A reaction system, a kit and a method for detecting abrin by LC-MS / MS

Abstract

The application provides a reaction system, a kit and a method for detecting abrin by using LC-MS / MS, and belongs to the technical field of toxin detection. The application mixes and incubates a mononucleotide substrate, a buffer, a BSA solution and a sample to be detected, then adds ammonia to terminate the reaction to obtain a reaction solution; the concentration of adenine is calculated by using LC-MS / MS detection and substituting the adenine peak area into a standard curve; when the number of de-adenine of the sample to be detected is greater than the average value of the number of de-adenine of a negative control plus 3 times of the standard deviation, the sample to be detected can be determined as a positive sample. The application uses magnetic beads coated with antibodies to enrich AT for detection, and the sensitivity can reach 0.391 ng / mL. The detection method of the application has high specificity and sensitivity, can only act on mononucleotides containing adenine, and does not have cross-reaction with other toxin detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of toxin detection, and particularly relates to an abrin reaction system, a kit and an abrin detection method using LC-MS / MS. BACKGROUND

[0002] Abrin (AT) is a highly toxic protein extracted from plant seeds, with a molecular weight of about 63-67 kDa, composed of A and B chains, connected by a disulfide bond. The B chain mediates the entry of the A chain into the cell, and the A chain has N-glycosidase activity, which can specifically cleave the adenine at position 4324 of ribosomal RNA, resulting in protein inactivation. Its chemical properties and structure are extremely similar to those of ricin (RT). Both toxins belong to type II ribosome inactivating proteins and have N-glycosidase activity. Studies have shown that the toxicity of AT is higher than that of RT, and the lethal dose for an adult is 0.1 μg / kg-1.0 μg / kg. Due to its extremely strong toxicity and easy availability, it can be used as a biological threat agent. Therefore, it is of great significance to develop a rapid and reliable method for detecting AT for national biological safety prevention and control.

[0003] Currently, the detection methods based on the enzyme activity of AT mainly focus on N-glycosidase activity, and mainly use mass spectrometry technology. Early studies found that when RT and AT exert their N-glycosidase activity in vitro, they require a double-stranded stem with a length of at least three base pairs and a loop containing a GAGA sequence near the center to exhibit detectable activity. It was later found that in addition to stem-loop nucleic acid substrates, AT can also act on single nucleotide chains. However, the length of the substrate on which AT can exert N-glycosidase activity has not been explored. SUMMARY

[0004] Therefore, the purpose of the present application is to provide an abrin reaction system, a kit and an abrin detection method using LC-MS / MS, which uses single nucleotides as detection substrates to establish an LC-MS / MS detection method for adenine, detects the content of adenine released in the reaction, and achieves the purpose of detecting toxins. This method not only reduces the detection cost, but also has good specificity and high sensitivity.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0006] The present application provides an abrin reaction system, which comprises a buffer solution, a single nucleotide substrate, a BSA solution and ammonia.

[0007] The single nucleotide substrate is ADP, ATP, AMP, dADP, dATP or dAMP.

[0008] A kit for detecting abrin, comprising the reaction system.

[0009] The application further provides a method for detecting abrin based on the reaction system, comprising the following steps:

[0010] 1) detecting a series of gradient-diluted adenine standard samples by LC-MS / MS, and drawing a standard curve with the concentration of adenine as the abscissa and the peak area of adenine as the ordinate;

[0011] 2) mixing and incubating the mononucleotide substrate, the buffer, the BSA solution and the sample to be detected, adding ammonia to terminate the reaction to obtain a reaction solution, detecting the reaction solution by LC-MS / MS, and calculating the concentration of adenine by substituting the peak area of adenine into the standard curve in step 1);

[0012] The sample to be detected is subjected to enrichment treatment by using antibody-coated magnetic beads before use.

[0013] When the de-adenine amount of the sample to be detected is greater than the average value of the de-adenine amount of the negative control plus 3 times the standard deviation, the sample to be detected is determined as a positive sample.

[0014] Preferably, the liquid chromatography condition of LC-MS / MS is as follows: the chromatographic column is a Waters -C18 reversed-phase column with an inner diameter × length of 2.1 mm × 150 mm and a particle size of 3.5 μm; the mobile phase is 10 mM ammonium formate solution with a pH of 4, and the organic phase is 100% methanol; the flow rate is 0.2 mL / min; the injection amount is 2 μL; the desolvation temperature is 250℃; and the source temperature is 150℃.

[0015] Preferably, the MRM mode parameter of LC-MS / MS is as follows: adenine m / z 136→m / z 92, m / z 136→m / z 119, and the orifice voltage is 20 V and the collision energy is 25, 20 eV.

[0016] Preferably, the incubation temperature in step 2) is 30-60℃, and the incubation time is 1-3 h.

[0017] Preferably, the buffer in step 2) is ammonium citrate + Mg 2+ + EDTA buffer, the pH of the ammonium citrate + Mg 2+ + EDTA buffer is 4.0-4.1, the final concentration of ammonium citrate in the ammonium citrate + Mg 2+ + EDTA buffer is 0.1-10 mM, and the concentration of Mg 2+ + EDTA in the ammonium citrate + Mg 2+The final concentration of ammonium citrate + Mg 2+ + EDTA buffer is 0.1-5 mM, and the final concentration of EDTA in the buffer is 0.1-10 mM.

[0018] Preferably, the final concentration of the BSA solution in step 2) is 8-12 μg / mL; the final concentration of the mononucleotide substrate is 3-7 μM; and the final concentration of the ammonia water is 15-25 mM.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The reaction principle of the present application is that AT exerts N-glycosidase activity to make the mononucleotide substrate shed adenine, and the content of the shed adenine is detected by establishing an adenine detection method of LC-MS / MS to achieve the purpose of detecting toxins. The positive determination criterion is that the number of adenine shed by the positive sample is greater than the average value of the number of adenine shed by the negative sample plus 3 times the standard deviation.

[0021] The present application uses mononucleotides ADP, ATP, AMP, dADP, dATP and dAMP as substrates, respectively, and uses ammonium citrate + Mg 2+ + EDTA buffer, bovine serum albumin and ammonia water to form a reaction system, and the optimal mononucleotide and the most suitable reaction temperature are screened to optimize the sensitivity of detection.

[0022] When the sample is directly added to the reaction system, the detection sensitivity of the method can reach 39 ng / mL; after enrichment of AT using coated antibody magnetic beads, the detection sensitivity can reach 0.391 ng / mL. The detection method of the present application has strong specificity and can only act on mononucleotides containing adenine, and does not have cross-reaction with other toxin detection. The sensitivity is high and is comparable to the sensitivity of the conventional AT acting on oligonucleotides containing adenine. In addition, the mononucleotide has a simple structure, is already commercialized and has low cost. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the chemical structural formula of the mononucleotide substrates ADP, ATP, AMP, dADP, dATP and dAMP;

[0024] Figure 2 is the mass spectrum graph of the adenine standard and its standard curve graph (wherein A is the mass spectrum graph of the adenine standard, and B is the standard curve graph);

[0025] Figure 3 is the reaction result graph of different types of substrates (note: H, 100 μg / mL; M, 50 μg / mL; L, 10 μg / mL);

[0026] Figure 4are reaction results at different reaction temperatures; (Note: H, 100 μg / mL; M, 50 μg / mL; L, 10 μg / mL);

[0027] Figure 5 is the detection sensitivity of AT without magnetic beads;

[0028] Figure 6 is the detection sensitivity of AT with magnetic beads;

[0029] Figure 7 is the detection sensitivity of milk samples;

[0030] Figure 8 is a single nucleotide specificity verification result chart (Note: from left to right, guanine, thymine, and uracil);

[0031] Figure 9 is a specificity verification result chart of different toxins. DETAILED DESCRIPTION

[0032] The application provides a reaction system of abrin, which comprises a buffer, a single nucleotide substrate, a BSA solution, and ammonia;

[0033] The single nucleotide substrate is ADP, ATP, AMP, dADP, dATP, or dAMP.

[0034] A kit for detecting abrin, comprising the reaction system.

[0035] The application further provides a method for detecting abrin based on the reaction system by using LC-MS / MS, comprising the following steps:

[0036] 1) detecting a series of gradient-diluted adenine standard samples by using LC-MS / MS, taking the concentration of adenine as the abscissa and the peak area of adenine as the ordinate, and drawing a standard curve;

[0037] 2) mixing the single nucleotide substrate, the buffer, the BSA solution, and a sample to be detected, incubating, adding ammonia to terminate the reaction to obtain a reaction solution, detecting the reaction solution by using LC-MS / MS, and calculating the concentration of adenine by substituting the peak area of adenine into the standard curve in step 1);

[0038] When the number of de-adenines in the sample to be detected is greater than the average value of the number of de-adenines in the negative control plus 3 times the standard deviation, the sample is determined to be positive.

[0039] In the application, the liquid chromatography condition of the LC-MS / MS is preferably as follows: the chromatographic column is a Waters -C18 reversed-phase column, inner diameter x length of 2.1 mm x 150 mm, particle size of 3.5 μm; mobile phase of 10 mM ammonium formate solution, pH 4, organic phase of 100% methanol; flow rate of 0.2 mL / min; injection volume of 2 μL; desolvation temperature of 250 °C; source temperature of 150 °C.

[0040] In the present application, the MRM mode parameters of the LC-MS / MS are as follows: adenine m / z 136→m / z 92, m / z 136→m / z 119, cone voltage of 20 V, and collision energy of 25, 20 eV.

[0041] In the present application, the sample to be tested is preferably subjected to enrichment treatment by using antibody-coated magnetic beads, the antibody is preferably AT rabbit polyclonal antibody, and the magnetic beads are preferably Dynabeads TM M-270 Streptavidin; the mass ratio of the antibody to the magnetic beads is preferably 1:95-105, further preferably 1:98-102, and more preferably 1:100. The amount of the magnetic beads used for each sample to be tested is preferably 25-35 μg, further preferably 27-32 μg, and more preferably 30 μg.

[0042] In the present application, the buffer is preferably ammonium citrate + Mg 2+ + EDTA buffer, the pH of the ammonium citrate + Mg 2+ + EDTA buffer is preferably 4.0-4.1, further preferably 4.02-4.08, and more preferably 4.05; the final concentration of ammonium citrate in the ammonium citrate + Mg 2+ + EDTA buffer is preferably 0.1-10 mM, further preferably 0.5-5 mM, and more preferably 1 mM; the final concentration of Mg 2+ in the ammonium citrate + Mg 2+ + EDTA buffer is preferably 0.1-5 mM, further preferably 0.2-1 mM, and more preferably 0.5 mM; and the final concentration of EDTA in the ammonium citrate + Mg 2+ + EDTA buffer is preferably 0.1-10 mM, further preferably 0.5-5 mM, and more preferably 1 mM.

[0043] In the present application, the final concentration of the BSA solution is preferably 8-12 μg / mL, further preferably 9-11 μg / mL, and more preferably 10 μg / mL; the final concentration of the mononucleotide substrate is preferably 3-7 μM, further preferably 4-6 μM, and more preferably 5 μM; and the final concentration of the ammonia is preferably 15-25 mM, further preferably 18-22 μM, and more preferably 20 μM.

[0044] In the present application, the temperature of the incubation is preferably 30-60℃, further preferably 32-58℃, and more further preferably 55℃; the time of the incubation is preferably 1-3h, further preferably 1.5-2.5h, and more further preferably 2h.

[0045] The technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0046] Main reagents and instruments used in the present application

[0047] (1) Main reagents

[0048] Dynabeads TM M-270 Streptavidin (65305): Thermo Fisher Scientific

[0049] Adenosine 5'-triphosphate (ATP, HY-B2176), Adenosine 5'-diphosphate (ADP, HY-W010918), Adenosine monophosphate (AMP, HY-A0181): purchased from MedChemExpress

[0050] 2'-Deoxyadenosine 5'-triphosphate (dATP, S18091), 2'-Deoxyadenosine 5'-diphosphate (dADP, S33011), 2'-Deoxyadenosine 5'-monophosphate (AMP, S18087): purchased from Shanghai Yuye Biotechnology Co., Ltd.

[0051] Bovine serum albumin (B2064), Ammonium citrate (25102): Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0052] Acetonitrile (HPLC grade) and methanol (HPLC grade) (>98%) were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and ultrapure water was obtained from China Hangzhou Wahaha Pure Water Co., Ltd.

[0053] Abrin and Abrin rabbit polyclonal antibody were prepared by the unit

[0054] Adenine (1012101) standard was purchased from Merck

[0055] Ammonia (1336-21-6): Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0056] (2) Main instruments

[0057] Biological safety cabinet: NUAIRE, USA

[0058] Vortex mixer: Klin-Bell Instrument Co., Ltd., Jiangsu, China

[0059] PCR workstation (Air Clean 600 PCR Workstation): AirClean Systems, USA

[0060] HulaMixerTM sample mixer (15920D), magnetic stand (12321D): Thermo Fisher Scientific

[0061] Triple quadrupole: Waters; Model: ACQUITY I-Class

[0062] Chromatographic column: ACQUITY UPLC@BEH Peptide BEH C18, Column, 1.7 μm, 2.1 mm x 150 mm (Waters)

[0063] Example 1

[0064] 1. The chemical structural formulas of different types of single nucleotide chain substrates ADP, ATP, AMP, dADP, dATP, and dAMP related to the experiment are as shown. Figure 1

[0065] 2. LC and MS conditions

[0066] (1) LC conditions

[0067] The liquid chromatography conditions were as follows: Waters -C18 reversed-phase column (2.1 mm x 150 mm, 3.5 μm); mobile phase was 10 mM ammonium formate solution, pH 4, and organic phase was 100% methanol; flow rate was 0.2 mL / min; injection volume was 2 μL.

[0068] Desolvation Temp.: 250 °C; Source Temp.: 150 °C.

[0069] (2) MS conditions​

[0070] Table 1 MRM detection parameter optimization results

[0071]

[0072]

[0073] Example 2

[0074] Establishment of adenine standard curve

[0075] The adenine standard was diluted with sterile water to 1.6, 8, 10, 20, 30, 80, 100 fmoL / μL, respectively, and detected under the LC-MS conditions of Example 1. The standard curve was drawn with the concentration of adenine as the abscissa and the peak area of adenine as the ordinate, and the linear equation was Y=24137*X-12400, R 2 =0.999. The experimental results are shown in Figure 2 .

[0076] Example 3

[0077] Optimization of reaction substrate

[0078] Different single nucleotides of Example 1 were used as substrates for the test, and AT was diluted to 100 μg / mL, 50 μg / mL, and 10 μg / mL, respectively, with sterile water as the negative control, and the reaction was carried out at 37°C and 55°C. The reaction system size was 50 μL, and the system included 10 μL of ammonium citrate (final concentration 1 mM) + Mg 2+ (terminal concentration 0.5 mM) + EDTA (terminal concentration 1 mM) buffer (pH=4.05), 10 μL of BSA solution with a final concentration of 10 μg / mL, 10 μL of single nucleotide substrate with a final concentration of 5 μM, 10 μL of AT with different concentrations, and 10 μL of 20 mM ammonia solution with a final concentration of 20 mM was added to terminate the reaction after the reaction. After the reaction system was configured and 10 μL of AT / sterile water was added, it was incubated at 55°C for 2 h. The ratio of the deprotonation content of the substrate treated with different concentrations of AT to the deprotonation content of the negative control was compared. The substrate with the highest ratio was the best reaction substrate.

[0079] The experimental results are shown in Figure 3 . Whether at 37°C or at 55°C, the deprotonation ratio of dADP was the highest, and the effect was better. Subsequent experiments were carried out with dADP as the substrate.

[0080] Example 4

[0081] Optimization of reaction temperature

[0082] The single nucleotide dADP was used as the substrate for the test, and the AT was diluted to 100 μg / mL, 50 μg / mL, and 10 μg / mL, respectively, with sterile water as the negative control, and the reaction was carried out at 37°C and 55°C, respectively. The reaction system size was 50 μL, and the system included 10 μL of ammonium citrate (final concentration 1 mM) + Mg 2+ (terminal concentration 0.5 mM) + EDTA (terminal concentration 1 mM) buffer (pH = 4.05), 10 μL of BSA solution with a final concentration of 10 μg / mL, 10 μL of single nucleotide substrate with a final concentration of 5 μM, 10 μL of AT with different concentrations, and 10 μL of ammonia with a final concentration of 20 mM was added after the reaction to terminate the reaction. After the reaction system was configured and 10 μL of AT / sterile water was added, it was incubated at 37°C / 55°C for 2 h. The ratio of the deprotonation content of the same substrate after the action of AT at different reaction temperatures to the deprotonation of the negative control was compared. The highest ratio was the best reaction temperature.

[0083] The experimental results are shown in Table 1. Figure 4 At 55°C, the deprotonation ratio of single nucleotide dADP was the highest in low, medium, and high concentrations of toxins, and the effect was better, so the best reaction temperature of dADP was 55°C.

[0084] Example 5

[0085] Optimization of detection sensitivity of samples directly added to the reaction system

[0086] The detection sensitivity of the detection method was directly explored using the optimized reaction system. AT was diluted with sterile water to 5000 ng / mL, 2500 ng / mL, 1250 ng / mL, 625 ng / mL, 312.5 ng / mL, 156.25 ng / mL, 78.125 ng / mL, 39 ng / mL, 19 ng / mL, 9.5 ng / mL, and 4.75 ng / mL, and reacted at 55°C for 2 h. The reaction system included 10 μL of ammonium citrate (final concentration 1 mM) + Mg 2+ (terminal concentration 0.5 mM) + EDTA (terminal concentration 1 mM) buffer (pH = 4.05), BSA solution with a final concentration of 10 μg / mL, dADP substrate with a final concentration of 5 μM, 10 μL of AT with different concentrations, and 10 μL of ammonia with a final concentration of 20 mM was added after the reaction to terminate the reaction, and the reaction system volume was 50 μL.

[0087] The experimental results are shown in Table 1. Figure 5 The detection sensitivity of AT was 39 ng / mL.

[0088] Example 6

[0089] Detection sensitivity of reaction system after sample enrichment by antibody-coated magnetic beads

[0090] Sample enrichment by antibody-coated magnetic beads, antibody: AT rabbit polyclonal antibody, magnetic beads: Dynabeads TM M-270 Streptavidin, 10 μg biotinylated antibody coated on 1 mg magnetic beads, 30 μg magnetic beads used for each sample. The volume of sample was 500 μL. After incubation of AT with corresponding magnetic beads for 1 h at room temperature, the supernatant was removed after 2-3 min of adsorption by magnetic stand. Then, 500 μL of sterile water was used to wash the magnetic beads for three times, and then the reaction system was added. The reaction system included 10 μL of ammonium citrate (final concentration of 1 mM) + Mg 2+ (0.5 mM) + EDTA (1 mM) buffer (pH = 4.05), BSA solution (10 μg / mL), dADP substrate (5 μM), 10 μL of AT with different concentrations, 10 μL of 20 mM ammonia solution was added to terminate the reaction after the reaction, and the volume of reaction system was 50 μL. After the antibody-coupled magnetic beads were evenly blown and sucked, they were incubated at 55 °C for 2 h. The concentrations of AT were set as 400 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, 1.563 ng / mL, 0.781 ng / mL, 0.391 ng / mL, 0.098 ng / mL, and sterile water as negative control, to explore the detection sensitivity of the detection method.

[0091] The experimental results are shown in Table 1. Figure 6 The detection sensitivity of AT after magnetic bead enrichment was 0.391 ng / mL.

[0092] Example 7

[0093] Detection sensitivity of complex sample

[0094] Sample enrichment by antibody-coated magnetic beads, antibody: AT rabbit polyclonal antibody, magnetic beads: Dynabeads TM M-270 Streptavidin, 10 μg biotinylated antibody coated on 1 mg magnetic beads, 30 μg magnetic beads used for each sample. The volume of sample was 500 μL. After incubation of AT with corresponding magnetic beads for 1 h at room temperature, the supernatant was removed after 2-3 min of adsorption by magnetic stand. Then, 500 μL of sterile water was used to wash the magnetic beads for three times, and then the reaction system was added. The reaction system included 10 μL of ammonium citrate (final concentration of 1 mM) + Mg 2+(0.5 mM final concentration) + EDTA (1 mM final concentration) buffer (pH = 4.05), a BSA solution with a final concentration of 10 μg / mL, a dADP substrate with a final concentration of 5 μM, 10 μL of AT with different concentrations, and 10 μL of ammonia with a final concentration of 20 mM were added to terminate the reaction after the reaction, and the reaction system had a volume of 50 μL. The antibody-coupled magnetic beads and the toxin were blown and sucked gently to be uniform, and then incubated at 55 °C for 2 h. The milk was used to prepare a simulated complex sample of AT, the AT was diluted with milk, and the concentrations were set to 400 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, 1.563 ng / mL, 0.781 ng / mL, 0.391 ng / mL, 0.195 ng / mL, 0.098 ng / mL, and sterile water was used as a negative control to explore the detection sensitivity of the detection method.

[0095] The experimental results are shown in Table 1. Figure 7 The detection sensitivity of the milk sample was 3.125 ng / mL.

[0096] Example 8

[0097] Specificity verification

[0098] 1. The standard samples of guanine, thymine and uracil were diluted to 10 μmol / L, and the detection of guanine, cytosine and thymine was detected according to the LC-MS / MS detection conditions in Example 1 to verify the specificity of AT. The AT was diluted to 10 μg / mL, and the mononucleotide substrate was diluted to 50 μmol / L, and then added to the reaction system for detection. The reaction system was the same as that in Example 7.

[0099] The experimental results are shown in Table 1. Figure 8 The AT cannot act on mononucleotides guanine, thymine and uracil, but only acts on adenine.

[0100] 2. AT, RT, DT, SEA and ETX toxins were used to verify the specificity of the detection method at the same time. All the toxins were diluted to 100 ng / mL, and the experimental steps of this experiment were consistent with those of the AT milk simulation sample in Example 7, except that different toxins were used.

[0101] The experimental results are shown in Table 1. Figure 9 RT, DT, SEA and ETX toxins have no cross-reaction with AT in the reaction process, which indicates that the detection method of the application has good specificity.

[0102] From the above examples, the present application takes single nucleotide as a detection substrate, establishes the detection method of adenine by LC-MS / MS, detects the content of the adenine dropped in the reaction, and achieves the purpose of detecting toxins, which not only reduces the detection cost, but also has good specificity and high sensitivity.

[0103] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for detecting abrin using LC-MS / MS, characterized in that, The method comprises the following steps: 1) detecting a series of gradient-diluted adenine standards by LC-MS / MS, and drawing a standard curve with the concentration of adenine as the abscissa and the peak area of adenine as the ordinate; 2) mixing and incubating a single nucleotide substrate, a buffer, a BSA solution and a sample to be tested, adding ammonia to terminate the reaction to obtain a reaction solution; detecting the reaction solution by LC-MS / MS, and calculating the concentration of adenine by substituting the peak area of adenine into the standard curve in step 1); The single nucleotide substrate is dADP.

2. The method of claim 1, wherein, The sample to be tested is subjected to enrichment treatment by using antibody-coated magnetic beads before use.

3. The method of claim 1, wherein, When the number of de-adenines in the sample to be tested is greater than the average value of the number of de-adenines in the negative control plus 3 times the standard deviation, the sample is determined to be positive.

4. The method of claim 1, wherein, The liquid chromatography condition of the LC-MS / MS is as follows: the chromatographic column is a Waters-C18 reversed-phase column with an inner diameter × length of 2.1 mm × 150 mm and a particle size of 3.5 μm; the mobile phase is 10 mM ammonium formate solution with pH 4, and the organic phase is 100% methanol; the flow rate is 0.2 mL / min; the injection amount is 2 μL; the desolvation temperature is 250℃; and the source temperature is 150℃.

5. The method of claim 1, wherein, The MRM mode parameters of the LC-MS / MS are as follows: adenine m / z 136→m / z 92, m / z 136→m / z 119, cone voltage 20 V, and collision energy 25, 20 eV.

6. The method of claim 1, wherein, The incubation temperature in step 2) is 30-60℃, and the incubation time is 1-3 h.

7. The method of claim 1, wherein, Step 2) the buffer is ammonium citrate + Mg 2+ + EDTA buffer, the ammonium citrate + Mg 2+ + EDTA buffer has a pH of 4.0 to 4.1, the ammonium citrate + Mg 2+ + EDTA buffer has a final concentration of ammonium citrate of 0.1 to 10 mM, the ammonium citrate + Mg 2+ + EDTA buffer has a final concentration of Mg 2+ of 0.1 to 5 mM, the ammonium citrate + Mg 2+ + EDTA buffer has a final concentration of EDTA of 0.1 to 10 mM.

8. The method of claim 1, wherein, The final concentration of the BSA solution in step 2) is 8-12 μg / mL; the final concentration of the single nucleotide substrate is 3-7 μM; and the final concentration of the ammonia is 15-25 mM.

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