Digestion-free plasma mass spectrum fragment DNA content detection method

Abstract

The invention relates to the technical field of biological detection, in particular to a digestion-free plasma mass spectrometry fragment DNA content detection method which comprises the following steps: S1, DNA sample purification: removing mononucleotide and small fragment nucleic acid with less than 10 basic groups by using a centrifugal tube with the molecular weight cutoff of 3KDa; carrying out interception treatment on the DNA sample subjected to gel electrophoresis purification, and realizing purification through multiple times of concentration and water replenishing; s2, setting liquid chromatography and mass spectrometry conditions: a liquid chromatography mobile phase is 10mM TRIS-HCL and 1mM EDTA, the pH setting range is 7-7.5, the pH is adjusted by using ammonia water, and the final optimization condition is 7.3; a molecular weight cut-off method is combined with a method of coupling size exclusion chromatography with inductively coupled plasma mass spectrometry to obtain a DNA fragment with higher purity, and the DNA fragment is used as a candidate standard substance DNA. And determining the content of the purified high-purity DNA fragment by using an inorganic phosphorus element standard curve to obtain the mass concentration information of the fragment.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a method for detecting DNA content in plasma mass spectrometry fragments without digestion. Background Art

[0002] In the fields of in vitro diagnosis, food testing, and forensic identification, the use of traceable DNA reference materials for value transfer is conducive to the healthy development of the industry. The preparation process of DNA reference materials usually involves the acquisition of pure DNA and the determination of its content. DNA purification methods usually undergo magnetic bead extraction and column purification, followed by further purification by gel electrophoresis, high-performance liquid chromatography, etc. How to obtain DNA candidates with higher purity and analyze their content is the key to obtaining DNA reference materials.

[0003] There are three types of methods for determining DNA content, namely, detection methods based on optical principles such as ultraviolet absorption and fluorescence emission, PCR detection methods based on sequence amplification, and mass spectrometry detection methods based on quality. Detection methods based on ultraviolet and fluorescence can quickly provide concentration information, but are easily interfered by impurity nucleic acids in the test system. PCR-based detection methods have high specificity and sensitivity, but nucleic acid amplification will produce false positive fluorescence signals and affect DNA quantification. Mass spectrometry methods based on quality can be divided into isotope dilution mass spectrometry and inductively coupled plasma mass spectrometry. The former has high sensitivity and the method is traceable, but the measurement process is greatly affected by the purity of the nucleic acid.

[0004] According to the publication number: CN110487923B, a method for detecting the content of linear plasmid DNA based on molecular exclusion chromatography-inductively coupled plasma mass spectrometry is disclosed. This technology discloses "a method for detecting the content of linear plasmid DNA, comprising the following steps: (1) purifying the linear plasmid DNA sample to be tested; (2) digesting the linear plasmid DNA to be tested that meets the requirements after purity testing; (3) using phosphorus standard substances as standards, and preparing a standard curve based on the characteristic value of the phosphorus standard substances and the corresponding mass spectrum peak area obtained by molecular exclusion chromatography-inductively coupled plasma mass spectrometry detection. line, and then subjecting the digested linear plasmid DNA sample to molecular exclusion chromatography-inductively coupled plasma mass spectrometry analysis, and using the obtained mass spectrum peak area corresponding to the linear plasmid DNA and the standard curve for quantitative analysis" and other technical solutions, which have the technical effects of "using molecular exclusion chromatography to perform purity analysis on linear plasmid DNA, which can effectively separate inorganic phosphorus, nucleotides, and oligonucleotides from plasmid DNA, and can effectively exclude phosphorus interference from non-plasmid DNA sources such as nucleotides, oligonucleotides, and inorganic phosphorus, thereby ensuring the accuracy and reliability of plasmid DNA quantification based on the quantification of phosphorus in plasmid DNA"; The DNA quantification method based on inductively coupled plasma mass spectrometry relies on a fixed stoichiometric ratio of DNA and the phosphorus element it contains, which is determined by the unique chemical structure of DNA. This method has high sensitivity and uses phosphorus standard substances to achieve traceability. It should be noted that DNA with longer fragments is prone to degradation during processing and measurement, which will result in a low measured phosphorus content, thus affecting the accuracy of DNA quantification; in addition, DNA is usually dissolved in a solution containing buffer substances, which may contain phosphorus or other substances that may interfere with the measurement. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for detecting the content of DNA fragments in plasma mass spectrometry without digestion, which adopts a molecular weight cutoff method combined with a size exclusion chromatography coupled to an inductively coupled plasma mass spectrometry method to obtain DNA fragments with higher purity for use as candidate standard substance DNA. The purified high-purity DNA fragments are measured using an inorganic phosphorus standard curve to obtain fragment mass concentration information.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for detecting DNA content in plasma mass spectrometry fragments without digestion, comprising the following steps: S1, DNA sample purification: Use a centrifuge tube with a molecular weight cutoff of 3KDa to remove single nucleotides and small nucleic acid fragments with less than 10 bases; and perform interception treatment on the DNA sample purified by gel electrophoresis, and purify it through multiple concentrations and water replenishment; S2, liquid chromatography and mass spectrometry conditions: the liquid chromatography mobile phase was 10 mM TRIS-HCL and 1 mM EDTA, the pH setting range was 7-7.5, ammonia was used to adjust the pH, and the final optimized condition was 7.3; S3, inductively coupled plasma mass spectrometry parameters: RF transmission power 1550W, RF voltage 1.80V; S4, drawing of standard curve of inorganic phosphorus element: prepare standard solutions of inorganic phosphorus element component analysis with mass concentrations of 0.1ppm, 0.5ppm, 1ppm, 5ppm and 10ppm, respectively, with an injection volume of 1ul, the chromatographic conditions are the same as above, and the injection is repeated three times or more; S5, DNA fragment content determination: The purified DNA fragments do not need to be digested. The DNA fragment samples are directly subjected to size exclusion chromatography and then enter the mass spectrometer for atomization, and then identified by inductively coupled plasma mass spectrometry.

[0007] Preferably, in S1, a DNA sample with a concentration of about 150 ng / ul and a volume of 500 ul is prepared, the concentration volume is set, 500 ul is concentrated to about 50 ul, the centrifugal speed is set to 13000 rpm, the centrifugal time is set to 10 min, and each time it is concentrated to 50 ul, it is supplemented to 500 ul with ultrapure water and concentrated again. The concentration times are more than 3 times to achieve purification.

[0008] Preferably, in S2, the mobile phase conditions are isocratic elution with salt concentration, a flow rate of 1 ml / min, and an injection volume of 4 ul.

[0009] Preferably, in S3, the sampling depth is 8.0 mm, the nebulizing gas is 0.8 L / min, the makeup gas is 25%, the quadrupole vacuum is 0.0015 Pa, the nebulizing chamber temperature is 2.0°C, the carrier gas flow rate is 1.0 L / min, the feedback power is 18 W, the peristaltic pump is 0.3 rps, the sampling depth is 8 mm, the data acquisition mode is TRA, the acquisition time is 12 min, and the integration time is 1.00 s.

[0010] Preferably, in S4, a standard curve is drawn with the mass concentration of phosphorus in the phosphorus standard solution as the abscissa and the peak area of ​​the mass spectrometry signal as the ordinate, a linear equation is fitted and the linear regression coefficient of the standard curve is calculated.

[0011] Preferably, in said S5, the fragment length is 311 bp.

[0012] Preferably, in S5, the identified result formula is: c=c s *f / P% Where: c is the concentration of undigested fragmented DNA, c s The mass concentration of phosphorus in the sample solution is calculated based on the standard curve formula, and f is the dilution multiple of DNA.

[0013] Preferably, P% is the percentage of phosphorus content in the DNA fragment, P%=2*bp*30.974 / DNA fragment molecular weight, bp represents the number of bases contained in the fragment.

[0014] The present invention provides a method for detecting the DNA content in plasma mass spectrometry fragments without digestion. Compared with the prior art, it has the following beneficial effects: 1. The technical solution of molecular weight cutoff centrifuge tube combined with size exclusion chromatography effectively removes impurities such as single nucleotides and small oligonucleotide fragments in DNA samples, significantly improving the purity of DNA fragments. After three consecutive molecular weight cutoff purifications, the purity of DNA fragments can reach more than 99%, which is crucial for the preparation of high-quality DNA standard materials. High-purity DNA fragments not only improve the accuracy of subsequent quantitative analysis, but also provide a more reliable basis for the transfer of quantitative values ​​in fields such as in vitro diagnosis, food testing, and forensic identification. In addition, this purification method is simple to operate and easy to control, providing strong support for the large-scale preparation of high-purity DNA standard materials.

[0015] 2. By introducing size exclusion chromatography coupled with inductively coupled plasma mass spectrometry, direct quantitative analysis of DNA fragments is achieved without digestion. This not only simplifies the determination process, but also reduces the errors that may be caused by the digestion process, thereby improving the accuracy and reliability of DNA content determination. In addition, this method is also applicable to DNA fragments of different lengths and has a wide range of applicability.

[0016] 3. The DNA fragments were quantified using the standard curve of inorganic phosphorus. Since the content of phosphorus in DNA is in a fixed ratio with its molecular weight, this method has high accuracy and sensitivity. At the same time, the accuracy and stability of the determination were further improved by optimizing the parameters of liquid chromatography and mass spectrometry. In addition, this method also avoids the influence of the purity of DNA fragments on the determination results, making the determination results more real and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a diagram of the method steps of the present invention; Figure 2 This is a sample test diagram without molecular weight cutoff auxiliary purification in the present invention; Figure 3 This is a sample test diagram after molecular weight cutoff-assisted purification in the present invention; Figure 4 This is a test diagram after three molecular weight cutoff assisted purification in the present invention; Figure 5 It is a quantitative characterization of the purification effect in the present invention; Figure 6 Figure 2 is a diagram of a method for calculating the content of a purified DNA sample in the present invention; Figure 7 It is the standard curve diagram of inorganic phosphorus element in the present invention; Figure 8 This is a test diagram of the peak time of the DNA fragment after size exclusion in the present invention; Fig. 9This is a result diagram of multiple measurements of the DNA fragment peak area without digesting the sample in the present invention. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] See also Figure 1-Figure 9 The present invention provides a technical solution: a method for detecting DNA content in plasma mass spectrometry fragments without digestion, comprising the following steps: S1, DNA sample purification: Use a centrifuge tube with a molecular weight cutoff of 3KDa to remove single nucleotides and small nucleic acid fragments with less than 10 bases; and perform interception treatment on the DNA sample purified by gel electrophoresis, and purify it through multiple concentrations and water replenishment; S2, liquid chromatography and mass spectrometry conditions: the liquid chromatography mobile phase was 10 mM TRIS-HCL and 1 mM EDTA, the pH setting range was 7-7.5, ammonia was used to adjust the pH, and the final optimized condition was 7.3; S3, inductively coupled plasma mass spectrometry parameters: RF transmission power 1550W, RF voltage 1.80V; S4, drawing of standard curve of inorganic phosphorus element: prepare standard solutions of inorganic phosphorus element component analysis with mass concentrations of 0.1ppm, 0.5ppm, 1ppm, 5ppm and 10ppm, respectively, with an injection volume of 1ul, the chromatographic conditions are the same as above, and the injection is repeated three times or more; S5, DNA fragment content determination: The purified DNA fragments do not need to be digested. The DNA fragment samples are directly subjected to size exclusion chromatography and then enter the mass spectrometer for atomization, and then identified by inductively coupled plasma mass spectrometry.

[0020] In this implementation scheme, purification of DNA standard substance candidates is of great significance for obtaining standard substances with higher purity; molecular weight cutoff auxiliary technology can effectively remove single nucleotide impurities with small molecular weight, which plays a key auxiliary role in improving the purity of standard substance candidates. By removing impurities through three consecutive molecular weight cutoff schemes, a DNA fragment sample with a purity of more than 99% can be obtained; the application of this technology makes it possible to use the inorganic phosphorus element standard curve for the quantitative process of DNA fragments without the need for fragment hydrolysis and other steps, thereby reducing the errors caused by the DNA hydrolysis process and the purity of the DNA fragments themselves, and realizing direct quantification of DNA fragment samples after injection, thereby increasing the accuracy of the quantitative method.

[0021] Specifically, in S1, a DNA sample with a concentration of about 150 ng / ul and a volume of 500 ul is prepared, the concentration volume is set, and 500 ul is concentrated to about 50 ul. The centrifugal speed is set to 13000 rpm, and the centrifugal time is set to 10 min. After each concentration to 50 ul, ultrapure water is used to make up to 500 ul, and it is concentrated again. The concentration times are more than 3 times to achieve purification.

[0022] In this embodiment, the technical scheme of molecular weight cutoff centrifuge tube combined with size exclusion chromatography is adopted to effectively remove impurities such as mononucleotides and small fragment oligonucleotides in the DNA sample, and significantly improve the purity of the DNA fragments. After three consecutive molecular weight cutoff purifications, the purity of the DNA fragments can reach more than 99%, which is crucial for the preparation of high-quality DNA standard materials. High-purity DNA fragments not only improve the accuracy of subsequent quantitative analysis, but also provide a more reliable basis for the transfer of quantity values ​​in the fields of in vitro diagnosis, food testing, and forensic identification. In addition, the purification method is simple to operate and easy to control, which provides strong support for the large-scale preparation of high-purity DNA standard materials.

[0023] Specifically, in S2, the mobile phase conditions are isocratic elution with salt concentration, a flow rate of 1 ml / min, and an injection volume of 4 ul.

[0024] Specifically, in S3, the sampling depth is 8.0 mm, the nebulizing gas is 0.8 L / min, the makeup gas is 25%, the quadrupole vacuum is 0.0015 Pa, the nebulizing chamber temperature is 2.0°C, the carrier gas flow rate is 1.0 L / min, the feedback power is 18 W, the peristaltic pump is 0.3 rps, the sampling depth is 8 mm, the data acquisition mode is TRA, the acquisition time is 12 min, and the integration time is 1.00 s.

[0025] In this example, the sample without molecular weight cut-off auxiliary purification was subjected to size exclusion chromatography and inductively coupled plasma mass spectrometry. Figure 2 As shown, the samples purified with the aid of molecular weight cutoff were subjected to size exclusion chromatography and inductively coupled plasma mass spectrometry. Figure 3 As shown, three molecular weight cut-offs assisted purification as Figure 4 As shown, it is finally concluded Figure 5 , single nucleotides are completely removed and the sample is pure enough in this case to be directly quantified based on the peak area of ​​the fragmented DNA.

[0026] Specifically, in S4, the mass concentration of phosphorus in the phosphorus standard solution is used as the abscissa, and the peak area of ​​the mass spectrometry signal is used as the ordinate to draw a standard curve, fit a linear equation, and calculate the linear regression coefficient of the standard curve.

[0027] In this embodiment, since the inorganic phosphorus standard curve is obtained based on the linear relationship between the phosphorus content and the mass spectrometry peak area, the linear equation of the inorganic phosphorus standard curve can be used as a method for calculating the content of the purified DNA sample; the five-point method is used to prepare the inorganic phosphorus component analysis standard curve, and the linear regression equation and R2 are obtained according to the horizontal and vertical coordinates. Figure 6 and Figure 7 As shown, the undigested fragmented DNA was directly quantified using the regression equation.

[0028] Specifically, in the S5, the fragment length is 311 bp.

[0029] In this embodiment, the identification results are as follows: Figure 8 As shown in the figure, the peak time of the fragmented DNA after size exclusion, that is, the fragmented DNA directly enters the mass spectrometer through the chromatography in the form of aggregated molecules. This process is significantly different from the injection after digestion into phosphorus element.

[0030] Specifically, in S5, the identified result formula is: c=c s *f / P% Where: c is the concentration of undigested fragmented DNA, c s The mass concentration of phosphorus in the sample solution is calculated based on the standard curve formula, and f is the dilution multiple of DNA.

[0031] Specifically, P% is the proportion of phosphorus content in the DNA fragment, P%=2*bp*30.974 / DNA fragment molecular weight, bp represents the number of bases contained in the fragment.

[0032] In this embodiment, according to the DNA fragment peak area, no digestion of the sample is required, and multiple measurements are performed to obtain the results as follows: Fig. 9 As shown in the figure, this quantitative process eliminates the possible errors caused by the sample digestion process and increases the accuracy of the method.

[0033] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0034] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting DNA content in plasma mass spectrometry fragments without digestion, characterized in that: The following steps are involved: S1, DNA sample purification: Use a centrifuge tube with a molecular weight cutoff of 3KDa to remove single nucleotides and small nucleic acid fragments with less than 10 bases; and perform interception treatment on the DNA sample purified by gel electrophoresis, and purify it through multiple concentrations and water replenishment; S2, liquid chromatography and mass spectrometry conditions: the liquid chromatography mobile phase was 10 mM TRIS-HCL and 1 mM EDTA, the pH setting range was 7-7.5, ammonia was used to adjust the pH, and the final optimized condition was 7.3; S3, inductively coupled plasma mass spectrometry parameters: RF transmission power 1550W, RF voltage 1.80V; S4, drawing of standard curve of inorganic phosphorus element: prepare standard solutions of inorganic phosphorus element component analysis with mass concentrations of 0.1ppm, 0.5ppm, 1ppm, 5ppm and 10ppm, respectively, with an injection volume of 1ul, the chromatographic conditions are the same as above, and the injection is repeated three times or more; S5, DNA fragment content determination: The purified DNA fragments do not need to be digested. The DNA fragment samples are directly subjected to size exclusion chromatography and then enter the mass spectrometer for atomization, and then identified by inductively coupled plasma mass spectrometry.

2. The method for detecting DNA content in plasma mass spectrometry fragments without digestion according to claim 1, characterized in that: In the S1, a DNA sample with a concentration of about 150 ng / ul and a volume of 500 ul is prepared, and the concentration volume is set to concentrate from 500 ul to about 50 ul, the centrifugal speed is set to 13000 rpm, and the centrifugal time is set to 10 min. After each concentration to 50 ul, ultrapure water is used to make up to 500 ul, and it is concentrated again. The concentration times are more than 3 times to achieve purification.

3. The method for detecting DNA content in plasma mass spectrometry fragments without digestion according to claim 1, characterized in that: In S2, the mobile phase conditions are isocratic elution with a salt concentration, a flow rate of 1 ml / min, and an injection volume of 4 ul.

4. The method for detecting DNA content in plasma mass spectrometry fragments without digestion according to claim 1, characterized in that: In the S3, the sampling depth is 8.0 mm, the nebulizing gas is 0.8 L / min, the makeup gas is 25%, the quadrupole vacuum is 0.0015 Pa, the nebulizing chamber temperature is 2.0°C, the carrier gas flow rate is 1.0 L / min, the feedback power is 18 W, the peristaltic pump is 0.3 rps, the sampling depth is 8 mm, the data acquisition mode is TRA, the acquisition time is 12 min, and the integration time is 1.00 s.

5. The method for detecting DNA content in plasma mass spectrometry fragments without digestion according to claim 1, characterized in that: In the S4, the mass concentration of phosphorus in the phosphorus standard solution is used as the abscissa and the peak area of ​​the mass spectrometry signal is used as the ordinate to draw a standard curve, fit a linear equation and calculate the linear regression coefficient of the standard curve.

6. The method for detecting DNA content in plasma mass spectrometry fragments without digestion according to claim 1, characterized in that: In the S5, the fragment length is 311 bp.

7. The method for detecting DNA content in plasma mass spectrometry fragments without digestion according to claim 1, characterized in that: In S5, the result formula identified is: c=c s *f / P% Where: c is the concentration of undigested fragmented DNA, c s The mass concentration of phosphorus in the sample solution is calculated based on the standard curve formula, and f is the dilution multiple of DNA.

8. The method for detecting DNA content in plasma mass spectrometry fragments without digestion according to claim 7, characterized in that: P% is the percentage of phosphorus content in the DNA fragment, P%=2*bp*30.974 / DNA fragment molecular weight, bp represents the number of bases contained in the fragment.

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