CN112816701B - Method for rapidly detecting ricin by colloid Jin Celiu chromatography and colloid Jin Celiu chromatography kit - Google Patents
Method for rapidly detecting ricin by colloid Jin Celiu chromatography and colloid Jin Celiu chromatography kit Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the field of toxin detection, and relates to a method for rapidly detecting ricin by using a colloid Jin Celiu chromatography method, colloid Jin Celiu chromatography test paper and a kit. The method comprises the following steps: (1) Incubating H1-AuNPs-FAM antibodies, FAM antibodies-AuNPs with a sample to be tested containing ricin; the sequence of H1 is shown in SEQ ID NO:1 is shown in the specification; the particle size of AuNPs is 2-10nm; (2) Adding the mixed liquid obtained in the step (1) to lateral flow chromatography test paper; the lateral flow chromatography test paper comprises a sample pad, a detection area, an absorption pad and an NC film with a plastic back card; the detection area comprises a T line combination area and a C line combination area; the T line bonding area is provided with a detection line, and the C line bonding area is provided with a control line; (3) Judging the detection result according to the color development values of the gold nano particles on the detection line and the control line. The invention establishes a colloidal Jin Celiu chromatography method for detecting ricin, and the method does not need to prepare corresponding antibodies, has simple operation and can meet the requirement of on-site detection.
Description
Technical Field
The invention belongs to the field of toxin detection, and particularly relates to a method for rapidly detecting ricin by a colloidal gold lateral flow chromatography method and a colloidal Jin Celiu chromatography kit for rapidly detecting ricin.
Background
Castor, latin brand name Ricinus communications l.native to sumatrix and kennia in northeast africa, are widely distributed throughout tropical regions of the world. The stems, leaves or seeds of castor plant contain toxic substances, mainly including ricin, castor reaction source, etc. Ricin (RT) can be dissolved in weak acid or water after chemical purification, and has stable physicochemical properties. In terms of chemical structure, ricin is a highly toxic plant protein consisting of two peptide chains of A, B. Active ricin enters eukaryotic cells through B-chain mediated endocytosis, followed by purification of specific adenine from 28S rRNA by a-chain, thereby inducing protein synthesis failure and eventually activating cell death pathways.
Ricin is a highly toxic protein with a toxicity 6000 times higher than cyanide. Ricin is not only attractive to scientific researchers due to its strong toxicity and availability, but also serves as a terrorist sharps, the well-known Markov umbrella being an example. Developing sensitive and efficient detection means is a major measure against threats. The traditional detection technology mainly comprises a radioimmunoassay technology, an enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography, immuno-PCR, a colloidal gold labeling technology and the like. However, these detection methods need to rely on expensive instruments and specialized technicians, which are not beneficial to field use and limit further application and popularization.
Colloidal gold immunochromatography (colloidal gold immune-chromatographic assay, gic a) is a field and rapid detection method developed at the end of the 20 th century, which combines the immunorecognition technology and the chromatography technology by using colloidal gold nanoparticles as a color developing element. The basic principle is that the specific recognition and combination of antigen and antibody are utilized to cause the color change of the colloidal gold on the lateral chromatography test strip. By utilizing the lateral chromatography test strip technology, the rapid determination of mercury ions in water, the hypersensitive detection of the metalaxyl-phosphorus in aquatic products by signal amplification and the like are realized.
The ricin has strong toxicity and is easy to obtain, and is a potential bioterrorism agent, so a method for quickly detecting the ricin is urgently needed to be established so as to ensure national public safety.
Disclosure of Invention
The invention aims to provide a method for rapidly detecting ricin by a colloidal gold lateral flow chromatography method and a colloidal Jin Celiu chromatographic kit.
In order to achieve the above object, a first aspect of the present invention provides a method for rapidly detecting ricin by colloidal gold lateral flow chromatography, comprising the steps of:
(1) Incubating an H1-AuNPs-FAM antibody with a sample to be tested containing ricin; the sequence of H1 is shown in SEQ ID NO:1 is shown in the specification; the particle size of AuNPs is 2-10nm;
5’-FAM-GAC CTA CTT ATG AAA AAA AAA AAA TTA TAT CTA TCT- Biotin-3’(SEQ ID NO：1)
(2) Adding the mixed liquid obtained in the step (1) to lateral flow chromatography test paper; the lateral flow chromatography test paper comprises a sample pad, a detection area, an absorption pad and an NC film with a plastic back card; the detection area comprises a T line combination area and a C line combination area; the T line bonding area is provided with a detection line, and the C line bonding area is provided with a control line; the secondary antibodies of the streptavidin and the anti-FAM antibody are respectively fixed on the detection line and the control line;
(3) Judging the detection result according to the color development values of the gold nano particles on the detection line and the control line.
According to the invention, the H1-AuNPs-FAM antibody may be obtained by incubating H1 with an AuNPs-FAM antibody.
The lateral flow chromatography test paper can be obtained commercially or by self-making.
According to the invention, the detection result can be interpreted in the step (3) either by visual observation or by imaging an instrument, such as Image J imaging. Visual inspection is typically a qualitative judgment and instrument imaging is typically a quantitative test.
Preferably, according to the present invention, the method further comprises the step of preparing a standard curve: according to the methods of the steps (1) and (2), detecting a series of samples to be detected containing ricin with known concentration, measuring a plurality of color values, and respectively plotting the concentration and the color values as the abscissa and the ordinate to obtain a standard curve. At this time, the concentration of ricin in the sample to be tested containing ricin may be calculated based on the standard curve.
The detection principle of the ricin lateral flow chromatography test paper is shown in figure 1. It comprises two steps: (1) incubating the H1-AuNPs-FAM antibody with ricin; (2) The results were read by lateral flow chromatography (lateral flow assay, LFA) test paper. According to the invention, ricin is taken as a target, and an oligonucleotide sequence H1 containing continuous adenine is designed according to the principle that an active ricin A chain can purify adenine and break an oligonucleotide sequence containing adenine, and when the target ricin exists, a plurality of continuous adenine on the H1 sequence can be cut, so that the H1 sequence breaks as shown in a figure 1. For LFA strip, as shown in fig. 1, it includes four parts: sample pad, detection zone (C line bonding zone, T line bonding zone), absorbent pad and NC film with plastic back card. The secondary antibodies of streptavidin and anti-FAM antibodies were immobilized on the detection and control lines of NC membrane, respectively. When the target object does not exist, the mixed liquid of the H1 sequence and the FAM antibody-AuNPs is dripped on a sample pad, the mixed liquid migrates to an absorption pad under the action of capillary force, and the H1 labeled with biotin and FAM is specifically combined with gold nanoparticles modified with the FAM antibody to form a compound. When these complexes pass through the detection line, they are captured by streptavidin immobilized on the detection line, producing a clearly visible signal on the detection line. Excess complex continues to migrate across the NC membrane and is captured by the secondary antibody of the anti-FAM antibody immobilized on the control line, thereby developing color. If ricin exists in the sample, after mixed incubation, the H1 sequence is broken and dissolved, so that the sandwich structure on the detection line is reduced or eliminated, and the color of the detection line is lightened or eliminated. In short, in the absence of the target ricin, two red lines ("on-turn on") were observed as negative results. With the increase of the concentration of the target object, the color of the detection line becomes lighter gradually, and when the concentration reaches a certain degree, only one red line is observed on the control line (off), and as a positive result, the red color on the control line is used for proving that the LFA test paper works normally.
The second aspect of the invention provides a colloidal Jin Celiu chromatographic kit for rapidly detecting ricin, which comprises the following components:
(1) Colloid Jin Celiu chromatographic test paper; the test paper comprises: sample pad, detection area, absorption pad and NC film with plastic back card; the detection area comprises a T line combination area and a C line combination area; the T line bonding area is provided with a detection line, and the C line bonding area is provided with a control line; the secondary antibodies of the streptavidin and the anti-FAM antibody are respectively fixed on the detection line and the control line;
(2) FAM antibodies-AuNPs; wherein the particle size of the AuNPs is 2-10nm;
(3) H1 nucleic acid strand; the sequence of H1 is shown in SEQ ID NO: 1.
In the kit of the present invention, FAM antibody-AuNPs and H1 chain may be provided separately or in the form of H1-AuNPs-FAM antibody.
The invention is based on a colloidal gold immunochromatography method, and according to the principle that adenine can be purified from a ricin A chain to break an oligonucleotide sequence containing adenine, a biotin and FAM co-modified oligonucleotide sequence H1 is designed as a recognition probe, a colloidal Jin Celiu chromatographic test strip with a modified FAM and biotin H1 sequence as a recognition element is developed, a rapid semi-quantitative detection method for ricin is established, visual semi-quantitative detection of ricin is realized, and sensitivity and specificity of the detection method are examined. The minimum detection value of the ricin colloid Jin Celiu chromatographic test strip is lower than 10ng/mL, and the detection requirement can be met. The detection method is simple to operate, does not need large-scale instruments and equipment, and can meet the requirements of field detection. In addition, the invention adopts the oligonucleotide chain as the recognition element, so that the ricin antibody does not need to be prepared, and the cost is reduced.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
FIG. 1 is a schematic diagram of the flow and principle of qualitative detection of ricin by the colloidal gold lateral flow test strip of the invention.
FIG. 2 shows the results of oligonucleotide sequence screening, wherein a is a polyacrylamide gel electrophoresis pattern, and b is a fluorescence spectrum of the oligonucleotide sequence, wherein Lane1: p1; lane2: p2; lane3: p3; lane4: p3+rt; lane5: a2; lane6: a2+rt; lane7: h1; lane8: h2+rt.
Fig. 3 shows the results of optimization of the concentration of H1 sequences, wherein 1 to 5 in turn represent the concentration of H1 as: 100nM, 50nM, 25nM, 10nM and 1nM.
FIG. 4 shows the results of optimizing the incubation time of ricin mixed with H1-AuNPs-FAM antibody, wherein 1-4 are added with 1 μg/mL ricin for incubation for 30min, 60min, 90min, 120min in sequence.
Figure 5 shows the visual sensitivity of the ricin colloid Jin Celiu chromatographic test strip. Wherein, 1-5 are 100ng/mL, 80ng/mL, 40ng/mL, 10ng/mL and 1ng/mL of ricin in turn; 6: blank control.
Fig. 6 shows a linear relationship between relative intensity and logarithmic ricin concentration.
Fig. 7 shows the specificity detection result of ricin colloid Jin Celiu chromatographic test strip, wherein 1: ricin; 2: abrin; 3: SEB;4: BSA;5: OVA;6: FB (FB) 1 The method comprises the steps of carrying out a first treatment on the surface of the 7: blank control.
FIG. 8 is a graph showing TEM characterization results of a 20nm AuNPs-FAM antibody.
FIG. 9 shows the results of detection of ricin by H1-AuNPs-FAM (20 nm). a: detecting the ricin by 5 mu L25 pM H1, wherein the ricin is 10 mu g/mL, 500ng/mL and 10ng/mL in sequence from 1 to 3; 4 is a blank; b: detecting the ricin by 5 mu L25 pM H1, wherein the ricin is 10 mu g/mL, 100ng/mL and 10ng/mL in sequence from 1 to 3; 4 is a blank.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the preferred embodiments of the present invention are described below, it should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein.
LX-200 palm centrifuge, ST70-2 micropore constant temperature oscillator, OSE-DB-02 refrigeration type five-section program controlled metal bath, constant temperature incubator, ricin standard (Beijing Han spectral medical institute of biological science), staphylococcus aureus enterotoxin B (SEB) (self-made), fumonisin standard (Shandong blue Biotechnology Co., ltd.), bovine Serum Albumin (BSA) and Ovalbumin (OVA) (Sigma-Aldrich), abrin lectin (Beijing, national institute of medical science and toxicant medicine), colloid Jin Celiu chromatography test stripHybrid detect). All nucleotides (ODNs) were purchased from Shanghai Biotechnology Inc. (Shanghai, china) and purified by High Performance Liquid Chromatography (HPLC) as follows:
TABLE 1 oligonucleotide sequences
In the invention, ricin is used as a target, an oligonucleotide P3 sequence containing continuous adenine, a modified biotin P1 sequence and a modified FAM P2 sequence are designed, and NUPACK software is used for fitting and optimizing analysis; designing an A1 sequence of modified biotin and an A2 sequence of modified FAM; the H1 sequence of the biotin and FAM which are modified simultaneously is designed, and the DNA sequence is screened through a polyacrylamide gel electrophoresis experiment and a SYBR Green1 fluorescence experiment.
Screening of sequences
The designed oligonucleotide sequences containing consecutive A are listed in Table 1, wherein the P1, P2 sequences are complementarily paired with the two ends of the P3 sequence, respectively, thereby forming a P1-P3-P2 complex, the 5 'end of P1 is modified with biotin, the 3' end of P2 is modified with FAM, and thus the complex can be captured by streptavidin on the detection line for color development. The A1 sequence can be complementarily matched with the A2 sequence, biotin is modified at the 3 'end of A1, FAM is modified at the 3' end of A2, and the FAM can be captured by streptavidin on a detection line to develop color. According to the designed sequence, ricin with the same concentration is respectively added into different oligonucleotide sequences under the same volume and concentration, and polyacrylamide gel electrophoresis is used for characterizing the cutting and dissolving capacity of the toxin to the oligonucleotide sequences. And (3) utilizing the principle that SYBR Green1 only dyes double-stranded DNA, examining sequence complementary pairing conditions, and finally screening out a proper oligonucleotide sequence.
Polyacrylamide gel electrophoresis is used to characterize the cleavage and dissolution capabilities of the target on the oligonucleotide sequences. As shown in FIG. 2 a, at the same volume and concentration, the same concentration of the target substance was added to the different oligonucleotide sequences, and a diffuse band appeared after adding the target substance, and the brightness was significantly reduced. It is illustrated that the target can cleave and solubilize oligonucleotide sequences containing successive adenine residues. The detection limit is too high when the P1-P3-P2 complex system is used for detection in the subsequent step, so that the system is not used. By utilizing the principle that SYBR Green1 only dyes double-stranded DNA, A1 and A2 sequences are mixed and incubated and then are dyed by SYBR Green1, and a fluorescence result (b of fig. 2) shows that A1 and A2 sequences can generate A1-A2 binding chains and A1 and A2 fold to form a double-stranded structure after being mixed and incubated, so that the cutting capability of toxin to the A2 sequences is affected and the color change of a detection line is interfered. Thus, the H1 sequence is ultimately selected as the recognition element.
Aunps particle size optimization
Gold nanoparticles (see figure 8 of a transmission electron microscope image of 20nm gold) of the FAM antibody modified by 20nm in the early stage are placed in a microporous constant temperature oscillator to be mixed and incubated with a 25pM H1 sequence, then a target is added for detection, 50 mu L of reaction liquid is dripped in a test strip sample area, and the test result is observed after standing for 10 min. The results are shown in FIG. 9. As can be seen from the results, the 20nm gold particles were not light even at a concentration of 10. Mu.g/mL of toxin. Therefore, experiments were subsequently performed with gold particles of 5 nm.
H1 sequence incubation concentration optimization
mu.L of 100nM, 50nM, 25nM, 10nM and 1nM of oligonucleotide sequence H1 were placed in a metal bath to incubate for 1H (4 runs were set: 5min at 95℃10min, 10min at 80℃10min at 37℃10 min), 10. Mu.L of 50ng/mL FAM-AuNPs and 85. Mu.L Tris-HCl buffer were added to the reaction tube to prepare 100. Mu.L of reaction system, 50. Mu.L of reaction solution was dropped in the test strip sample zone, and the experimental result was observed at rest for 10 min.
The H1 sequence of the FAM and biotin is a recognition element in the lateral flow strip, and can be specifically combined with streptavidin immobilized on the detection line of the test strip, and a sandwich structure formed on the detection line can generate obvious visible signals. Therefore, the H1 sequence addition concentration has a larger influence on the color of the detection line. In the present invention, the color changes of the detection line and control line were observed with the addition of different concentrations of H1 sequences (100 nM, 50nM, 25nM, 10nM and 1 nM), and as a result, as shown in FIG. 3, it was found from FIG. 3 that the optimal H1 concentration was added to be 10nM.
Optimization of incubation time for adding target
Adding ricin standard substances with different concentrations into a reaction tube containing 5 mu L H1-AuNPs-FAM antibody, placing the reaction tube in a microporous constant temperature oscillator, respectively incubating at 700rpm at 37 ℃ for 30min, 60min, 90min and 120min, dripping 50 mu L of reaction solution into a test strip sample area after the reaction is finished, and standing for 10min to observe an experimental result.
When the target ricin exists, the H1 sequence containing adenine is cut and dissolved, so that the H1 sequence is broken, and the color of a detection line becomes light or disappears. Therefore, the incubation time of the added target object and the H1 sequence has a great influence on the detection effect. In the invention, the color change of the detection line and the control line under the condition of different incubation time (30 min, 60min, 90min and 120 min) of the H1 sequence with the same concentration and the target object is observed, and the result is shown in fig. 4, and as can be seen from fig. 4, the optimal incubation time for mixing the added target object with the H1 sequence is 120min.
Determination of detection range
Ricin was diluted to 100ng/mL, 80ng/mL, 40ng/mL, 10ng/mL, 1ng/mL with Tris-HCl buffer and each concentration was assayed 3 times. Sample treatment was used as a negative control. And adding 5 mu L H-AuNPs-FAM antibody mixture into 95 mu L of ricin with different concentrations, mixing and incubating for 120min, dripping 50 mu L of reaction liquid into a test strip sample area after the reaction is finished, standing for 10min to observe experimental results, and analyzing the results by using image J software.
Ricin was diluted to 100ng/mL, 80ng/mL, 40ng/mL, 10ng/mL and 1ng/mL with Tris-HCl buffer and detected with colloidal Jin Celiu chromatographic test strips. The results are shown in FIG. 5, where the sensitivity of direct visual inspection is 10ng/mL.
Standard Curve fitting
Based on the above results, analysis was performed by image J software, with the logarithm of the ricin concentration (100 ng/mL, 80ng/mL, 40ng/mL, 10ng/mL, 1 ng/mL) as the abscissa, the relative intensity detected at each concentration as the ordinate, the relative intensity representing the integrated density ratio of the detection line and the control line, and the error bars representing the standard deviation of the three measurements. As shown in fig. 6, the linear equation is y= -0.1563x+0.9521; r is R 2 = 0.98647. The linear relationship is good in the range of 1-100 ng/mL.
Specific detection
Detecting abrin, bovine Serum Albumin (BSA), ovalbumin (OVA), staphylococcus aureus enterotoxin B and fumonisin by using the prepared colloidal Jin Celiu chromatographic test strip under the same experimental conditions. Repeating for 3 times, and judging the specificity of the test strip.
Ovalbumin (OVA), fetal Bovine Serum Albumin (BSA), abrin, staphylococcus aureus enterotoxin B (SEB), fumonisin (FB) with Tris-HCl buffer 1 ) Diluted to 100ng/mL and detected by a gel Jin Celiu chromatographic test strip. As shown in FIG. 7, it is clear from FIG. 7 that only the color of the detection line of ricin was relatively light, while the bands of other proteins and toxins were not significantly changed from those of the control group.
Repeatability detection
Within the linear detection range, 3 concentrations (100 ng/mL, 60ng/mL, 20 ng/mL) were detected, medium, and low, each concentration was measured 6 times, the concentration was calculated as a fitted curve, and the repetition rate (CV) was calculated as the ratio of the variance (STDEVA) to the AVERAGE (AVERAGE). The results are shown in Table 2.
TABLE 2 repeated detection of ricin colloid Jin Celiu chromatography test strips
| Actual concentration (ng/mL) | AVERAGE(ng/mL) | STDEVA | CV(％) |
| 100 | 103.1609 | 17.52 | 16.98 |
| 60 | 65.391 | 7.185 | 10.99 |
| 20 | 22.446 | 2.73 | 12.17 |
The invention uses the oligonucleotide sequences of modified biotin and FAM as recognition probes to combine with colloidal gold particles of labeled FAM antibody; and (3) coating streptavidin on the nitrocellulose membrane as a detection line, coating a secondary antibody of an anti-FAM antibody as a control line, establishing a ricin naked eye detection method, evaluating the specificity and repeatability of the ricin naked eye detection method, performing data processing analysis by using image J software, and drawing a detection curve. The gel Jin Celiu chromatographic test strip can complete detection within 120min, and has the advantages of 1ng/mL detectable at the lowest, good specificity and repeatability. The invention establishes a colloidal Jin Celiu chromatography method for detecting ricin, and the method does not need to prepare corresponding antibodies, has simple operation and can meet the requirement of on-site detection.
The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described.
Sequence listing
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Claims (7)
1. A method for rapidly detecting ricin by a colloidal gold lateral flow chromatography, which is characterized by comprising the following steps:
(1) Incubating an H1-AuNPs-FAM antibody with a sample to be tested containing ricin; the sequence of H1 is SEQ ID NO:1 is shown in the specification; the particle size of AuNPs was 5nm; the concentration of H1 in the sample to be tested is 10nM; the incubation time is 120min;
(2) Adding the mixed liquid obtained in the step (1) to lateral flow chromatography test paper; the lateral flow chromatography test paper comprises a sample pad, a detection area, an absorption pad and an NC film with a plastic back card; the detection area comprises a T line combination area and a C line combination area; the T line bonding area is provided with a detection line, and the C line bonding area is provided with a control line; the secondary antibodies of the streptavidin and the anti-FAM antibody are respectively fixed on the detection line and the control line;
(3) Judging the detection result according to the color development values of the gold nano particles on the detection line and the control line.
2. A method for rapid detection of ricin by colloidal Jin Celiu chromatography according to claim 1, wherein the H1-AuNPs-FAM antibody is obtained by incubating H1 with an AuNPs-FAM antibody.
3. The method for rapid detection of ricin by colloidal Jin Celiu chromatography according to claim 1, wherein the means for interpreting the detection result in step (3) is visual observation or instrumental imaging.
4. A method for rapid detection of ricin by colloidal Jin Celiu chromatography according to claim 1, wherein the method further comprises the step of preparing a standard curve: according to the methods of the steps (1) and (2), a series of samples to be detected containing ricin with known concentration are detected, a plurality of color values are detected, and the concentration and the color values are respectively plotted as the horizontal and vertical coordinates to obtain a standard curve.
5. The method for rapid detection of ricin by colloidal Jin Celiu chromatography of claim 4, further comprising: and calculating the concentration of the ricin in the sample to be detected containing the ricin based on the standard curve.
6. A colloidal Jin Celiu chromatographic kit for rapidly detecting ricin, which comprises the following components:
(1) Colloid Jin Celiu chromatographic test paper; the test paper comprises: sample pad, detection area, absorption pad and NC film with plastic back card; the detection area comprises a T line combination area and a C line combination area; the T line bonding area is provided with a detection line, and the C line bonding area is provided with a control line; the secondary antibodies of the streptavidin and the anti-FAM antibody are respectively fixed on the detection line and the control line;
(2) FAM antibodies-AuNPs; wherein the particle size of the AuNPs is 5nm;
(3) H1 nucleic acid strand; the sequence of H1 is SEQ ID NO: 1.
7. The kit of claim 6, wherein FAM antibodies-AuNPs and H1 chains are provided in the form of H1-AuNPs-FAM antibodies.
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