CN113564231B

CN113564231B - Nucleic acid diluent

Info

Publication number: CN113564231B
Application number: CN202110872095.6A
Authority: CN
Inventors: 王新涛; 蒋威; 郑静; 肖啸; 杜增民; 王利群; 赵阳; 吴侠; 陈晨; 郑浩
Original assignee: Faith Pharmaceutical Technology Suzhou Co ltd
Current assignee: Shanghai Mianyi Biotechnology Co ltd
Priority date: 2021-07-30
Filing date: 2021-07-30
Publication date: 2023-03-24
Anticipated expiration: 2041-07-30
Also published as: CN113564231A

Abstract

The present disclosure relates to a nucleic acid diluent comprising 1 × TE, F68, and DNA selected from the group consisting of: lambda DNA, pUC19 plasmid DNA, pUC18 plasmid DNA, pBR322 plasmid DNA, denatured protamine DNA, pUC57 plasmid DNA, pTZ19R DNA, escherichia coli DNA, and sssDNA. The nucleic acid diluent disclosed by the invention can be used for diluting a standard substance and a sample in quantitative PCR, and has the functions of protecting and stabilizing the standard substance and the sample. The present disclosure also relates to a standard solution prepared using the nucleic acid diluent and a quantitative PCR method using the standard solution.

Description

Nucleic acid diluent

Technical Field

The present disclosure is in the field of life analysis. In particular, the present disclosure relates to a nucleic acid diluent that can be used to quantify the dilution of standards and samples in PCR. The present disclosure also relates to a standard solution prepared using the nucleic acid diluent and a quantitative PCR method using the standard solution.

Background

Quantitative PCR is a method by which the amount of PCR product can be determined, and the initial template copy number or concentration of a sample can be quantified with high accuracy over a wide concentration range. The quantitative PCR has the advantages of simple operation, high sensitivity, good repeatability and the like, and is widely applied to various fields of life science research, such as gene differential expression analysis, SNP detection, allele detection, drug development, clinical diagnosis, transgenic research and the like.

The quantitative PCR analysis method utilizes a group of standard products diluted in a gradient to make a standard curve and establishes a linear relation between a Ct value and concentration. And after the Ct value of the sample to be detected is obtained, the quantity of the sample to be detected can be converted by using the standard curve. Typically, water or TE is used as a diluent to perform a gradient dilution of the standard or a dilution of the sample to be tested. However, the thus-prepared standard substance or sample (particularly, a standard substance or sample having a low concentration) is often unstable and cannot be stored for a long period of time. Standards or samples diluted using conventional dilutions fail only a few hours after preparation, thus requiring the diluted standards and samples to be freshly prepared prior to each quantitative PCR experiment.

Therefore, in order to enable the diluted standard and/or sample to be stably stored for a long period of time, it is necessary to obtain a diluent having an action of stabilizing the standard and/or sample.

Disclosure of Invention

In view of the above-mentioned deficiencies of the prior art, in a first aspect, the present disclosure provides a nucleic acid diluent comprising 1 × TE, F68, and a DNA selected from the group consisting of: lambda DNA, pUC19 plasmid DNA, pUC18 plasmid DNA, pBR322 plasmid DNA, denatured milt DNA, pUC57 plasmid DNA, pTZ19R DNA, E.coli DNA, and sssDNA (minus-strand-stop DNA). The nucleic acid diluent can be used as a diluent of a standard substance or a sample, and has the functions of protecting and stabilizing the standard substance or the sample. The standard or sample diluted with the diluent can be stored for a long time without fail.

In one embodiment, the concentration of F68 is 0.001 to 0.05%, preferably 0.002 to 0.04%, more preferably 0.003 to 0.03%. In a preferred embodiment, the concentration of F68 is 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, or 0.05%. When F68 is in the above concentration range, better protection and stabilization may be provided.

In one embodiment, the concentration of DNA is 3-30 ng/. Mu.l, preferably 4-25 ng/. Mu.l, more preferably 5-20 ng/. Mu.l.

In a preferred embodiment, the concentration of DNA is 3 ng/. Mu.l, 4 ng/. Mu.l, 5 ng/. Mu.l, 6 ng/. Mu.l, 7 ng/. Mu.l, 8 ng/. Mu.l, 9 ng/. Mu.l, 10 ng/. Mu.l, 15 ng/. Mu.l, 20 ng/. Mu.l, 25 ng/. Mu.l or 30 ng/. Mu.l. When the DNA is in the above concentration range, a better stabilizing effect can be provided, which is advantageous for reducing adsorption loss of the standard or sample on the container.

In a preferred embodiment, the DNA is Lambda DNA.

In a second aspect, the present disclosure provides a standard solution, wherein the standard solution is obtained by diluting a standard stock solution with the above-mentioned nucleic acid diluent, and the concentration of the standard solution is lower than that of the standard stock solution.

In one embodiment, the standard is a standard for DNA sample quantification or a standard for RNA sample quantification. In one embodiment, the standard for DNA sample quantification is selected from the group consisting of genomic DNA, plasmid, and PCR product. In one embodiment, the standard for RNA sample quantification is selected from the group consisting of in vitro transcribed RNA, PCR products, and plasmids.

In one embodiment, the ratio of the concentration of the standard solution to the concentration of the standard stock solution is 1 ¹ To 1 × 10 ¹² (e.g., 1 ¹ 、1:1×10 ² 、1:1×10 ³ 、1:1×10 ⁴ 、1:1×10 ⁵ 、1:1×10 ⁶ 、1:1×10 ⁷ 、1:1×10 ⁸ 、1:1×10 ⁹ 、1:1×10 ¹⁰ 、1:1×10 ¹¹ Or 1 × 10 ¹² ) Preferably, the ratio of the concentration of the standard solution to the concentration of the standard stock solution is 1 × 10 ⁵ To 1 × 10 ¹² (e.g., 1 ⁵ 、1:1×10 ⁶ 、1:1×10 ⁷ 、1:1×10 ⁸ 、1:1×10 ⁹ 、1:1×10 ¹⁰ 、1:1×10 ¹¹ Or 1 ¹² )。

In one embodiment, the standard solution is freshly prepared or stored for a period of time.

In one embodiment, the period of time may be one day to one year (e.g., one day, one week, one month, three months, or one year).

In a third aspect, the present disclosure provides a method for determining a concentration of a target molecule using quantitative PCR, comprising: carrying out gradient dilution on the stock solution of the standard substance by using the nucleic acid diluent to prepare a standard curve of quantitative PCR; and obtaining the concentration of the target molecule using the standard curve. Alternatively, the target molecule is diluted with the nucleic acid diluent described above.

In one embodiment, the target molecule is RNA or DNA.

Drawings

FIG. 1 shows a standard curve obtained by diluting a stock solution of a standard substance with ultrapure water (diluent 1) in a gradient manner, and the Ct value of the amplification curve is used to prepare the standard curve by immediately performing quantitative PCR on the diluted stock solution of the standard substance.

FIG. 2 shows a standard curve obtained by diluting a stock solution of a standard substance with ultrapure water (diluent 1) in a gradient manner, and the diluted stock solution of the standard substance is left at 4 ℃ for about 1 hour and then subjected to quantitative PCR to prepare a standard curve from the Ct value of the amplification curve.

FIG. 3 shows a standard curve obtained by diluting a stock solution of a standard substance with ultrapure water (dilution 1) in a gradient manner, and the diluted stock solution of the standard substance is left at 4 ℃ overnight and then subjected to quantitative PCR to prepare a standard curve using the Ct value of the amplification curve.

FIG. 4 shows a standard curve obtained by diluting a stock solution of a standard substance with diluent 5 (1 XTE +0.005% F68+6 ng/. Mu.l Lambda DNA) in a gradient manner, and the diluted solution of the standard substance is immediately subjected to quantitative PCR to prepare a standard curve using the Ct value of the amplification curve.

FIG. 5 shows a standard curve obtained by diluting the stock solution of the standard substance with diluent 5 (1 XTE +0.005% F68+6 ng/. Mu.l Lambda DNA) in a gradient manner, and the diluted solution of the standard substance is stored at 4 ℃ for one week and then subjected to quantitative PCR to prepare a standard curve using the Ct value of the amplification curve.

FIG. 6 shows a standard curve obtained by diluting the stock solution of the standard substance with diluent 5 (1 XTE +0.005% F68+6 ng/. Mu.l Lambda DNA) in a gradient manner, and the diluted solution of the standard substance is stored at 4 ℃ for one month and then subjected to quantitative PCR to prepare a standard curve using the Ct value of the amplification curve.

Detailed Description

Unless defined otherwise, 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 disclosure belongs.

The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") in this context.

It is known in the art that quantitative PCR can be used to quantify deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in a sample. Using sequence specific primers, the concentration (i.e., copy number) of a particular DNA or RNA sequence can be determined. Quantification is achieved by detecting the amount of amplified product in each stage of the PCR cycle.

As used in the art, ct values refer to the number of PCR cycles at which the fluorescence value reaches a threshold value.

Herein, a standard curve is established using serially diluted standard solutions for determining the initial amount of template of interest in a sample to be tested, or evaluating amplification efficiency. As is known in the art, the accuracy of the quantification of the sample to be tested is directly related to the quality of the standard curve. A standard curve is plotted with the logarithm of the known serially diluted concentration as the abscissa (x-axis) and the Ct value corresponding to this concentration as the ordinate (y-axis). From the standard curve, information about the reaction performance and various reaction parameters including slope, y-intercept and correlation coefficient can be derived. The concentration selected in the standard curve is determined according to the concentration of the target molecule to be detected, and should cover the possible concentration range of the sample to be detected.

Herein, the indicators for evaluating the effect of the quantitative PCR reaction include: amplification efficiency, R ² Value and recovery.

Herein, the correlation coefficient (R) of the standard curve, as known in the art ² ) It is a statistical term that describes the degree of correlation between two values and is a key parameter in assessing PCR efficiency. R ² The values reflect the linearity of the standard curve. R ² The closer to 1, the better the linearity. It is generally accepted that R of the standard curve ² At values greater than 0.999, the confidence in the correlation between the two values is good.

Herein, as known in the art, the slope of the standard curve reflects the amplification efficiency of the PCR reaction, and is calculated by the formula: amplification efficiency =10 ^(-1/slope) -1. To obtain accurate and reproducible results, the amplification efficiency should be as close to 100% as possible, forThe slope should be-3.32. This amplification was considered acceptable when the amplification efficiency was between 90-110%, when the slope of the standard curve was-3.58 to-3.10.

Herein, as known in the art, the sample recovery is calculated as follows: sample recovery = number of copies of sample after recovery/number of copies of sample before recovery x 100%. The closer the recovery is to 100%, the higher the accuracy.

In a preferred embodiment, a nucleic acid diluent of the present disclosure comprises 1 × TE, F68, and Lambda DNA.

In this context, as known in the art, a typical formulation of a 1 × TE buffer is: 10mM Tris (pH adjusted to 8.0 using HCl) and 1mM EDTA. Commercially available 1 XTE, for example, 1 XTE available from Allantin reagent Co., ltd can be used.

As used herein, "F68" refers to polyether F68, which may also be referred to as Pluronic F68. Commercially available F68 may be used. In one embodiment, the concentration of F68 is 0.001 to 0.05%, preferably 0.002 to 0.04%, more preferably 0.003 to 0.03%. In a preferred embodiment, the concentration of F68 is 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, or 0.05%.

In this context, "Lambda DNA", also referred to as Lambda DNA, is a linear double-stranded phage DNA comprising a 12bp single-stranded complementary 5' end, derived from a Bacteriophage Escherichia coli (Bacteriophage Lambda cI857 Sam 7). Commercially available Lambda DNA, for example, commercially available from therofisher (cat. SD 0011) can be used. In one embodiment, the Lambda DNA concentration is 3-30 ng/. Mu.l, preferably 4-25 ng/. Mu.l, more preferably 5-20 ng/. Mu.l. In a preferred embodiment, the Lambda DNA concentration is 3 ng/. Mu.l, 4 ng/. Mu.l, 5 ng/. Mu.l, 6 ng/. Mu.l, 7 ng/. Mu.l, 8 ng/. Mu.l, 9 ng/. Mu.l, 10 ng/. Mu.l, 15 ng/. Mu.l, 20 ng/. Mu.l, 25 ng/. Mu.l, or 30 ng/. Mu.l.

In one embodiment, the nucleic acid diluent of the present disclosure is prepared by mixing commercially available 1 × TE, F68, and Lambda DNA.

As can be seen from the description herein, the nucleic acid dilutions of the present disclosure have the effect of protecting and stabilizing the nucleic acid standards, and can be used to dilute the standard stock solutions in a gradient to make a standard curve for quantitative PCR. The standard solution diluted with the nucleic acid diluent of the present disclosure can be stored for a longer period of time without being inactivated. Meanwhile, the nucleic acid diluent disclosed by the invention also has the functions of protecting and stabilizing a nucleic acid sample, and can be used for preparing a sample to be detected.

In one embodiment, a standard solution of the present disclosure is obtained by diluting a standard stock solution with a nucleic acid diluent of the present disclosure. Therefore, the standard solution of the present disclosure is a diluted standard solution obtained by diluting a standard stock solution with the nucleic acid diluent of the present disclosure, and the concentration of the diluted standard solution is lower than that of the standard stock solution.

In one embodiment, the standard is a standard for DNA sample quantification or a standard for RNA sample quantification. In one embodiment, the standard for DNA sample quantification is selected from the group consisting of genomic DNA, plasmid, and PCR product. In one embodiment, the standard for RNA sample quantification is selected from the group consisting of in vitro transcribed RNA, PCR products, and plasmids. Methods for preparing standards are known in the art, and can be prepared by methods commonly used in the art, such as enzymatic cleavage, PCR generated fragments, or linearized plasmids.

In order to prepare a standard curve, the stock solution of the standard substance is diluted in a gradient manner by adopting a multiple proportion dilution method. In one embodiment, a standard stock solution of known concentration is subjected to a multiple continuous gradient dilution of at least 5 orders of magnitude (e.g., 5 orders of magnitude, 6 orders of magnitude, 7 orders of magnitude, or 8 orders of magnitude) to obtain standard solutions of different concentrations. In one embodiment, the ratio of the concentration of the standard solution to the concentration of the standard stock solution is 1 ¹ 、1:1×10 ² 、1:1×10 ³ 、1:1×10 ⁴ 、1:1×10 ⁵ 、1:1×10 ⁶ 、1:1×10 ⁷ 、1:1×10 ⁸ 、1:1×10 ⁹ 、1:1×10 ¹⁰ 、1:1×10 ¹¹ Or 1 × 10 ¹² . In a preferred embodiment, the ratio of the concentration of the standard solution to the concentration of the standard stock solution is 1 ⁵ 、1:1×10 ⁶ 、1:1×10 ⁷ 、1:1×10 ⁸ 、1:1×10 ⁹ 、1:1×10 ¹⁰ 、1:1×10 ¹¹ Or 1 ¹² 。

In one embodiment, the standard solution of the present disclosure is freshly prepared. The freshly prepared standard solution refers to a standard solution obtained after dilution with a diluent, which is not stored. In one embodiment, the standard solution of the present disclosure is stored for a period of time (which may be any length of time, e.g., one day, one week, one month, three months, one year). The standard solution after being stored for a certain period of time means a standard solution obtained after being diluted with a diluent and stored for a certain period of time. The freshly prepared standard solution or the standard solution after being stored for a period of time is stable and can be prepared into an ideal standard curve.

As used herein, "ideal standard curve" or "good standard curve" refers to the R of the standard curve ² Values greater than 0.999 and the slope of the standard curve is in the range of-3.58 to-3.10.

As known in the art, quantitative PCR methods typically include the following steps: extracting a sample; designing a primer; preparing a standard substance; carrying out PCR reaction; and (6) carrying out data analysis.

In one embodiment, the present disclosure relates to a method of determining the concentration of a target molecule using quantitative PCR, comprising: carrying out gradient dilution on a standard substance stock solution by using the nucleic acid diluent disclosed by the invention, and making a standard curve of quantitative PCR; and obtaining the concentration of the target molecule using the standard curve. In one embodiment, the target molecule is RNA or DNA. In one embodiment, the target molecule is diluted using a nucleic acid diluent of the present disclosure.

In one embodiment, the methods of the present disclosure may employ relative or absolute quantitation. The choice of the quantitative method depends on the experimental objective in the actual measurement.

Relative quantitation refers to comparing the expression of a gene of interest in one sample (treated) with the expression of the same gene in another sample (untreated) in quantitative PCR. The results are expressed as fold-change (increase or decrease) in the expression level of the treated sample relative to the expression level of the untreated sample. Calculation methods for relative quantification include standard curve method and comparative CT method. For relative quantitation, any stock solution of RNA or DNA containing the appropriate target molecule can be used as a standard. The data obtained using relative quantitation is the relative abundance of the target molecule, not the exact copy number. Relative quantitation methods are suitable for studying gene expression.

The absolute quantification method is to use a standard with a known initial copy number to form a standard curve in quantitative PCR, and to calculate the initial concentration of an unknown sample from the standard curve by measuring the Ct value of the unknown sample. For absolute quantitation, plasmid DNA or in vitro transcribed RNA is typically used to prepare standards. Absolute quantitation methods can measure the actual copy number of a target molecule, requiring a highly accurate standard curve. Absolute quantitation is commonly used to determine the genomic titer of viral particles.

In one embodiment, the quantitative PCR is performed using a PCR instrument selected from the group consisting of: ABI7000, 7300, 7500, 7700, 7900HT, stepOnePlusTM, stepOneNet, PRISM @ StepOneTM series, CFX96 of BIO-RAD, iCycler iQ5@, myiQ @ MJ Research Chromo4TM Opticon series, stratagene MxTM series, roche LightCycler @ series, eppendorf Masercycler @, cort Robet-Gene series, cepheid SmartCycler @ and BIOER LineGene series.

In one embodiment, a probe or non-probe may be used to indicate an increase in PCR amplification products. Probes such as TaqMan @ probes and molecular beacons, which utilize probes that specifically hybridize to a target sequence to indicate an increase in amplification product. Non-probes such as SYBR @ Green I or specially designed primers (e.g., LUX @ primers) that indicate an increase in product by a fluorescent dye.

The present disclosure is described in further detail below with reference to the accompanying drawings and examples. The following examples are merely illustrative of the present disclosure and are not intended to limit the scope of the present disclosure. The experimental procedures, in which the specific conditions are not indicated in the examples, are carried out according to the conventional conditions known in the art or according to the conditions recommended by the manufacturer.

Examples

Example 1: stabilization of standard with diluent

In this example, the stabilizing effect of the diluent of the present disclosure (exemplified by 1 × TE + F68+ Lambda DNA) on a standard (exemplified by a DNA standard) was verified. Table 1 lists the composition of each dilution.

TABLE 1 composition of the dilutions

The DNA standard stock solutions were subjected to gradient dilutions (10-fold serial gradient dilution) as follows using the dilutions of Nos. 1 to 7 described above, respectively:

1) 1v of standard substance stock solution i +9v of diluent to obtain standard substance solution ii;

2) 1v of a standard solution ii +9v of a diluent to obtain a standard solution iii;

3) 1v of a standard solution iii +9v of a diluent to obtain a standard solution iv;

4) 1v of a standard solution iv +9v diluent to obtain a standard solution v;

5) Diluting the 1v standard solution v +9v to obtain a standard solution vi;

6) 1v of standard solution vi +9v of diluent to obtain standard solution vii;

then, quantitative PCR (instrument: roche 480) was performed on the standard solutions ii to vii three times for each experiment, and the average of three Ct values was calculated. Then, a standard curve is drawn with the log values of the concentrations of the standards ii to vii as the abscissa and the average Ct value corresponding to each concentration as the ordinate, and an equation of the standard curve is fitted.

As shown in FIGS. 1 and 2, a good calibration curve (R) was obtained when the standard solution diluted with ultrapure water (diluent 1) was used immediately after dilution and for about 1 hour ² Up to 0.999, amplification efficiency of more than 90%). However, as shown in FIG. 3, the R of the standard curve obtained after overnight for the thus diluted standard solution ² Is only that0.99865, slope-4.11, calculated amplification efficiency of only 75%, indicating that accurate quantification cannot be performed.

Similarly, the results show that the standard solutions diluted with 1 × TE (dilution 2) and commercially available dilutions (dilutions 3 and 4) did not yield satisfactory amplification efficiency (less than 90%) after overnight, and accurate quantification was not possible.

In contrast, as shown in FIGS. 4 to 6, the standard solution gradient-diluted with diluent 5 (1 XTE +0.005% F68+6ng/ul Lambda DNA) not only obtained a good standard curve when used immediately after dilution (FIG. 4, R ² Up to 0.999 with an amplification efficiency of 101%), and maintaining a fairly excellent stability, R of the standard curve, after one week of storage (FIG. 5), even after one month (FIG. 6) ² All reach 0.999, and the amplification efficiency is about 100%.

Further, the stock DNA standard solution was subjected to gradient dilution using diluent 6 (1 XTE +0.001% F68+3ng/ul Lambda DNA) and diluent 7 (1 XTE +0.05% F68+30ng/ul Lambda DNA) as diluents, respectively, to obtain standard curves after different storage times (immediately, one week, one month). The results showed that a good standard curve (not shown) was still obtained after long-term storage of one month for the diluted standard solutions, confirming that both diluent 6 and diluent 7 had the effect of stabilizing the standard.

In addition, the standard solutions were graded diluted with

dilutions

5, 6 and 7, respectively, for up to 3 months. The results show that the standard solutions diluted with the

dilutions

5, 6 and 7 gradients are very stable during 3 months of storage, and an ideal standard curve (not shown) can be obtained, the R of which is the R of the standard curve ² All reach 0.999, and the amplification efficiency is close to 100%.

The above results show that when the stock standard is diluted with water or other diluent (e.g., commercially available diluent), the resulting calibration curve is inaccurate (R) after storage for a period of time ² Lower values and amplification efficiencies deviate from acceptable ranges). In contrast, a standard solution diluted with a gradient of

diluent

5, 6 or 7 may remain stable for storage periods up to three months, thereby achieving the desired standard curve.

It can be seen that a nucleic acid diluent of the present disclosure comprising three specific components, 1 × TE, F68, and Lambda DNA, can serve to stabilize the standard. The standard solution prepared using the nucleic acid diluent of the present disclosure can be stably stored for up to several months. Therefore, the standard solution disclosed by the invention does not need to be prepared again in each experiment, so that the operation flow of quantitative PCR is simplified, and the detection time is saved.

Example 2: stabilization of nucleic acid samples with dilutions

In this example, the stabilizing effect of the dilution (exemplified by 1 × TE + F68+ Lambda DNA) of the present disclosure on a nucleic acid sample (exemplified by a DNA sample) was verified. Table 2 lists the sample recovery with different dilutions.

Taking a proper amount of a sample to be detected (a laboratory sample with virus S01201001), and respectively adding 120 pg/mu l, 60 pg/mu l and 30 pg/mu l of standard substances with the same volume as the sample to be detected as a sample labeling group. Both samples and standards were diluted with diluent 5 or commercially available diluent (Shenke kit, diluent lots 06301 and 0901).

TABLE 2 sample recovery

The results show that the recovery of dilution 5 (1 × TE + F68+ Lambda DNA) of the present disclosure is close to 100%, and is more accurate and reliable than the dilutions of the commercial kits.

While the present disclosure has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing is a more detailed description of the disclosure than is possible with reference to the specific embodiments, and that no limitation to the specific embodiments of the disclosure is intended. Various changes in form and detail, including simple deductions or substitutions, may be made by those skilled in the art without departing from the spirit and scope of the present disclosure.

Claims

1. A nucleic acid diluent consisting of 1 XTE, 0.001-0.005% F68 and 3-6 ng/. Mu.l Lambda DNA.

2. The nucleic acid diluent according to claim 1, wherein the concentration of Lambda DNA is 4 to 6ng/μ l.

3. The nucleic acid diluent according to claim 1 or 2, wherein the concentration of Lambda DNA is 5 to 6 ng/. Mu.l.

4. Use of the nucleic acid diluent of any one of claims 1 to 3 in diluting a stock standard solution, comprising diluting the stock standard solution with the nucleic acid diluent of any one of claims 1 to 3 to obtain a standard solution, the standard solution having a lower concentration than the stock standard solution, the standard being a standard for DNA sample quantification.

5. The use according to claim 4, wherein the standard for DNA sample quantification is selected from the group consisting of genomic DNA, plasmid and PCR product.

6. Use according to claim 4 or 5, wherein the ratio of the concentration of the standard solution to the standard stock solution is 1 ¹ To 1 × 10 ¹² 。

7. Use according to claim 4 or 5, wherein the ratio of the concentration of the standard solution to the standard stock solution is 1 ⁵ To 1 × 10 ¹² 。

8. Use according to claim 4 or 5, wherein the standard solution is freshly prepared or stored for a period of time.

9. The use of claim 8, wherein the period of time is one day to one year.

10. The use of claim 8, wherein the period of time is one day, one week, one month, three months or one year.

11. A method for non-diagnostic purposes of determining the concentration of a target molecule using quantitative PCR, comprising: performing gradient dilution on a standard stock solution by using the nucleic acid diluent according to any one of claims 1 to 3 to prepare a standard curve of quantitative PCR; and obtaining the concentration of the target molecule by using the standard curve, wherein the target molecule is DNA.