Method for detecting nucleic acid by nuclear magnetic resonance phosphorus spectrum

Abstract

The invention relates to the technical field of analysis and detection, and particularly provides a method for detecting nucleic acid through nuclear magnetic resonance phosphorus spectrums.The method comprises the following steps that 1, a sample to be detected is dissolved in a solvent without phosphorus element, detection liquid is obtained, certain detection conditions are set, nuclear magnetic resonance phosphorus spectrum detection is conducted on the detection liquid, and a nuclear magnetic phosphorus spectrogram is obtained; determining the peak position of the phosphorus-containing substance in the sample; and (2) if the sample in the step (1) has a peak at-3 to-5 ppm, proving that nucleic acid exists in the sample. The method has good performance in the aspects of accuracy, repeatability, stability, nondestructive testing and the like, and can provide effective technical support for large-scale detection of nucleic acid.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for detecting nucleic acid using nuclear magnetic resonance phosphorus spectrum. Background Art

[0002] Nucleic acid is a biological macromolecule formed by the polymerization of many nucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the carrier of genetic information, which contains the genetic instructions required for the growth, development, reproduction and other life activities of organisms. The DNA molecule has a double helix structure, which maintains its stability and the accuracy of genetic information through the principle of base pairing. RNA plays an important role in the expression of genetic information. For example, messenger RNA (mRNA) is responsible for transferring the genetic information in DNA to the ribosome, and siRNA can specifically bind to the target gene mRNA to achieve gene silencing, which can be used to inhibit the expression of disease-related genes and study gene functions and signal pathways. Nucleic acids play an extremely critical role in the life activities of organisms, and are of vital importance to inheritance, variation, protein synthesis, gene expression regulation and other aspects.

[0003] Nucleic acids and phosphates are widely distributed in organisms. However, the phosphate group in nucleic acids is very similar to the phosphate radical in phosphates. Therefore, when identifying nucleic acids, phosphates will greatly interfere with the test results, making it difficult to identify. Although existing methods such as complexometric titration and mass spectrometry can achieve the purpose of detection, they will also damage the samples.

[0004] Traditional methods for detecting nucleic acids mainly include agarose gel electrophoresis, restriction endonuclease digestion analysis, polymerase chain reaction, etc. These methods have the problems of complex operation, high cost, and complex data analysis. They will also damage the test samples and make them unusable. Therefore, a more effective method for detecting nucleic acids is needed. Summary of the invention

[0005] Phosphate groups are important components of nucleic acids. Nucleic acids are biological macromolecules formed by the polymerization of many nucleotides. The basic unit of nucleic acids contains phosphate groups. Phosphate groups form phosphodiester bonds between nucleotides, connecting nucleotides one by one to form the backbone structure of nucleic acids. Therefore, the phosphorus atoms in nucleic acids have a more special chemical environment, and nuclear magnetic resonance phosphorus spectroscopy (NMRP) 31 P NMR is very sensitive to the chemical environment of phosphorus atoms. The inventors of this application have found that 31P NMR analyzes nucleic acid samples and detects the phosphate groups in nucleic acids. It does not require complicated sample processing and will not damage the samples during the detection process. With the continuous development of technology, it may be possible to achieve in situ real-time monitoring of nucleic acids in organisms or chemical reaction systems.

[0006] Based on the above situation, the purpose of the present invention is to provide a method for detecting nucleic acids by nuclear magnetic resonance phosphorus spectroscopy, which can overcome the problems of complex operation and sample damage in nucleic acid detection in the prior art, and can more simply and non-destructively detect the nucleic acids present in the sample.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0008] A method for detecting nucleic acid by nuclear magnetic resonance phosphorus spectroscopy, the method comprising the following steps:

[0009] (1) dissolving a sample to be tested in a solvent that does not contain phosphorus to obtain a test solution, performing nuclear magnetic resonance phosphorus spectrum detection on the test solution to obtain a nuclear magnetic resonance phosphorus spectrum, and determining the peak position of the phosphorus-containing substance in the sample;

[0010] (2) If the peak position of the sample in step (1) is around -3 to -5 ppm, it proves that nucleic acid exists in the sample;

[0011] When performing nuclear magnetic resonance phosphorus spectrum detection in step (1), a zgig pulse sequence is used, the test temperature is 270-280K; the pulse width is 9-10μs; D1 is 3-5s; the spectrum width is 80000-82000Hz, preferably 81521.7Hz; the radio frequency center frequency is 161-161.5MHz; LB=3Hz, the number of acquisition points is 60000-66000, preferably 65536; the number of sampling accumulation times is 8000-9000 times, preferably 8192 times;

[0012] Furthermore, the sample to be detected is any one or more of samples containing phosphorus, such as nucleic acid, phosphate, blood, etc.

[0013] Furthermore, in step (1), the nucleic acid is DNA, mRNA, siRNA or miRNA.

[0014] Furthermore, the solvent is any one of water, deuterated water, deuterated methanol, deuterated acetonitrile, deuterated dimethyl sulfoxide, citrate buffer, anhydrous ethanol and other solvents that do not contain phosphorus, preferably citrate buffer.

[0015] Furthermore, the citrate buffer is a citrate buffer with a pH of 3 and a 0.05M value.

[0016] Furthermore, when the nuclear magnetic resonance phosphorus spectrum is detected, the frequency of the nuclear magnetic resonance instrument used is 400-600 MHz, preferably 400 MHz.

[0017] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0018] The present invention uses nuclear magnetic resonance phosphorus spectroscopy to detect the presence of nucleic acids in samples, and can 31 The different peak positions of P can be used to identify nucleic acids and their common interferences, phosphates. By optimizing the test parameters, the detection method is simple to operate, has good repeatability, high accuracy, non-destructive detection, and can be used routinely. In addition, compared with some other detection methods that require nucleic acid labeling, nuclear magnetic resonance phosphorus spectroscopy does not require the introduction of additional markers, avoiding the interference that may be caused by the labeling process and the impact on the properties of the sample, and can provide technical support for detecting the presence of nucleic acids in samples of different concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the nuclear magnetic resonance phosphorus spectrum of the disodium hydrogen phosphate solution in Example 1;

[0020] Figure 2 This is the nuclear magnetic resonance phosphorus spectrum of the pEGFP plasmid solution in Example 1. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is described in detail below in conjunction with specific embodiments and drawings.

[0022] The main reagents and materials used in the following examples are described as follows:

[0023] The nuclear magnetic resonance phosphorus spectrum was detected using a 400 MHz nuclear magnetic resonance instrument (Bruker, Switzerland) and a 10 mm nuclear magnetic tube (purchased from Wilmad, USA).

[0024] 0.5 M citrate buffer was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., pH=3.

[0025] The high-purity plasmid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. DP116.

[0026] RNase-free ddH2O was purchased from Shenggong Biotechnology Co., Ltd. Disodium hydrogen phosphate was purchased from Sinopharm Group.

[0027] Preparation of 50 mM citrate buffer: Take 300 μL of pH=3 0.5 M citrate buffer and add it to 2.7 mL RNase-free ddH2O, mix well, and dilute to obtain 50 mM citrate buffer.

[0028] Example 1 A method for detecting nuclei by nuclear magnetic resonance phosphorus spectroscopy, the specific steps are as follows:

[0029] (1) Prepare the sodium hydrogen phosphate solution for the control group: weigh 286.8 mg Na2HPO4 and dissolve it in 10 mL deionized water to obtain a 0.2 M sodium hydrogen phosphate solution for the control group. Take 2 mL as the control group.

[0030] (2) Preparation of pEGFP plasmid solution for the experimental group: Use a high-purity plasmid extraction kit to extract the EGFP-expressing plasmid pEGFP from Escherichia coli, and then dilute it to 1 mg / mL with 50 mM citrate buffer for subsequent experiments. Take 1.5 mL of the above 1 mg / mL pEGFP plasmid solution, add 50 mM citrate buffer to make it up to 2 mL, mix well to obtain the pEGFP plasmid detection solution, which is used as the experimental group.

[0031] (3) The prepared disodium hydrogen phosphate solution of the control group and the pEGFP plasmid solution of the experimental group were subjected to nuclear magnetic resonance phosphorus spectrum detection to obtain the nuclear magnetic resonance spectra of disodium hydrogen phosphate and the pEGFP plasmid.

[0032] The sampling parameters of the nuclear magnetic resonance phosphorus spectrum of the control group are as follows: the zg30 pulse sequence is used, the test temperature is 278K, the pulse width is 9.9μs, D1 is 1s, the spectrum width is 49019.6Hz, the RF center frequency is 161.9931762MHz, LB=3Hz, the number of acquisition points is 32768, the number of sampling accumulation times is 64 times, the number of empty scans is 0 times, and the gain is 1. The phosphorus spectrum obtained is as follows Figure 1 shown.

[0033] The sampling parameters of the experimental group's nuclear magnetic resonance phosphorus spectrum are as follows: the zgig pulse sequence is used, the test temperature is 278K, the pulse width is 9.9μs, D1 is 4s, the spectrum width is 81521.7Hz, the RF center frequency is 161.9931762MHz, LB=3Hz, the number of acquisition points is 65536, the number of sampling accumulation times is 8192 times, the number of empty scans is 0 times, and the gain is 2050. The phosphorus spectrum obtained is as follows Figure 2 shown.

[0034] Analysis of results: After nuclear magnetic resonance phosphorus spectrum detection, the peak position of the target peak of the phosphorus spectrum of the disodium hydrogen phosphate solution in the control group was -0.27ppm, and the peak position distribution range of the pEGFP plasmid solution in the experimental group was -3 to -5ppm. There was a certain difference in the peak position, which indicated that the chemical environment of the phosphorus atom of the phosphate group in nucleic acid was different from that of the phosphorus atom in phosphate. Nuclear magnetic resonance phosphorus spectrum can be used to identify nucleic acids and phosphates, and detect the presence of nucleic acids in samples.

Claims

1. A method for detecting nucleic acid using nuclear magnetic resonance phosphorus spectroscopy, the method comprising the following steps: (1) dissolving the sample to be tested in a solvent that does not contain phosphorus to obtain a test solution, performing nuclear magnetic resonance phosphorus spectrum detection on the test solution to obtain a nuclear magnetic resonance phosphorus spectrum, and determining the peak position of the phosphorus-containing substance in the sample; (2) If the sample in step (1) has a peak at -3 to -5 ppm, it indicates the presence of nucleic acid in the sample; When performing nuclear magnetic resonance phosphorus spectrum detection in step (1), a zgig pulse sequence is used, the test temperature is 270-280 K; the pulse width is 9-10 μs; D1 is 3-5 s; the spectrum width is 80000-82000 Hz; the RF center frequency is 161-161.5 MHz; LB = 3 Hz, the number of acquisition points is 60000-66000; and the number of sampling accumulation times is 8000-9000 times.

2. The method according to claim 1, characterized in that The sample to be detected is nucleic acid, phosphate and / or blood.

3. The method according to claim 1, characterized in that The nucleic acid is DNA, mRNA, siRNA or miRNA.

4. The method according to claim 1, characterized in that The solvent is any one of water, deuterated water, deuterated methanol, deuterated acetonitrile, deuterated dimethyl sulfoxide, citrate buffer, and anhydrous ethanol.

5. The method according to claim 1, characterized in that When the nuclear magnetic resonance phosphorus spectrum is detected, the frequency of the nuclear magnetic resonance instrument used is 400-600 MHz.

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