude sample to obtain a reaction working solution;

(3) and (b) performing quantitative and/or qualitative analysis by PCR instrument amplification using the reaction working solution.

Preferably, in step (1), the volume of the blood sample is 10-50 μ L; more preferably, the volume dose of the sample-releasing agent is equal to the volume dose of the blood sample.

Preferably, in the step (2), water such as deionized water is used to make up to the target volume of the reaction working solution; more preferably, the target volume of the reaction working solution is twice that of the crude sample direct amplification reagent. That is, the crude sample Direct amplification reagent was added at twice the concentration (i.e., 2X Direct qPCR Master Mix).

Preferably, in step (3), the PCR machine is preferably a qPCR machine and the analysis is preferably a quantitative analysis.

Preferably, in the step (2), the volume of the crude sample is 5 to 40% of the volume of the reaction working solution (i.e., the volume of the entire amplification reaction solution), and more preferably, 10 to 30% of the volume of the reaction working solution.

Compared with the prior art, the invention has at least the following beneficial technical effects:

(1) the invention provides a sample releasing agent and a direct amplification reagent which can directly carry out fluorescence quantitative PCR analysis on DNA in a blood sample, and avoids the complicated steps of nucleic acid extraction.

(2) The combination of the sample releasing agent and the direct amplification reagent provided by the invention solves the interference of hemoglobin on the acquisition of a fluorescence signal, and makes the direct and accurate analysis of the DNA of the blood sample possible.

Detailed Description

The present application is further described below with reference to examples.

In the following description, different "one embodiment" or "an embodiment" may not necessarily refer to the same embodiment, in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art. Various embodiments may be replaced or combined, and other embodiments may be obtained according to the embodiments without creative efforts for those skilled in the art.

Examples

For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the materials are commercially available, unless otherwise specified.

Preparation example 1:

the present preparation provides a sample release agent comprising the following components: 20mM Tris, 25mM sodium bicarbonate, 10mM cyanoguanidine isosulfate, 0.05% sodium dodecyl diphenyl ether disulfonate, 0.5% Brij30, 2% sucrose, and balance water, and the pH of the sample release agent was adjusted to 11.5 using sodium hydroxide.

Preparation example 2:

the preparation example provides a Direct amplification reagent (2X Direct qPCR Master Mix) for blood lysis crude samples, comprising the following components: 60mM Tris-HCl, 75mM KCl, 250. mu.M dNTPs, 250. mu.g/mL BSA, 2.5mM MgCl₂0.5% Brij52, 2U of hot start DNA polymerase and 2mM tetramethylammonium chloride and balance water (deionized water) in a total volume of 100 mL.

Example 1: the process of direct amplification of blood genomic DNA and amplification-versus-nucleic acid extraction after nucleic acid extraction was as follows: 200. mu.L of the anticoagulated blood sample was subjected to nucleic acid extraction using QIAamp DNA Micro Kit (Qiagen, Cat #56304), and finally eluted using 200. mu.L of Buffer AE, and the DNA product (hereinafter sometimes referred to as nucleic acid extraction product) was placed in a 4-degree refrigerator for use.

Pretreatment of the direct amplification process was as follows: 50 mu L of the anticoagulation sample is added with 50 mu L of the nucleic acid releasing agent of the preparation example 1, and after vortex mixing is carried out for 1min, a crude sample product (hereinafter sometimes referred to as a crude sample) is obtained and placed in a refrigerator at 4 ℃ for standby.

The nucleic acid extraction product and the crude sample were tested using the crude sample Direct amplification reagent (2X Direct qPCR Master Mix) provided in preparation example 2, the RNase P reference gene and EX9-4 gene were selected for amplification respectively, ABI 7500 Fast was selected for quantification, and the sample loading was tested using 4 μ L template. The primers and probes required for amplification are shown below:

qPCR reaction system:

components	Volume (μ L)
		2X Direct qPCR Master Mix	10
Primer F (10. mu.M)	0.5
		Primer R (10. mu.M))	0.5
Probe P (10. mu.M)	0.5
		Deionized water	4.5
Stencil sheet	4
		Total volume	20

The amplification procedure was as follows:

the results are shown in the following table:

TABLE 1 comparison of direct amplification of crude samples of blood genomic DNA with amplification of nucleic acid extract products

From the results in table 1, it can be seen that the difference between the Ct values of the genomic DNA (crude sample) in the blood sample treated with the nucleic acid releasing agent and the DNA product (nucleic acid extraction product) extracted from the nucleic acid is less than 2 by the fluorescence quantitative PCR detection, which can be used for not only qualitative analysis but also accurate quantitative analysis, whereas the method developed by the present invention requires only 1-2min for sample pretreatment to release the nucleic acid, whereas the nucleic acid extraction kit using Qigen takes about 40 minutes per sample on average. Thus, the direct blood DNA amplification technology developed by the present invention greatly saves time, but does not reduce the sensitivity of detection.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

It should be noted that the above embodiments can be freely combined as necessary. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Suzhou Chalkbrook science and technology Co., Ltd

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Claims

1. A sample-releasing agent, wherein said sample-releasing agent comprises: tris, sodium bicarbonate, guanidine isothiocyanate, sodium dodecyl diphenyl ether disulfonate, Brij30, sucrose and water.

2. The sample release agent according to claim 1, characterized in that:

the sample-releasing agent comprises: 1-50mM Tris, 1-50mM sodium bicarbonate, 1-20mM guanidine isothiocyanate, 0.01-0.2% sodium dodecyl diphenyl ether disulfonate, 0.01-2% Brij30, 0.5-10% sucrose and the balance water; more preferably, the pH of the sample-releasing agent is 9.0 to 12.5;

more preferably, the sample-releasing agent comprises: 10-20mM Tris, 5-40mM sodium bicarbonate, 2-10mM guanidine isothiocyanate, 0.02-0.1% sodium dodecyl diphenyl ether disulfonate, 0.02-1% Brij30, 1-5% sucrose and the balance water; more preferably, the pH of the sample-releasing agent is 10.0 to 12.0;

3. A reagent for direct amplification of a crude sample, comprising: Tris-HCl, KCl, dNTPs, BSA, MgCl₂Brij52, hot start DNA polymerase and tetramethylammonium chloride.

4. The reagent for direct amplification of a crude sample according to claim 3, wherein:

the crude sample direct amplification reagent comprises: 10-150mM Tris-HCl, 10-150mM KCl, 100-₂0.1-1% Brij52, 1-5U of hot start DNA polymerase and 1-20mM tetramethylammonium chloride;

preferably, the crude sample direct amplification reagent comprises: 20-100mM Tris-HCl, 25-100mM KCl, 200-₂0.2-0.5% Brij52, 2-5U of hot start DNA polymerase and 1-5mM tetramethylammonium chloride;

more preferably, the crude sample direct amplification reagent comprises: 60mM Tris-HCl, 75mM KCl, 250. mu.M dNTPs, 250. mu.g/mL BSA, 2.5mM MgCl₂0.5% Brij52, 2U of hot start DNA polymerase and 2mM tetramethylammonium chloride.

5. A direct amplification kit, comprising:

(1) a reagent for formulating the sample-releasing agent of claim 1 or 2; and/or

(2) A reagent for preparing the direct amplification reagent of crude sample according to claim 3 or 4.

6. The kit of claim 5, wherein:

a reagent for formulating the sample-releasing agent of claim 1 or 2 selected from at least one or more of the group consisting of Tris, sodium bicarbonate, cyanoguanidine isosulfate, sodium dodecyldiphenyloxide disulfonate, Brij30 and sucrose; preferably, the reagent for formulating the sample-releasing agent of claim 1 or 2 comprises Tris, sodium bicarbonate, cyanoguanidine isosulfate, sodium dodecyldiphenyloxide disulfonate, Brij30 and sucrose; it is further preferred that the reagent for formulating the sample-releasing agent according to claim 1 or 2 further comprises water such as deionized water and/or sodium hydroxide for adjusting pH;

it is also preferred that the reagent used to prepare the reagent for direct amplification of a crude sample according to the second aspect of the present invention is selected from Tris-HCl, KCl, dNTPs, BSA, MgCl₂Brij52, hot start DNA polymerase and tetramethylammonium chloride; more preferably, the reagents used to formulate the crude sample direct amplification reagent according to the second aspect of the invention comprise Tris-HCl, KCl, dNTPs, BSA, MgCl₂Brij52, hot start DNA polymerase and tetramethylammonium chloride.

7. A method for the quantitative and/or qualitative analysis of DNA in a blood sample, characterized in that it employs: (1) the sample-releasing agent of claim 1 or 2; and/or

(2) The crude sample direct amplification reagent according to claim 3 or 4.

8. The method of claim 7, wherein:

the method is carried out using the kit of claim 5 or 6.

9. Method according to claim 7 or 8, characterized in that it comprises the following steps:

10. The method according to any one of claims 7 to 9, characterized in that:

in the step (1), the volume dosage of the blood sample is 10-50 mu L; more preferably, the volume dose of the sample-releasing agent is equal to the volume dose of the blood sample;

in the step (2), water such as deionized water is used to make up to the target volume of the reaction working solution; more preferably, the target volume of the reaction working solution is twice that of the crude sample direct amplification reagent;

in step (3), the PCR instrument is a qPCR instrument and the analysis is a quantitative analysis.

It is also preferable that, in the step (2), the volume of the crude sample is 5 to 40%, more preferably 10 to 30% of the volume of the reaction working solution.