Reaction system of ricin, kit and method for detecting ricin by utilizing LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry)

Abstract

The invention provides a ricin reaction system, a kit and a method for detecting ricin by using LC-MS / MS, and belongs to the technical field of toxin detection. The method comprises the following steps: detecting a series of gradient diluted adenine standard substances by utilizing LC-MS / MS, and drawing a standard curve by taking the concentration of adenine as an abscissa and the corresponding peak area of adenine as an ordinate; 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; detecting the reaction liquid by using LC-MS / MS, 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. 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 ricin reaction system, a kit and a method for detecting ricin by using LC-MS / MS. Background Art

[0002] Ricin toxin (RT) is a highly toxic protein extracted from plant seeds. It consists of two chains, A and B, connected by a disulfide bond. The B chain can mediate the entry of the A chain into cells. The A chain has N-glycosidase activity and can specifically cut adenine at position 4324 of ribosomal RNA, thereby inactivating the protein and exerting a toxic effect. Due to its extremely strong toxicity and easy access, it can be classified as a bioterrorist agent. Therefore, the ability to quickly and accurately detect RT is of great significance to national biosafety prevention and control and to maintaining national security and social stability.

[0003] At present, the detection methods based on RT enzyme activity are mainly centered around 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 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, RT can also act on single nucleotide chains. Since longer adenine-containing oligonucleotide chain substrates themselves will undergo different degrees of deadenination, shortening the length of the oligonucleotide chain substrate can improve this phenomenon. However, no scholar has explored the limit length of nucleic acid substrates 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 ricin reaction system, a kit and a method for detecting ricin using LC-MS / MS. A LC-MS / MS adenine detection method is established using a mononucleotide as a detection substrate to detect the adenine content released by the reaction, thereby achieving the purpose of detecting ricin. 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 ricin reaction system, comprising antibody-coated magnetic beads, a buffer, a mononucleotide substrate, a BSA solution and ammonia water;

[0007] The antibody is a 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: adding fusion-promoting markers TRX and B4 antibodies in sequence on a pET 28a vector with NcoI and XhoI as restriction sites, a connecting peptide is added between the TRX and B4 antibodies, and the purification marker His is added after the XhoI restriction site;

[0008] The amino acid sequence of the fusion-promoting marker 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 connecting peptide is shown in SEQ ID NO: 3;

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

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

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

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

[0015] 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;

[0016] 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;

[0017] The sample to be tested is enriched by using antibody-coated magnetic beads before use;

[0018] 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.

[0019] 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℃.

[0020] 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.

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

[0022] Preferably, the buffer is ammonium citrate + Mg 2+ +EDTA buffer, the ammonium citrate +Mg 2+ The pH of the +EDTA buffer is 4.0-4.1, and the ammonium citrate +Mg 2+ The final concentration of ammonium citrate in the +EDTA buffer is 0.5-2 mM. 2+ +Mg in EDTA buffer 2+ The final concentration is 0.5-2 mM, ammonium citrate + Mg 2+ The final concentration of EDTA in the +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; and the final concentration of the 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 make the mononucleotide substrate shed adenine, and 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.

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

[0027] When the sample is directly added to the reaction system, the detection sensitivity of the method can reach 4.75ng / mL; after the detection using magnetic beads coated with antibodies to enrich RT, the sensitivity can reach 0.781ng / mL. 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 conventional RT detection methods for oligonucleotides containing adenine. In addition, mononucleotides have a simple structure, are already commercialized, and are low in cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the chemical formula of the mononucleotide substrates ADP, ATP, AMP, dADP, dATP, and dAMP;

[0029] 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);

[0030] 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);

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

[0032] Figure 5 It is the detection sensitivity of RT without adding magnetic beads;

[0033] Figure 6 is the detection sensitivity of RT plus magnetic beads;

[0034] Figure 7 is the sensitivity of the milk sample test;

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

[0036] Fig. 9 It is the antibody specificity verification. DETAILED DESCRIPTION

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

[0038] The antibody is a 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: adding fusion-promoting markers TRX and B4 antibodies in sequence on a pET 28a vector with NcoI and XhoI as restriction sites, a connecting peptide is added between the TRX and B4 antibodies, and the purification marker His is added after the XhoI restriction site;

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

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

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

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

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

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

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

[0046] 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;

[0047] 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;

[0048] The sample to be tested is enriched by using antibody-coated magnetic beads before use;

[0049] 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.

[0050] 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℃.

[0051] 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.

[0052] In the present invention, the sample to be tested is preferably enriched using antibody-coated magnetic beads before use, and the magnetic beads are preferably Dynabeads TM M-270Streptavidin 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.

[0053] 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 to 4.1, more preferably 4.02 to 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+ +Mg in EDTA buffer 2+ The final concentration 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 8-12 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.

[0055] 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.

[0056] 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.

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

[0058] (1) Main reagents

[0059] Dynabeads TM M-270Streptavidin (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 Clean600 PCR 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] Column: ACQUITY UPLC@BEH Peptide BEH C18, Column, 1.7μm, 2.1mm×150mm (Waters).

[0076] Example 1

[0077] 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.

[0078] 2. LC and MS conditions

[0079] (1)LC conditions

[0080] 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.

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

[0082] (2)MS conditions

[0083] Table 1 MRM detection parameter optimization results

[0084]

[0085]

[0086] Example 2

[0087] Establishment of standard curve of adenine standard

[0088] 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.

[0089] Example 3

[0090] Optimization of reaction substrates

[0091] Experiments were conducted using different mononucleotides as substrates. RT was diluted to 100 μg / mL, 50 μg / mL, and 10 μg / mL, respectively. Sterile water was used as a negative control and the reaction was carried out at 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 1mM) + EDTA (final concentration of 0.5mM) 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 RT of 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 RT / sterile water was added and incubated at 55°C for 2h. The depurination content of different concentrations of RT 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.

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

[0093] Example 4

[0094] Reaction temperature optimization

[0095] The mononucleotide dADP was used as the substrate for the experiment. RT 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 1mM) + EDTA (final concentration of 0.5mM) buffer (pH = 4.05), 10μL of BSA solution with a final concentration of 10μg / mL, 10μL of mononucleotide substrate dADP with a final concentration of 5μM, 10μL of AT of 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 RT / sterile water was added and placed at 37℃ / 55℃ for 2h. The ratio of depurination content after RT at different reaction temperatures acted on the same substrate and the depurination ratio of the negative control was compared. The temperature with the highest ratio was the optimal reaction temperature.

[0096] Experimental results: Figure 4 As shown. At 55°C, the depurination ratio of the mononucleotide dADP is the highest in low and medium concentrations of toxins, and the effect is better. Although the depurination ratio of the mononucleotide dADP is not the highest in high concentrations of toxins, it is not much different from the ratio at 37°C. Overall, the best conditions at low and medium concentrations are taken as the standard, so 55°C is selected as the best reaction temperature for dADP.

[0097] Example 5

[0098] Optimization of detection sensitivity by adding samples directly into the reaction system

[0099] The optimized reaction system was directly used to explore the detection sensitivity of the detection method. RT was diluted with sterile water to 10000ng / ml, 5000ng / mL, 2500ng / mL, 1250ng / mL, 625ng / mL, 312.5ng / mL, 156.25ng / mL, 78.125ng / mL, 39ng / mL, 19ng / mL, 9.5ng / mL, 4.75ng / mL, and reacted at 55℃ for 2h. The reaction system included 10μL of ammonium citrate (final concentration was 1mM) + Mg 2+ (final concentration is 1mM) + EDTA (final concentration is 0.5mM) buffer (pH=4.05), BSA solution with a final concentration of 10μg / mL, dADP substrate with a final concentration of 5μM, 10μL of AT with 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 volume was 50μL.

[0100] Experimental results: Figure 5 As shown in Figure 2, the detection sensitivity of RT was 4.75 ng / mL.

[0101] Example 6

[0102] Detection sensitivity of the reaction system after sample enrichment by antibody-coated magnetic beads

[0103] Antibody-coated magnetic beads are used for sample enrichment, wherein the antibody is a 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: fusion-promoting marker TRX and B4 antibody are sequentially added to the pET 28a vector with NcoI and XhoI as restriction sites, a connecting peptide is added between the TRX and B4 antibodies, and the purification marker His is added after the XhoI restriction site; the amino acid sequence of the fusion-promoting marker 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 connecting peptide is shown in SEQ ID NO:3, specifically GGGGSGGGGSGGGGS; the amino acid sequence of the purification tag His is shown in SEQ ID NO:4, specifically HHHHHH. Dynabeads were used as 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 incubation with the corresponding magnetic beads at room temperature for 1h, the magnetic stand is adsorbed for 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 is 1mM) + EDTA (final concentration is 0.5mM) buffer (pH = 4.05), BSA solution with a final concentration of 10μg / mL, dADP substrate with a final concentration of 5μM, 10μL of RT of different concentrations, 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 pipetted evenly and incubated at 55℃ for 2h. The concentrations of RT were set to 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, and sterile water was used as a negative control to explore the detection sensitivity of the detection method.

[0104] Experimental results: Figure 6 The detection sensitivity after RT and magnetic bead enrichment was 0.781 ng / mL.

[0105] Example 7

[0106] Detection sensitivity in complex samples

[0107] Antibody-coated magnetic beads are used for sample enrichment, the antibody is a 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: on the pET 28a vector, NcoI and XhoI are used as restriction sites to sequentially add fusion-promoting markers TRX and B4 antibodies, a connecting peptide is added between the TRX and B4 antibodies, and the purification marker His is added after the XhoI restriction site; the amino acid sequence of the fusion-promoting marker 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: NO:2, specifically EVQLQASGGGLVQAGGSLRLSCVHSGSPLRSSAMAWFRQAPGKDREFVATINFSGSLAKYTDSVKGRFTISRDNDQNTVYLQMNSLKAEDAAVYYCAAAPAWDRLEYAPRAASDFVSWGPGTQVTVFAEPKTPKPQP; the amino acid sequence of the connecting peptide is shown in SEQ ID NO:3, specifically GGGGSGGGGSGGGGS; the amino acid sequence of the purification tag His is shown in SEQ ID NO:4, specifically HHHHHH. Dynabeads were used as 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 incubation with the corresponding magnetic beads at room temperature for 1h, adsorb on the magnetic stand for 3min, and remove the supernatant. Wash the magnetic beads three times with 500μL of sterile water, and then add the reaction system, which includes 10μL of ammonium citrate (final concentration is 1mM) + Mg 2+(final concentration of 1mM) + EDTA (final concentration of 0.5mM) buffer (pH = 4.05), BSA solution with a final concentration of 10μg / mL, dADP substrate with a final concentration of 5μM, 10μL of RT of different concentrations, 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 magnetic beads coupled to the antibody and the toxin were gently blown and evenly incubated at 55℃ for 2h. The simulated complex sample of RT was made with milk. RT was diluted with milk, and the concentrations were set to 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, 0.098ng / mL, respectively. Sterile water was used as a negative control to explore the detection sensitivity of the detection method.

[0108] Experimental results: Figure 7 The detection sensitivity of milk samples 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, and guanine, uracil and thymine were detected according to the LC-MS / MS detection method of Example 1 to verify the specificity of RT. RT was diluted to 10 μg / mL, and the mononucleotide substrate was diluted to 50 μmol / L respectively. The reaction system was added for detection, and the reaction system was the same as that of Example 7.

[0112] Experimental results: Figure 8 RT cannot 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 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.

[0114] Experimental results: Fig. 9 These toxins had no cross-reaction with RT during the reaction, indicating that this detection method has good specificity.

[0115] As can be seen from the above examples, 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 ricin, which not only reduces the detection cost but also has good specificity and high sensitivity.

[0116] 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 ricin reaction system, characterized in that: Includes antibody-coated magnetic beads, buffer, mononucleotide substrate, BSA solution and ammonia water; The antibody is a 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: adding fusion-promoting markers TRX and B4 antibodies in sequence on a pET 28a vector with NcoI and XhoI as restriction sites, a connecting peptide is added between the TRX and B4 antibodies, and the purification marker His is added after the XhoI restriction site; The amino acid sequence of the fusion-promoting marker TRX is shown in SEQ ID NO: 1; The amino acid sequence of the B4 antibody is shown in SEQ ID NO: 2; The amino acid sequence of the connecting peptide is shown in SEQ ID NO: 3; The amino acid sequence of the purification tag His is shown in SEQ ID NO: 4; The mononucleotide substrate is ADP, ATP, AMP, dADP, dATP or dAMP.

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

3. A method for detecting ricin 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 Reversed 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 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 The buffer is ammonium citrate + Mg 2+ +EDTA buffer, the ammonium citrate +Mg 2+ The pH of the +EDTA buffer is 4.0-4.1, and the ammonium citrate +Mg 2+ The final concentration of ammonium citrate in the +EDTA buffer is 0.5-2 mM. 2+ +Mg in EDTA buffer 2+ The final concentration is 0.5-2 mM, ammonium citrate + Mg 2+ The final concentration of EDTA in the +EDTA buffer 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; and the final concentration of the ammonia water is 15-25 mM.

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