A reaction system, kit for ricin, and method for detecting ricin using LC-MS / MS

Abstract

The present invention provides a reaction system for ricin, a kit and a method for detecting ricin by LC-MS / MS, belonging to the technical field of toxin detection. In the present invention, a series of serially diluted adenine standards are detected by LC-MS / MS. With the concentration of adenine as the abscissa and the corresponding peak area of adenine as the ordinate, a standard curve is plotted; after mixing and incubating a single nucleotide substrate, a buffer solution, a BSA solution and a sample to be tested, ammonia water is added to terminate the reaction to obtain a reaction solution; the reaction solution is detected by LC-MS / MS, and the concentration of adenine is calculated by substituting the peak area of adenine into the standard curve; when the number of de-adenylated adenine in the sample to be tested is greater than the average value of the number of de-adenylated adenine in the negative control plus 3 times the standard deviation, it can be determined as a positive sample. The detection method of the present invention has strong specificity and high sensitivity, can only act on single nucleotides containing adenine, and does not cross-react with other toxin detections.

Description

Technical Field

[0001] The present invention belongs to the technical field of toxin detection, and particularly relates to a reaction system for ricin toxin, a kit, and a method for detecting ricin toxin using LC-MS / MS. Background Art

[0002] Ricin toxin (RT) is a highly toxic protein extracted from plant seeds, consisting of two chains, A and B, connected by a disulfide bond. Chain B can mediate the entry of chain A into cells, and chain A has N-glycosidase activity, which can specifically cleave the adenine at the 4324th position of ribosomal RNA, resulting in protein inactivation and exerting toxic effects. Due to its extremely high toxicity and easy availability, it can be classified as a bioterror agent. Therefore, the ability to quickly and accurately detect RT is of great significance for national biosecurity prevention and control, maintaining national security, and social long-term stability.

[0003] Currently, the detection methods based on the enzyme activity of RT mainly focus on N-glycosidase activity and are mainly based on mass spectrometry technology. Early studies found that when RT exerts its N-glycosidase activity in vitro, it requires a double-stranded helix stem with a length of at least three base pairs and a loop containing the GAGA sequence near the center to exhibit detectable activity. Later, it was found that in addition to the nucleic acid substrate with a stem-loop structure, RT can also act on single-stranded nucleotide chains. Since the longer oligonucleotide chain substrate containing adenine itself will undergo varying degrees of de-adenylation, shortening the length of the oligonucleotide chain substrate can improve this phenomenon. However, no scholars have explored the issue of the limit length of the nucleic acid substrate on which RT can exert N-glycosidase activity. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a reaction system for ricin toxin, a kit, and a method for detecting ricin toxin using LC-MS / MS. By establishing a detection method for adenine using LC-MS / MS with single nucleotides as the detection substrate, the content of adenine shed in the reaction is detected to achieve the purpose of detecting ricin toxin, which not only reduces the detection cost but also has good specificity and high sensitivity.

[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a reaction system for ricin toxin, comprising antibody-coated magnetic beads, a buffer solution, a single nucleotide substrate, a BSA solution, and ammonia water;

[0007] The antibody is an Nco I-TRX-B4-Xho I-HIS recombinant nanobody, and the preparation method of the Nco I-TRX-B4-Xho I-HIS recombinant nanobody is as follows: on the pET 28a vector, the fusion-promoting label TRX and the B4 antibody are sequentially added with NcoI and XhoI as restriction enzyme sites, a linker peptide is added between the TRX and the B4 antibody, and the purification label His is added behind the XhoI restriction enzyme site;

[0008] The amino acid sequence of the fusion-promoting label TRX is shown in SEQ ID NO:1;

[0009] The amino acid sequence of the B4 antibody is shown in SEQ ID NO:2;

[0010] The amino acid sequence of the linker peptide is shown in SEQ ID NO:3;

[0011] The amino acid sequence of the purification label His is shown in SEQ ID NO:4;

[0012] The single nucleotide substrate is ADP, ATP, AMP, dADP, dATP or dAMP.

[0013] The present invention also provides a kit for detecting ricin, comprising the reaction system described above.

[0014] The present invention also provides a method for detecting ricin by LC-MS / MS based on the reaction system described above, comprising the following steps:

[0015] 1) Detect the serial diluted adenine standards by LC-MS / MS, take the concentration of adenine as the abscissa and the corresponding peak area of adenine as the ordinate to draw a standard curve;

[0016] 2) After mixing and incubating the single nucleotide substrate, buffer, BSA solution and the sample to be tested, add ammonia water to terminate the reaction to obtain a reaction solution; detect the reaction solution by LC-MS / MS, and substitute the adenine peak area into the standard curve described in step 1) to calculate the concentration of adenine;

[0017] Before use, the sample to be tested is enriched by antibody-coated magnetic beads;

[0018] When the number of de-adenylated of the sample to be tested is greater than the average value of the number of de-adenylated of the negative control plus 3 times the standard deviation, it can be determined as a positive sample.

[0019] Preferably, the liquid chromatography conditions of the LC-MS / MS are as follows: the chromatographic column is Waters Reverse-phase column, inner diameter × length is 2.1 mm × 150 mm, particle size is 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 volume is 2 μL; the desolvation temperature is 250 °C; the source temperature is 150 °C.

[0020] Preferably, the MRM mode parameters of the LC-MS / MS are as follows: for adenine, m / z 136 → m / z 92, m / z 136 → m / z 119, the cone voltage is 20 V, and the collision energies are 25 and 20 eV.

[0021] Preferably, the incubation temperature is 30 - 60 °C, and the incubation time is 1 - 3 h.

[0022] Preferably, the buffer 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 0.5 - 2 mM, the final concentration of Mg 2+ in the ammonium citrate + Mg 2+ + EDTA buffer is 0.5 - 2 mM, and the final concentration of EDTA in the ammonium citrate + Mg 2+ + EDTA buffer is 0.3 - 0.8 mM.

[0023] Preferably, the final concentration of the BSA solution is 8 - 12 μg / mL; the final concentration of the mononucleotide substrate is 3 - 7 μM; the final concentration of ammonia water is 15 - 25 mM.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The reaction principle of the present invention is that RT exerts N-glycosidase activity to cause the adenine of the mononucleotide substrate to fall off. By establishing a detection method for adenine by LC-MS / MS, the content of adenine fallen off in the reaction is detected to achieve the purpose of detecting toxins. The positive determination criterion is that the number of adenine removed from the positive sample is greater than the average value of the number of adenine removed from the negative sample plus 3 times the standard deviation.

[0026] The present invention 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 screens out the best mononucleotide and the optimal reaction temperature to optimize the detection sensitivity. The composition components of this method system include ammonium citrate (final concentration is 1 mM) + Mg 2+(Final concentration: 1 mM) + EDTA (Final concentration: 0.5 mM) buffer (pH = 4.05), single nucleotide substrate (dADP), 10 μg / mL bovine serum albumin, 100 mM ammonia water. The reaction system was incubated at 55 °C for 2 h and then detected.

[0027] When the sample is directly added to the reaction system in the present invention, the detection sensitivity of this method can reach 4.75 ng / mL; after enrichment RT using magnetic beads coated with antibodies and then detection, the sensitivity can reach 0.781 ng / mL. The present invention has strong specificity, can only act on single nucleotides containing adenine, and does not cross-react with other toxin detections. It has high sensitivity, comparable to that of conventional RT acting on oligonucleotides containing adenine detection methods. In addition, the single nucleotide has a simple structure, is already commercialized and has a low cost. Description of the Drawings

[0028] Figure 1 are the chemical structural formulas of single nucleotide substrates ADP, ATP, AMP, dADP, dATP, dAMP;

[0029] Figure 2 is the mass spectrum and standard curve of adenine standard (where A is the mass spectrum of adenine standard, and B is the standard curve);

[0030] Figure 3 is the reaction result diagram of different types of substrates (Note: H, 100 μg / mL; M, 50 μg / mL; L, 10 μg / mL);

[0031] Figure 4 is the reaction result diagram at different reaction temperatures; (Note: H, 100 μg / mL; M, 50 μg / mL; L, 10 μg / mL);

[0032] Figure 5 is the detection sensitivity without magnetic beads in RT;

[0033] Figure 6 is the detection sensitivity with magnetic beads in RT;

[0034] Figure 7 is the detection sensitivity of milk samples;

[0035] Figure 8 is the verification result of single nucleotide specificity (where A is guanine, B is thymine, and C is uracil);

[0036] Figure 9 is the verification of antibody specificity. Detailed Embodiments

[0037] The present invention provides a reaction system for ricin, comprising antibody-coated magnetic beads, a buffer, a mononucleotide substrate, a BSA solution, and ammonia water;

[0038] The antibody is an Nco I-TRX-B4-Xho I-HIS recombinant nanobody. The preparation method of the Nco I-TRX-B4-Xho I-HIS recombinant nanobody is as follows: on the pET 28a vector, the fusion-promoting label TRX and the B4 antibody are sequentially added with NcoI and XhoI as restriction enzyme sites. A linker peptide is added between the TRX and the B4 antibody, and the purification label His is added behind the XhoI restriction enzyme site;

[0039] The amino acid sequence of the fusion-promoting label TRX is as shown in SEQ ID NO:1, specifically MGSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADE YQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQ LKEFLDANLA;

[0040] The amino acid sequence of the B4 antibody is as shown in SEQ ID NO:2, specifically EVQLQASGGGLVQAGGSLRLSCVHSGSPLRSSAMAWFRQAPGKDREFVA TINFSGSLAKYTDSVKGRFTISRDNDQNTVYLQMNSLKAEDAAVYYCAAAPAWDRLEYAPRAASDFVSWGPGTQVTVFAEPKTPKPQP;

[0041] The amino acid sequence of the linker peptide is as shown in SEQ ID NO:3, specifically GGGGSGGGGSGGGGS;

[0042] The amino acid sequence of the purification label His is as shown in SEQ ID NO:4, specifically HHHHHH;

[0043] The mononucleotide substrate is ADP, ATP, AMP, dADP, dATP, or dAMP.

[0044] The present invention also provides a kit for detecting ricin, comprising the above-mentioned reaction system.

[0045] The present invention also provides a method for detecting ricin using LC-MS / MS based on the above-mentioned reaction system, comprising the following steps:

[0046] 1) Detect a series of serially diluted adenine standards using LC-MS / MS. Plot a standard curve with the adenine concentration on the abscissa and the corresponding peak area of adenine on the ordinate.

[0047] 2) After mixing and incubating the single nucleotide substrate, buffer, BSA solution, and the sample to be tested, add ammonia water to terminate the reaction to obtain a reaction solution. Detect the reaction solution using LC-MS / MS, and substitute the adenine peak area into the standard curve described in step 1) to calculate the adenine concentration.

[0048] Before use, the sample to be tested is preferably enriched using antibody-coated magnetic beads.

[0049] When the number of de-adenylated bases in the sample to be tested is greater than the average value of the number of de-adenylated bases in the negative control plus 3 times the standard deviation, it can be determined as a positive sample.

[0050] In the present invention, the liquid chromatography conditions of the LC-MS / MS are preferably as follows: the chromatographic column is a Waters 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 a 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 volume is 2 μL; the desolvation temperature is 250 °C; the source temperature is 150 °C.

[0051] In the present invention, the MRM mode parameters of the LC-MS / MS are: for adenine, m / z 136 → m / z 92, m / z 136 → m / z 119, the cone voltage is 20 V, and the collision energy is 25, 20 eV.

[0052] In the present invention, before use, the sample to be tested is preferably enriched using antibody-coated magnetic beads. 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, more preferably 1:98 - 102, and even more preferably 1:100. In the present invention, the usage amount of magnetic beads for each sample to be tested is preferably 25 - 35 μg, more preferably 27 - 32 μg, and even more preferably 30 μg.

[0053] In the present invention, the buffer is preferably ammonium citrate + Mg 2+ + EDTA buffer, and the pH of the ammonium citrate + Mg 2+ + 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 0.5 to 2 mM, more preferably 0.8 to 1.5 mM, and even more preferably 1 mM; ammonium citrate + Mg 2+ The final concentration of Mg 2+ in the +EDTA buffer is preferably 0.5 to 2 mM, more preferably 0.8 to 1.5 mM, and even more preferably 1 mM; ammonium citrate + Mg 2+ The final concentration of EDTA in the +EDTA buffer is preferably 0.3 to 0.8 mM, more preferably 0.4 to 0.7 mM, and even more preferably 0.5 mM.

[0054] In the present invention, the final concentration of the BSA solution is preferably 8 to 12 μg / mL, more preferably 9 to 11 μg / mL, and even more preferably 10 μg / mL; the final concentration of the mononucleotide substrate is preferably 3 to 7 μM, more preferably 4 to 6 μM, and even more preferably 5 μM; the final concentration of the ammonia water is preferably 15 to 25 mM, more preferably 18 to 22 μM, and even more preferably 20 μM.

[0055] In the present invention, the incubation temperature is preferably 30 to 60 °C, more preferably 32 to 58 °C, and even more preferably 55 °C; the incubation time is preferably 1 to 3 h, more preferably 1.5 to 2.5 h, and even more preferably 2 h.

[0056] The technical solutions provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

[0057] Main reagents and instruments used in the present invention

[0058] (1) Main reagents

[0059] Dynabeads TM M-270 Streptavidin (65305): Thermo Fisher Scientific;

[0060] Adenosine 5'-triphosphate (ATP, HY-B2176), Adenosine 5'-diphosphate (ADP, HY-W010918), Adenosine monophosphate (AMP, HY-A0181): purchased from MedChemExpress;

[0061] 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'-Deoxyadenosine5'-monophosphate, dAMP, S18087): purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0062] dTTP solution (AG11014), dGTP solution (AG11015), and UTP solution (AG11017): purchased from Hunan Aikerui Bioengineering Co., Ltd.;

[0063] Bovine serum albumin (B2064), ammonium citrate (25102): Sigma-Aldrich (Shanghai) Trading Co., Ltd.;

[0064] Acetonitrile (HPLC grade) and methanol (HPLC grade) (>98%) were purchased from Thermo Fisher Scientific (Waltham, MA, USA);

[0065] Ultrapure water was obtained from Wahaha Pure Water Company, Hangzhou, China;

[0066] Ricin is prepared by this unit;

[0067] Adenine (1012101) standard was purchased from Merck;

[0068] ammonia( 1336-21-6 ): Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0069] (2) Main instruments

[0070] Biological safety cabinet: NUAIRE, USA;

[0071] Vortex mixer: Jiangsu Haimen Qilin Bell Instrument Co., Ltd.

[0072] PCR workstation (Air Clean600PCR Workstation): AirClean Systems, USA;

[0073] HulaMixer TM Sample mixer (15920D), magnetic stand (12321D): Thermo Fisher Scientific;

[0074] Triple Quadrupole: Waters Corporation; Model: ACQUITY I-Class;

[0075] Chromatographic column: ACQUITY UPLC@BEH Peptide BEH C18, Column, 1.7μm, 2.1mm×150mm (Waters).

[0076] Example 1

[0077] 1. Substrates of single nucleotide chains of different categories related to the experiment: ADP, ATP, AMP, dADP, dATP, dAMP, and their chemical structural formulas are as Figure 1 shown.

[0078] 2. LC and MS conditions

[0079] (1) LC conditions

[0080] The liquid chromatography conditions are as follows: The chromatographic column is a Waters reversed-phase column (2.1mm×150mm, 3.5μm); The mobile phase is a 10mM ammonium formate solution with a pH of 4, and the organic phase is 100% methanol; The flow rate is 0.2mL / min; The injection volume is 2μL.

[0081] Desolvation Temp.: 250°C; Source Temp.: 150°C.

[0082] (2) MS conditions

[0083] Table 1 Optimization results of MRM detection parameters

[0084]

[0085]

[0086] Example 2

[0087] Establishment of the standard curve of adenine standard

[0088] The adenine standard was diluted to 1.6, 8, 10, 20, 30, 80, 100 fmoL / μL with sterile water respectively, and detected under the LC-MS conditions of Example 1. Taking the concentration of adenine as the abscissa and the corresponding peak area of adenine as the ordinate, a standard curve was plotted, and the linear equation obtained was Y = 24137*X - 12400, R 2 = 0.999. The experimental results are as Figure 2 shown.

[0089] Example 3

[0090] Optimization of reaction substrates

[0091] Experiments were carried out separately with different single nucleotides as substrates. RT was diluted to 100 μg / mL, 50 μg / mL, and 10 μg / mL respectively, and sterile water was used as a negative control. The reaction was carried out at 55 °C. The reaction system had a volume of 50 μL, and the system included 10 μL of ammonium citrate (final concentration 1 mM) + Mg 2+ (final concentration 1 mM) + EDTA (final concentration 0.5 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 RT at different concentrations. After the reaction, 10 μL of ammonia water with a final concentration of 20 mM was added to terminate the reaction. The reaction system was prepared and 10 μL of RT / sterile water was added, then incubated at 55 °C for 2 h. The ratio of the depurination content of RT at different concentrations acting on the same substrate to the depurination of the negative control was compared, and the substrate with the highest ratio was the optimal reaction substrate.

[0092] Experimental results: As Figure 3 shown. At 55 °C, the depurination ratios of dADP and dATP were similar and better than the other groups. The response values of dADP and dATP were not very different, but the negative value of dADP was lower. So finally, we decided to use dADP as the substrate for subsequent experiments.

[0093] Example 4

[0094] Optimization of reaction temperature

[0095] An experiment was carried out with the single nucleotide dADP as the substrate. RT was diluted to 100 μg / mL, 50 μg / mL, and 10 μg / mL respectively, and sterile water was used as a negative control. The reaction was carried out at 37 °C and 55 °C respectively. The reaction system had a volume of 50 μL, and the system included 10 μL of ammonium citrate (final concentration 1 mM) + Mg 2+ (final concentration 1 mM) + EDTA (final concentration 0.5 mM) buffer (pH = 4.05), 10 μL of BSA solution with a final concentration of 10 μg / mL, 10 μL of single nucleotide substrate dADP with a final concentration of 5 μM, 10 μL of RT at different concentrations. After the reaction, 10 μL of ammonia water with a final concentration of 20 mM was added to terminate the reaction. The reaction system was prepared and 10 μL of RT / sterile water was added, then incubated at 37 °C / 55 °C for 2 h. The ratio of the depurination content of RT at different reaction temperatures acting on the same substrate to the depurination of the negative control was compared, and the temperature with the highest ratio was the optimal reaction temperature.

[0096] Experimental results: As Figure 4As shown in the figure. At 55 °C, the depurination ratio of the single nucleotide dADP is the highest in low and medium concentrations of the toxin, with better effects. Although the depurination ratio of the single nucleotide dADP is not the highest in high concentrations of the toxin, it does not differ much from the ratio at 37 °C. Overall, based on the optimal conditions of low and medium concentrations, 55 °C is therefore selected as the optimal reaction temperature for dADP.

[0097] Example 5

[0098] Optimization of the detection sensitivity of samples directly added to the reaction system

[0099] Directly use the optimized reaction system to explore the detection sensitivity of the detection method. RT was diluted to 10000 ng / ml, 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, 4.75 ng / mL with sterile water respectively, and reacted at 55 °C for 2 h. The reaction system includes 10 μL of ammonium citrate (final concentration 1 mM) + Mg 2+ (final concentration 1 mM) + EDTA (final concentration 0.5 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 different concentrations of RT. After the reaction, 10 μL of ammonia water with a final concentration of 20 mM was added to terminate the reaction, and the volume of the reaction system was 50 μL.

[0100] Experimental results: As Figure 5 shown. The detection sensitivity of RT is 4.75 ng / mL.

[0101] Example 6

[0102] Detection sensitivity of the reaction system after samples are enriched by antibody-coated magnetic beads

[0103] Sample enrichment is carried out using antibody-coated magnetic beads. The antibody is the Nco I-TRX-B4-Xho I-HIS recombinant nanobody. The preparation method of the Nco I-TRX-B4-Xho I-HIS recombinant nanobody is as follows: The fusion-promoting tag TRX and the B4 antibody are sequentially added to the pET 28a vector with NcoI and XhoI as restriction enzyme sites. A linker peptide is added between the TRX and the B4 antibody, and the purification tag His is added behind the XhoI restriction enzyme site. The amino acid sequence of the fusion-promoting tag TRX is as shown in SEQ ID NO:1, specifically MGSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA. The amino acid sequence of the B4 antibody is as shown in SEQ ID NO:2, specifically EVQLQASGGGLVQAGGSLRLSCVHSGSPLRSSAMAWFRQAPGKDREFVATINFSGSLAKYTDSVKGRFTISRDNDQNTVYLQMNSLKAEDAAVYYCAAAPAWDRLEYAPRAASDFVSWGPGTQVTVFAEPKTPKPQP. The amino acid sequence of the linker peptide is as shown in SEQ ID NO:3, specifically GGGGSGGGGSGGGGS. The amino acid sequence of the purification tag His is as shown in SEQ ID NO:4, specifically HHHHHH. The magnetic beads used are Dynabeads TM M-270 Streptavidin. The coating amount of the antibody and the magnetic beads is 10 μg of biotinylated antibody coated on 1 mg of magnetic beads. The amount of magnetic beads used for each sample is 30 μg. The volume of the sample is 500 μL. After incubating by inversion with the corresponding magnetic beads at RT for 1 h at room temperature, adsorb with a magnetic stand for 3 min and remove the supernatant. Subsequently, the reaction system is added. The reaction system includes 10 μL of ammonium citrate (final concentration 1 mM) + Mg 2+(Final concentration: 1 mM) + EDTA (Final concentration: 0.5 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 RT at different concentrations. After the reaction, 10 μL of ammonia water with a final concentration of 20 mM was added to terminate the reaction, and the volume of the reaction system was 50 μL. The magnetic beads conjugated with antibodies and the toxin were gently pipetted up and down to mix evenly and then incubated with inversion at 55 °C for 2 h. The concentrations of RT were set at 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.

[0104] Experimental results: As Figure 6 shown. The detection sensitivity after enrichment of RT with magnetic beads was 0.781 ng / mL.

[0105] Example 7

[0106] Detection sensitivity of complex samples

[0107] Sample enrichment is carried out with antibody-coated magnetic beads. The antibody is the Nco I-TRX-B4-Xho I-HIS recombinant nanobody. The preparation method of the Nco I-TRX-B4-Xho I-HIS recombinant nanobody is as follows: The fusion-promoting tags TRX and B4 antibody are sequentially added to the pET 28a vector with NcoI and XhoI as restriction enzyme sites. A linker peptide is added between the TRX and B4 antibody, and the purification tag His is added behind the XhoI restriction enzyme site. The amino acid sequence of the fusion-promoting tag TRX is as shown in SEQ ID NO:1, specifically MGSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA. The amino acid sequence of the B4 antibody is as shown in SEQ ID NO:2, specifically EVQLQASGGGLVQAGGSLRLSCVHSGSPLRSSAMAWFRQAPGKDREFVATINFSGSLAKYTDSVKGRFTISRDNDQNTVYLQMNSLKAEDAAVYYCAAAPAWDRLEYAPRAASDFVSWGPGTQVTVFAEPKTPKPQP. The amino acid sequence of the linker peptide is as shown in SEQ ID NO:3, specifically GGGGSGGGGSGGGGS. The amino acid sequence of the purification tag His is as shown in SEQ ID NO:4, specifically HHHHHH. The magnetic beads used are Dynabeads TM M-270 Streptavidin. The coating amount of the antibody and the magnetic beads is 10 μg of biotinylated antibody per 1 mg of magnetic beads coated. The amount of magnetic beads used for each sample is 30 μg. The volume of the sample is 500 μL. After RT and inversion incubation with the corresponding magnetic beads at room temperature for 1 h, the magnetic rack is used to adsorb for 3 min, and the supernatant is removed. The magnetic beads are washed three times with 500 μL of sterile water, and then the reaction system is added. The reaction system includes 10 μL of ammonium citrate (final concentration 1 mM) + Mg 2+(Final concentration: 1 mM) + EDTA (Final concentration: 0.5 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 RT at different concentrations. After the reaction, 10 μL of ammonia water with a final concentration of 20 mM was added to terminate the reaction, and the volume of the reaction system was 50 μL. The magnetic beads conjugated with antibodies and the toxin were gently pipetted and mixed evenly, and then incubated at 55 °C with inversion for 2 h. Simulated complex samples of RT were prepared with milk. RT was diluted with milk, and the concentrations were set to 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.

[0108] Experimental results: As Figure 7 shown. The detection sensitivity of the milk sample was 1.563 ng / mL.

[0109] Example 8

[0110] Specificity verification

[0111] 1. The standards of guanine, thymine, and uracil were diluted to 10 μmol / L respectively. According to the LC-MS / MS detection method in Example 1, guanine, uracil, and thymine were detected for the specificity verification of RT. RT was diluted to 10 μg / mL respectively, and the single nucleotide substrates were diluted to 50 μmol / L respectively for detection. They were added to the reaction system for detection, and the reaction system was the same as that in Example 7.

[0112] Experimental results: As Figure 8 shown. RT could not act on the single nucleotides guanine, thymine, and uracil, but only on adenine.

[0113] 2. RT, AT, DT, SEA, and ETX toxins were used simultaneously for the specificity verification of this detection method. All toxins were diluted to 100 ng / mL respectively. The experimental steps in this experiment were the same as those in the experiment of the RT milk simulated sample in Example 7, only the toxins used were different.

[0114] Experimental results: As Figure 9 shown. These toxins had no cross-reaction with RT during the reaction, indicating that the specificity of this detection method was good.

[0115] As can be seen from the above embodiments, the present invention uses single nucleotides as detection substrates to establish a detection method for adenine by LC-MS / MS, and detects the content of adenine shed in the detection reaction to achieve the purpose of detecting ricin. This not only reduces the detection cost, but also has good specificity and high sensitivity.

[0116] The above description is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A reaction system for ricin, characterized in that, It includes antibody-coated magnetic beads, buffer, mononucleotide substrates, BSA solution and ammonia water; The antibody is Nco I-TRX-B4-Xho I-HIS recombinant nanobody, and the preparation method of the Nco I-TRX-B4-Xho I-HIS recombinant nanobody is as follows: on the pET 28a vector, the fusion-promoting tags TRX and B4 antibody are sequentially added with NcoI and XhoI as restriction enzyme sites, a linker peptide is added between the TRX and B4 antibody, and the purification tag His is added behind the XhoI restriction enzyme site; The amino acid sequence of the fusion-promoting tag TRX is as shown in SEQ ID NO:1; The amino acid sequence of the B4 antibody is as shown in SEQ ID NO:2; The amino acid sequence of the linker peptide is as shown in SEQ ID NO:3; The amino acid sequence of the purification tag His is as shown in SEQ ID NO:4; The mononucleotide substrates are ADP, ATP, AMP, dADP, dATP or dAMP.

2. A kit for detecting ricin, characterized in that, It includes the reaction system described in claim 1.

3. A method for detecting ricin using LC-MS / MS in the reaction system according to claim 1, characterized in that It includes the following steps: 1) Detect the serial dilution of adenine standard with LC-MS / MS. Using the concentration of adenine as the abscissa and the corresponding peak area of adenine as the ordinate, draw a standard curve; 2) After mixing and incubating the mononucleotide substrates, buffer, BSA solution and the sample to be tested, add ammonia water to terminate the reaction to obtain a reaction solution; detect the reaction solution with LC-MS / MS, and substitute the adenine peak area into the standard curve described in step 1) to calculate the concentration of adenine; Before use, the sample to be tested is enriched with antibody-coated magnetic beads; When the amount of de-adenylation of the sample to be tested is greater than the average value of the amount of de-adenylation of 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 Xtarre®-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 volume is 2 μL; the desolvation temperature is 250 °C; the source temperature is 150 °C.

5. The method according to claim 3, wherein The MRM mode parameters of the LC-MS / MS are: for adenine, m / z 136→m / z 92, m / z 136→m / z 119, the cone voltage is 20 V, and the collision energy is 25, 20 eV.

6. The method according to claim 3, wherein The incubation temperature is 30~60 °C, and the incubation time is 1~3 h.

7. The method according to claim 3, wherein The buffer solution is ammonium citrate + Mg 2+ + EDTA buffer solution. The pH of the ammonium citrate + Mg 2+ + EDTA buffer solution is 4.0 - 4.

1. The final concentration of ammonium citrate in the ammonium citrate + Mg 2+ + EDTA buffer solution is 0.5 - 2 mM. The final concentration of Mg 2+ in the ammonium citrate + Mg 2+ + EDTA buffer solution is 0.5 - 2 mM. The final concentration of EDTA in the ammonium citrate + Mg 2+ + EDTA buffer solution is 0.3 - 0.8 mM.

8. The method according to claim 3, characterized in that, The final concentration of the BSA solution is 8~12 μg / mL; the final concentration of the mononucleotide substrate is 3~7 μM; the final concentration of the ammonia water is 15~25 mM.

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