Reaction system of abrus precatorius toxin, kit and method for detecting abrus precatorius toxin by utilizing LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry)

Abstract

The invention provides a reaction system and a kit of abrus precatorius toxin and a method for detecting the abrus precatorius toxin by utilizing LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry), and belongs to the technical field of toxin detection. The method comprises the following steps: mixing and incubating a mononucleotide substrate, a buffer solution, a BSA solution and a to-be-detected sample, and adding ammonia water to terminate the reaction to obtain a reaction solution; and carrying out LC-MS / MS detection, and substituting the adenine peak area into the standard curve to calculate the concentration of adenine. When the deadenine quantity of the to-be-detected sample is greater than the sum of the average value of the deadenine quantity of the negative control and three times of the standard deviation, the to-be-detected sample can be judged as a positive sample. According to the invention, the antibody-coated magnetic beads are used for detection after AT enrichment, and the sensitivity can reach 0.391 ng / mL. The detection method provided by the invention is strong in specificity and high in sensitivity, can only act on mononucleotide containing adenine, and does not generate cross reaction with other toxin detection.

Description

Technical Field

[0001] The invention belongs to the technical field of toxin detection, and in particular relates to a reaction system and a kit for abrinus toxin and a method for detecting abrinus toxin by utilizing LC-MS / MS. Background Art

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

[0003] At present, the detection methods based on AT enzyme activity are mainly centered around N-glycosidase activity and are mainly based on mass spectrometry technology. Early studies found that when RT and AT exert their N-glycosidase activity in vitro, they need a double helix stem of at least three base pairs in length and a loop containing a GAGA sequence near the center to show detectable activity. Later, it was discovered that in addition to nucleic acid substrates with stem-loop structures, 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 of the invention

[0004] In view of this, the purpose of the present invention is to provide a reaction system, a kit and a method for detecting abrin toxin using LC-MS / MS. A LC-MS / MS adenine detection method is established using mononucleotide as a detection substrate, and the content of adenine shed in the reaction is detected to achieve the purpose of detecting toxins. This not only reduces the detection cost, but also has good specificity and high sensitivity.

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

[0006] The present invention provides a reaction system of abrin toxin, the reaction system comprising a buffer, a mononucleotide substrate, a BSA solution and ammonia water;

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

[0008] A kit for detecting abrin toxin comprises the reaction system.

[0009] The present invention also provides a method for detecting abrin toxin by using LC-MS / MS based on the reaction system, comprising the following steps:

[0010] 1) Detect the serially diluted adenine standard by LC-MS / MS, and draw a standard curve with the concentration of adenine as the abscissa and the corresponding peak area of ​​adenine as the ordinate;

[0011] 2) After the mononucleotide substrate, buffer, BSA solution and the sample to be tested are mixed and incubated, ammonia water is added to terminate the reaction to obtain a reaction solution; the reaction solution is detected by LC-MS / MS, and the adenine peak area is substituted into the standard curve described in step 1) to calculate the concentration of adenine;

[0012] The sample to be tested is enriched by using antibody-coated magnetic beads before use;

[0013] When the number of deadenines in the sample to be tested is greater than the average number of deadenines in the negative control plus 3 times the standard deviation, it can be determined as a positive sample.

[0014] Preferably, the liquid chromatography conditions of the LC-MS / MS are: the chromatographic column is Waters -C18 reverse phase column, inner diameter × length 2.1mm × 150mm, particle size 3.5μm; mobile phase is 10mM, pH4 ammonium formate solution, organic phase is 100% methanol; flow rate is 0.2mL / min; injection volume is 2μL; desolvation temperature is 250℃; source temperature is 150℃.

[0015] Preferably, the MRM mode parameters of the LC-MS / MS are: adenine m / z 136→m / z 92, m / z 136→m / z119, cone voltage is 20V, and collision energy is 25, 20eV.

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

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

[0018] Preferably, in step 2), the final concentration of the BSA solution 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 invention has the following beneficial effects:

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

[0021] The present invention uses mononucleotide ADP, ATP, AMP, dADP, dATP, and dAMP as substrates, and uses ammonium citrate + Mg 2+ +EDTA buffer, bovine serum albumin and ammonia water constitute the reaction system, and the best single nucleotide and the optimal reaction temperature are screened to optimize the sensitivity of the detection.

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

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

[0024] Figure 2 is a mass spectrum of an adenine standard and a standard curve thereof (wherein A is a mass spectrum of an adenine standard and B is a standard curve);

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

[0026] Figure 4It is the reaction result diagram at different reaction temperatures; (Note: H, 100μg / mL; M, 50μg / mL; L, 10μg / mL);

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

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

[0029] Figure 7 is the sensitivity of the milk sample test;

[0030] Figure 8 This is a graph of single nucleotide specificity verification results (Note: from left to right are guanine, thymine, and uracil);

[0031] Fig. 9 This is a graph showing the specificity validation results for different toxins. DETAILED DESCRIPTION

[0032] The present invention provides a reaction system of abrin toxin, the reaction system comprising a buffer, a mononucleotide substrate, a BSA solution and ammonia water;

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

[0034] A kit for detecting abrin toxin comprises the reaction system.

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

[0036] 1) Detect the serially diluted adenine standard by LC-MS / MS, and draw a standard curve with the concentration of adenine as the abscissa and the corresponding peak area of ​​adenine as the ordinate;

[0037] 2) After the mononucleotide substrate, buffer, BSA solution and the sample to be tested are mixed and incubated, ammonia water is added to terminate the reaction to obtain a reaction solution; the reaction solution is detected by LC-MS / MS, and the adenine peak area is substituted into the standard curve described in step 1) to calculate the concentration of adenine;

[0038] When the number of deadenines in the sample to be tested is greater than the average number of deadenines in the negative control plus 3 times the standard deviation, it can be determined as a positive sample.

[0039] In the present invention, the liquid chromatography conditions of the LC-MS / MS are preferably: the chromatographic column is Waters -C18 reverse phase column, inner diameter × length 2.1mm × 150mm, particle size 3.5μm; mobile phase is 10mM, pH4 ammonium formate solution, organic phase is 100% methanol; flow rate is 0.2mL / min; injection volume is 2μL; desolvation temperature is 250℃; source temperature is 150℃.

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

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

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

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

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

[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

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

[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 trisodium salt (2'-Deoxyadenosine 5'-triphosphate, dATP, S18091), 2'-Deoxyadenosine 5'-diphosphate sodium salt (2'-Deoxyadenosine 5'-diphosphate, dADP, S33011), 2'-Deoxyadenosine 5'-monophosphate (2'-Deoxyadenosine 5'-monophosphate, AMP, S18087): purchased from Shanghai Yuanye 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). Ultrapure water was obtained from Wahaha Pure Water Company, Hangzhou, China.

[0053] Abrin and Abrin rabbit polyclonal antibody were prepared by our 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: Jiangsu Haimen Qilin Bell Instrument Co., Ltd.

[0059] PCR workstation (Air Clean600 PCR Workstation): AirClean Systems, USA

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

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

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

[0063] Example 1

[0064] 1. Experimental related different types of single nucleotide chain substrates ADP, ATP, AMP, dADP, dATP, dAMP, their chemical structures are as follows Figure 1 shown.

[0065] 2. LC and MS conditions

[0066] (1)LC conditions

[0067] Liquid chromatography conditions are: chromatographic column is Waters -C18 reverse phase column (2.1 mm×150 mm, 3.5 μm); the mobile phase was 10 mM ammonium formate solution at pH 4, and the organic phase was 100% methanol; the flow rate was 0.2 mL / min; and the 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 standard curve of adenine standard

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

[0076] Example 3

[0077] Optimization of reaction substrates

[0078] The experiments were carried out using different mononucleotides in Example 1 as substrates. AT was diluted to 100 μg / mL, 50 μg / mL, and 10 μg / mL, respectively. Sterile water was used as a negative control, and the reactions were 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 was 1 mM) + Mg 2+ (final concentration of 0.5mM) + EDTA (final concentration of 1mM) buffer (pH = 4.05), 10μL of BSA solution with a final concentration of 10μg / mL, 10μL of a single nucleotide substrate with a final concentration of 5μM, 10μL of AT at different concentrations, and after the reaction, 10μL of ammonia water with a final concentration of 20mM was added to terminate the reaction. The reaction system was configured and 10μL of AT / sterile water was added and incubated at 55℃ for 2h. The depurination content of different concentrations of AT acting on the same substrate was compared with the ratio of depurination of the negative control. The substrate with the highest ratio was the best reaction substrate.

[0079] Experimental results: Figure 3 Whether at 37°C or 55°C, the depurination ratio of dADP is the highest and the effect is better. Subsequent experiments were conducted using dADP as a substrate.

[0080] Example 4

[0081] Reaction temperature optimization

[0082] The mononucleotide dADP was used as the substrate for the experiment. AT was diluted to 100 μg / mL, 50 μg / mL, and 10 μg / mL, respectively. Sterile water was used as the negative control. 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 was 1 mM) + Mg 2+ (final concentration of 0.5mM) + EDTA (final concentration of 1mM) buffer (pH = 4.05), 10μL of BSA solution with a final concentration of 10μg / mL, 10μL of a single nucleotide substrate with a final concentration of 5μM, 10μL of AT at different concentrations, and after the reaction, 10μL of ammonia water with a final concentration of 20mM was added to terminate the reaction. The reaction system was configured and 10μL of AT / sterile water was added and placed at 37℃ / 55℃ for 2h. The ratio of depurination content after AT at different reaction temperatures acted on the same substrate and the depurination of the negative control was compared. The temperature with the highest ratio was the optimal reaction temperature.

[0083] Experimental results: Figure 4 As shown. At 55°C, the depurination ratio of the mononucleotide dADP is the highest among the toxins at low, medium and high concentrations, and the effect is better, so the optimal reaction temperature of dADP is 55°C.

[0084] Example 5

[0085] Optimization of detection sensitivity by adding samples directly into the reaction system

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

[0087] Experimental results: Figure 5 As shown in Figure 2, the detection sensitivity of AT was 39 ng / mL.

[0088] Example 6

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

[0090] Samples were enriched using antibody-coated magnetic beads, using AT rabbit polyclonal antibody and Dynabeads magnetic beads. TM M-270 Streptavidin streptavidin, the coating amount of antibody and magnetic beads is 1mg magnetic beads coated with 10μg biotinylated antibody, and the amount of magnetic beads used for each sample is 30μg. The volume of the sample is 500μL. After AT and the corresponding magnetic beads are flipped and incubated for 1h at room temperature, the magnetic stand is adsorbed for 2-3min, and the supernatant is removed. Then the reaction system is added. The reaction system includes 10μL of ammonium citrate (final concentration is 1mM) + Mg 2+ (final concentration of 0.5mM) + EDTA (final concentration of 1mM) buffer (pH = 4.05), BSA solution with a final concentration of 10μg / mL, dADP substrate with a final concentration of 5μM, 10μL of different concentrations of AT, after the reaction, add 10μL of ammonia water with a final concentration of 20mM to terminate the reaction, and the reaction system volume is 50μL. The antibody-coupled magnetic beads and toxins were gently blown and evenly incubated at 55℃ for 2h. The concentrations of AT were set to 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 12.5ng / mL, 6.25ng / mL, 3.125ng / mL, 1.563ng / mL, 0.781ng / mL, 0.391ng / mL, 0.195ng / mL, and 0.098ng / mL, respectively. Sterile water was used as a negative control to explore the detection sensitivity of the detection method.

[0091] Experimental results: Figure 6 The detection sensitivity of AT after enrichment with magnetic beads was 0.391 ng / mL.

[0092] Example 7

[0093] Detection sensitivity in complex samples

[0094] Antibody-coated magnetic beads were used for sample enrichment. The antibody used was AT rabbit polyclonal antibody and the magnetic beads used were Dynabeads. TM M-270 Streptavidin streptavidin, the coating amount of antibody and magnetic beads is 1mg magnetic beads coated with 10μg biotinylated antibody, and the amount of magnetic beads used for each sample is 30μg. The volume of the sample is 500μL. After AT and the corresponding magnetic beads are flipped and incubated for 1h at room temperature, the magnetic stand is adsorbed for 2-3min, and the supernatant is removed. The magnetic beads are washed three times with 500μL of sterile water, and then the reaction system is added. The reaction system includes 10μL of ammonium citrate (final concentration is 1mM) + Mg 2+(final concentration 0.5mM) + EDTA (final concentration 1mM) buffer (pH = 4.05), BSA solution with a final concentration of 10μg / mL, dADP substrate with a final concentration of 5μM, 10μL of AT of different concentrations, after the reaction, add 10μL of ammonia water with a final concentration of 20mM to terminate the reaction, the reaction system volume is 50μL. Gently pipette the antibody-coupled magnetic beads and toxins evenly, then incubate at 55℃ for 2h. A simulated complex sample of AT was made with milk. AT was diluted with milk, and the concentrations were set to 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 12.5ng / mL, 6.25ng / mL, 3.125ng / mL, 1.563ng / mL, 0.781ng / mL, 0.391ng / mL, 0.195ng / mL, and 0.098ng / mL, respectively. Sterile water was used as a negative control to explore the detection sensitivity of the detection method.

[0095] Experimental results: Figure 7 The detection sensitivity of milk samples was 3.125 ng / mL.

[0096] Example 8

[0097] Specificity verification

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

[0099] Experimental results: Figure 8 AT cannot act on the single nucleotides guanine, thymine, and uracil, but only on adenine.

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

[0101] Experimental results: Fig. 9 As shown, RT, DT, SEA, and ETX toxins had no cross-reaction with AT during the reaction process, indicating that the detection method of the present invention has good specificity.

[0102] It can be seen from the above embodiments that the present invention uses a single nucleotide as a detection substrate to establish an LC-MS / MS adenine detection method to detect the adenine content released by the reaction to achieve the purpose of detecting toxins, which not only reduces the detection cost but also has good specificity and high sensitivity.

[0103] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A reaction system of abrinus toxin, characterized in that: The reaction system comprises a buffer, a mononucleotide substrate, a BSA solution and ammonia water; The mononucleotide substrate is ADP, ATP, AMP, dADP, dATP or dAMP.

2. A kit for detecting abrin toxin, characterized in that: The method comprises the reaction system according to claim 1.

3. A method for detecting abrin toxin using LC-MS / MS based on the reaction system of claim 1, characterized in that: The following steps are involved: 1) Detect the serially diluted adenine standard by LC-MS / MS, and draw a standard curve with the concentration of adenine as the abscissa and the corresponding peak area of ​​adenine as the ordinate; 2) After the mononucleotide substrate, buffer, BSA solution and the sample to be tested are mixed and incubated, ammonia water is added to terminate the reaction to obtain a reaction solution; The reaction solution was tested by LC-MS / MS, and the adenine peak area was substituted into the standard curve in step 1) to calculate the concentration of adenine; The sample to be tested is enriched by using antibody-coated magnetic beads before use; When the number of deadenines in the sample to be tested is greater than the average number of deadenines in the negative control plus 3 times the standard deviation, it can be determined as a positive sample.

4. The method according to claim 3, characterized in that The liquid chromatography conditions of the LC-MS / MS are as follows: the chromatographic column is Waters -C18 reverse phase column, inner diameter × length 2.1mm × 150mm, particle size 3.5μm; mobile phase is 10mM, pH4 ammonium formate solution, organic phase is 100% methanol; flow rate is 0.2mL / min; injection volume is 2μL; desolvation temperature is 250℃; source temperature is 150℃.

5. The method according to claim 3, characterized in that: The MRM mode parameters of the LC-MS / MS were: adenine m / z 136→m / z 92, m / z 136→m / z 119, cone voltage was 20 V, and collision energy was 25, 20 eV.

6. The method according to claim 3, characterized in that Step 2) The incubation temperature is 30-60° C. and the incubation time is 1-3 hours.

7. The method according to claim 3, characterized in that Step 2) The buffer is ammonium citrate + Mg 2+ +EDTA buffer, the ammonium citrate +Mg 2+ +EDTA buffer pH 4.0 ~ 4.1, ammonium citrate + Mg 2+ The final concentration of ammonium citrate in the +EDTA buffer is 0.1-10 mM, ammonium citrate + Mg 2+ +Mg in EDTA buffer 2+ The final concentration is 0.1-5 mM, ammonium citrate + Mg 2+ The final concentration of EDTA in the +EDTA buffer is 0.1-10 mM.

8. The method according to claim 3, characterized in that Step 2) The final concentration of the BSA solution 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.

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