CN112666349A - Liquid phase chip for detecting PRV antibody - Google Patents
Liquid phase chip for detecting PRV antibody Download PDFInfo
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Abstract
The invention discloses a liquid phase chip for detecting PRV antibodies, belonging to the technical field of biology. The liquid phase chip for detecting PRV comprises coupled magnetic beads coupled with PRV-gB protein, a biotin-labeled detection antibody and a reporter molecule. The liquid phase chip detection technology disclosed by the invention can detect a large number of samples at high flux, is sensitive, accurate and quick, and can meet the requirements of clinical quick diagnosis and large-scale quick detection of import and export quarantine. The method can be used for the epidemic investigation and the pathogen detection of PRV and the immune tracking monitoring of vaccines, and has important significance for the detection, the prevention and the control of pseudorabies.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a liquid phase chip for detecting PRV antibodies.
Background
Pseudorabies (Aujeszky's disease, AD), is a highly infectious acute viral infection caused by Pseudorabies virus (PRV) and characterized by the major pathogenic factors of neurological and respiratory diseases. Pseudorabies virus (PRV), also known as Aujeszky's virus (ADV) or porcine herpesvirus type 1 (SuHV 1), is a double-stranded DNA virus of the varicella herpesvirus genus (varicelovirus) belonging to the herpesviridae (herpesviridae) sub-family α -herpesviridae (alphaherpesviridae). The PRV genome is about 145-150Kbp in overall length, and the whole genome consists of 4 parts, namely a unique long segment (UL), a unique short segment (US), an internal repeat segment (IR) and a terminal repeat segment (TR). The PRV genome encodes 16 total envelope proteins, including 11 glycoproteins, gB, gC, gD, gE, gG, gH, gI, gK, gL, gM, gN, wherein gB, gD, gH and gL are glycoprotein genes essential for replication of all herpes viruses. The gB gene mainly forms the envelope structure of the pseudorabies virus, and the sequence is highly conserved. Mainly responsible for promoting fusion of the viral envelope with the host cell membrane, binding and activation into the host cell membrane by the receptors of the gH/gL complex, are essential for penetration of the virus into cells, intracellular release, and diffusion of the virus between cells.
Pigs are the only natural host for PRV and also the storage host for PRV, but PRV has a wide host range and is highly susceptible to most mammals, including ruminants, rodents, and carnivores, in addition to being non-susceptible to primates and humans, and pigs, cattle, sheep, goats, cats, dogs, etc. may be infected. PRV is extremely infectious, releasing large amounts of virus from the body secretions and excretions of infected pigs. PRV is transmitted primarily by direct contact, but may also be transmitted by air, contaminated water. PRV infection is characterized primarily by respiratory, reproductive and nervous system symptoms, and pigs of various ages are susceptible to infection, the characteristics of which depend on the age of the pig and the virulence of the strain. When PRV infects piglets, the piglets will have nervous system symptoms such as shivering, convulsion, limb discordance, paralysis and the like, the death rate is extremely high, and the piglets usually die within 24 to 36 hours after the disease attack; when a pregnant sow is infected with PRV, the symptoms of PRV infection may cause abortion or delivery of a dead and weak piglet in addition to neurological symptoms, and the weak piglet may also die shortly after birth; when PRV infects boars, the virus stays in semen for 12 days, and mating or insemination is carried out at the stage, so that fertilized sows are sick, and pregnancy is failed; when PRV infects older pigs, the symptoms of the infection are manifested primarily as respiratory symptoms such as coughing, sneezing, rhinitis and dyspnea, and a life-long incubation period is established in the peripheral nervous system of the pig to maintain the pig in a state of persistent infection, which in turn can reinfect other healthy pigs under certain conditions. Thus, PRV infection is a devastating disease of swine herds and causes enormous economic losses worldwide.
The pseudorabies is a virulent highly infectious viral infectious disease which has great harm to the global pig industry, the degree of the pseudorabies is second to foot-and-mouth disease and swine fever, the pseudorabies is listed as a B-type infectious disease by the world animal health organization, and the pseudorabies is one of necessary inspection items when animal products are imported and exported. Although several attenuated or inactivated live vaccines have been developed to protect pigs against PRV infection, there are still reports showing that PRV has an outbreak in domestic and wild pigs, and our understanding of the extent of PRV infection in wild populations and the extent of PRV threat to domestic pigs is still inadequate. At present, PRV epidemiology in China is quite complex, and in order to better prevent, control and detect porcine pseudorabies, it is very important to develop related research of porcine pseudorabies detection technology.
Laboratory diagnosis of viral infections mainly comprises virology detection and serology detection, wherein the serology detection can also be used for evaluating the vaccine immunization effect. Currently, the PRV serological detection technology mainly includes serum neutralization test (SN), immunoperoxidase monolayered assay (IPMA), indirect immunofluorescent antibody test (IFA), enzyme-linked immunosorbent assay (ELISA), and the like, wherein ELISA is widely used for detecting PRV antibodies. However, ELISA can not simultaneously detect a large amount of various pathogens, and has weak specificity, which is difficult to meet the requirement of high-throughput quarantine.
The liquid-phase chip technology (Multi-analysis profiling solution for unknown x, xMAP technology) is a novel detection technology for detecting pathogenic antibodies invented by Luminex company in 1995, is a novel high-throughput detection technology, integrates a laser technology, a digital signal processing technology, a flow cytometry technology and a traditional chemical technology, and has the advantages of simplicity in operation, wide application range, high throughput, multiple targets, high speed, good repeatability, high sensitivity, good specificity and the like. The analysis base of the liquid phase chip technology is that polystyrene microspheres marked with fluorescent dyes with different colors are provided, each fluorescent microsphere has a unique number, and the microspheres can be divided into 100 types according to different proportions of adding two different fluorescein. The microspheres are connected with carboxyl groups, and can be covalently coupled with proteins. The coupled microspheres can be used to capture specific antibodies in serum, and the microspheres can be incubated with a fluorescent secondary antibody to bind the captured serum antibodies later. After reaction, the single microsphere substrate passes through a detection channel, red fluorescence on the microsphere substrate and green fluorescence on the reporter molecule are simultaneously detected by red laser and green laser, the red laser is used for determining the color of the beads, and the green laser is used for detecting the fluorescence intensity of the combined secondary antibody through a detection system. The liquid-phase chip detection method is suitable for simultaneously and rapidly detecting various pathogens, is simple to operate, has high accuracy and is superior to the traditional method. At present, no report for detecting PRV specific antibody in serum by using antibody liquid phase chip technology exists.
Disclosure of Invention
The present invention is directed to a liquid phase chip for detecting PRV antibodies, which solves the above problems of the prior art.
In order to achieve the purpose, the invention provides the following scheme: the invention provides a liquid phase chip for detecting PRV antibody, which contains coupled magnetic beads, biotin-labeled detection antibody and reporter molecule; the coupled magnetic beads are coupled with PRV-gB protein.
Further, the detection antibody is a biotin-labeled secondary antibody.
Further, the reporter molecule is streptavidin marked by fluorescein.
Further, the streptavidin marked by the fluorescein is SA-PE.
Further, the preparation method of the coupled magnetic beads comprises the following steps: and adding PRV-gB protein after the blank magnetic beads are activated, and incubating overnight at 4 ℃ in a dark place to obtain the coupled magnetic beads.
Further, the blank magnetic bead activation specifically comprises the following steps: suspending blank magnetic beads by using microsphere cleaning fluid, activating in microsphere activating solution, adding carboxyl activating agent and protective agent for carboxyl activation, and oscillating and incubating at room temperature in a dark place.
Further, the carboxyl activating agent is EDC, and the protective agent is sulfo-NHS.
The invention discloses the following technical effects: according to the invention, a Luminex 200 liquid phase chip detection platform is utilized to couple PRV-gB protein with fluorescent microspheres, the amount of coupled protein is optimized, a threshold value determination test, a sensitivity test, a repeatability test, a specificity test and a consistency test are carried out, and a PRV single liquid phase chip detection method with perfect repeatability, high sensitivity and good specificity is successfully established. The PRV antibody liquid chip detection kit established by the invention can automatically detect a large number of samples at high flux, is sensitive, accurate and quick, and can meet the requirements of clinical quick diagnosis and large number of quick detections of import and export quarantine. The method can be used for the epidemic investigation and the pathogen detection of PRV and the immune tracking monitoring of vaccines, and has important significance for the detection, the prevention and the control of pseudorabies.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 is a graph of coupling efficiency verification;
FIG. 2 shows the median fluorescence intensity measurements of positive and negative sera;
FIG. 3 shows fluorescence values for different protein concentrations;
FIG. 4 shows the results of ELISA kit detection at different dilutions of serum;
FIG. 5 shows the results of liquid-phase chip detection of serum at different dilutions;
FIG. 6 shows the result of specificity test of the PRV antibody liquid phase chip detection method.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
Antigen and serum: prokaryotically expressed and purified Pseudorabies virus gB protein (PRV-gB) was provided by south beige customs, 32 Specific pathogen free pig sera (SPF pig serum) were provided by harbin veterinary institute; positive virus sera for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Vesicular Stomatitis Virus (VSV) are both provided by kunming customs; both positive virus serum of Pseudorabies virus (PRV) and 64 porcine serum samples were stored in the preventive veterinary focus laboratory of the university of celand agriculture.
Main reagents and consumables: Bio-Plex coupling kit, Bio-Plex carboxyl magnetic beads, 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1- (3-dimethylamino propyl) -3-ethylcarbodiimide hydrochloride, EDC) and N-hydroxythiosuccinimide (N-hydroxythiosuccinimide sodium salt, sulfo-NHS), Bio-Plex detection reaction kit (Bio-Rad, Shanghai) were purchased from Berle biomedical products (Shanghai) Limited; biotinylated anti-His Antibody (6X-His Tag Monoclonal Antibody, Biotin) was purchased from Invitrogen (USA); biotin-labeled secondary antibodies (coat Anti-Pig IgG H & L, Biotin) (Abcam, Shanghai) were purchased from Ebos (Shanghai) trade company, Inc.; PRV ELISA detection kits were purchased from Idex (IDEXX) biotechnology limited; the Bradford protein concentration determination kit was purchased from Beijing Tiangen Biotech, Inc.
Example 1 microsphere activation and protein coupling
1.1 microsphere activation and protein coupling
100. mu.L of microspheres (about 1.25X 10)6One) is transferred into a coupling reaction tube, mixed evenly and counted by a blood counting chamber, and the actual measurement initial concentration of the microspheres is recorded. The microspheres were placed on a magnetic separation rack and left for 30s, then centrifuged to remove the supernatant, and then 100. mu.L of microsphere cleaning solution was added and the microspheres were suspended in 80. mu.L of microsphere activation solution. Then 10. mu.L of 50mg/mL EDC was added, followed by the rapid addition of 10. mu.L of 50mg/mL sulfo-NHS. The coupling reaction tube was incubated for 20min at room temperature with shaking in the dark. Two separate washes of PBS were added and the microspheres resuspended. The concentration of PRV-gB protein was quantified using the Bradford protein concentration assay kit, and then the protein was coupled to activated fluorescent microspheres. PRV-gB protein was added, the volume was adjusted to 500. mu.L with PBS, and the coupled reaction tube was incubated overnight at 4 ℃ in the absence of light. The coupled microspheres were centrifuged and washed with PBS, and then added with blocking buffer, spun at room temperature in the dark, and blocked for 30 min. After washing, the microspheres were resuspended in 150. mu.L of storage buffer, and the coupling yield was calculated by counting with a hemocytometer. The initial concentration of the microspheres was found to be 6.7X 106Per ml, microsphere concentration after coupling was 5.9X 106And/ml, coupling yield is 88.06%, sealing the microsphere sealing film, packaging with aluminum foil in a dark place, and storing at 4 ℃.
1.2 coupling efficiency verification
Labeling 2 centrifuge tubes, one for negative control, approximately 1X 10 addition per tube4A coupled microsphere. The test tubes were added with diluted biotin-labeled anti-His antibody and the negative control tubes were added with the same volume of PBS. Incubate at room temperature in dark for 10 min. Fully dispersing the microspheres and then placing the microspheres in a magnetic separatorStanding off the rack to completely precipitate the microspheres and removing the supernatant. Diluted PE-labeled Streptavidin (Streptavidin-PE, SA-PE) was added to the test tube, the same volume of assay buffer (assay buffer) was added to the negative control tube, and incubation was carried out for 10min at room temperature in the dark. After being fully dispersed, the microspheres are placed on a magnetic separation frame for standing, and after the microspheres are completely precipitated, the supernatant is removed. The microspheres were resuspended in 125. mu.L of microsphere storage buffer and transferred to a 96-well plate and sent to the instrument for detection.
The fluorescence value of the negative control (background) must not exceed 100 MFI; the coupling was considered successful if the fluorescence value of the coupled microspheres exceeded 2000 MFI. As shown in fig. 1, the average fluorescence value of the Negative Control (NC) was 20.5, less than 100; the mean fluorescence value of the test wells (PRV) was 9365.5, which was greater than 2000, demonstrating successful coupling of PRV-gB protein to the microspheres.
Example 2 establishment of PRV antibody liquid phase chip detection method
The microspheres successfully coupled in example 1 were diluted to 1X 105Per ml, 50. mu.L of diluted microspheres (5000 per well) were added per well. The 96-well plate is placed on a magnetic plate and kept stand, and the supernatant is thrown off after the microspheres are completely precipitated. The microspheres were washed, 50. mu.L of negative and positive serum was added to each well of the sample well, and 50. mu.L of assay buffer was added to the background control well. Sealing the sealing plate in dark and incubating for 1h at room temperature with shaking. After incubation, the 96-well plate was placed on a magnetic plate and allowed to stand, allowing the microspheres to completely precipitate, and then the supernatant was spun off. Washing the microspheres, adding 50 μ L of biotin-labeled detection antibody (biotin-labeled secondary antibody) 1ug/ml into each well, sealing the sealing plate in dark, and incubating for 30-45min with shaking at room temperature. After incubation, the 96-well plate was placed on a magnetic plate and allowed to stand for 1min to allow the microspheres to completely precipitate and the supernatant was discarded. Washing the microspheres, adding 50 μ L of 1X SA-PE into each well, sealing and pasting a sealing plate in a dark place, and performing shaking incubation at room temperature for 10-15 min. After incubation, the 96-well plate was placed on a magnetic plate and allowed to stand for 1min to allow the microspheres to completely precipitate and the supernatant was discarded. Washing the microspheres, adding 125 μ L of assay buffer resuspension microspheres into each well, shaking and mixing uniformly, and sending into an instrument (Luminex 200) for detection.
And (4) result judgment standard: the mean of the fluorescence values of the negative controls plus a Standard Deviation (SD) of 3 times gave a value above which a positive result was judged and below which a negative result was judged.
As shown in table 1 and fig. 2, the average value of the fluorescence intensity of the Background Control (BC) was 45, the average value of the fluorescence intensity of the negative serum (NC) was 41, the standard deviation of the fluorescence intensity of the negative control was 2.7, and the result of adding 3 times the standard deviation SD to the average value of the fluorescence values of the negative serum was 49.1. The results of the positive serum and the negative serum are judged to be correct and are consistent with the expectation. The PRV single liquid phase chip detection method is successfully established initially.
TABLE 1 median fluorescence intensity measurements of positive and negative sera
Example 3 determination of the concentration of protein optimally bound by the PRV antibody liquid phase chip detection method
By testing 4 different concentrations (6,8,10, 12. mu.g) of PRV-gB protein with 1.25X 106Coupling each microsphere, and determining the optimal combined protein concentration detected by the single liquid phase chip. As shown in Table 2 and FIG. 3, the microspheres were successfully coupled at a protein concentration of 6. mu.g, but the fluorescence intensity was close to the critical value (2000). When the coupled protein concentration is 8,10 and 12 mug, the coupling of the microspheres is successful, the coupling efficiency is high, when the coupled protein concentration is 8,10 and 12 mug, the fluorescence intensity difference is not large, the coupled protein of the microspheres is close to saturation, and the protein usage amount is saved on the premise of high coupling efficiency, so that the optimal binding protein concentration is determined to be 8 mug/1.25 multiplied by 106And (3) microspheres.
TABLE 2 fluorescence values for different protein concentrations
Example 4 determination of threshold value of PRV antibody liquid phase chip detection method
The threshold is defined as the sum of the mean plus 3 times the standard deviation, also called the cutoff. To determine the threshold for the single liquid phase chip detection method of PRV, 32 SPF pig serum samples were diluted 1:100 with sample diluent (sample diluent) and the mean MFI values and standard deviations were obtained following the procedure described above in example 2. As can be derived from table 3, the threshold for PRV detection is 164.8. In the subsequent test and detection of detecting specific antibody in serum by using the single liquid phase chip detection method of PRV, the sample can be effectively identified by using the threshold value, and the sample can be judged to be positive if the threshold value is exceeded.
TABLE 3 threshold values for PRRSV antibody detection
Example 5 reproducibility test of antibody liquid-phase chip detection method
In order to prove the repeatability of the detection method, a PRV liquid phase chip detection method is used for detecting samples, and repeated tests within 3 batches and repeated tests among 3 batches are respectively carried out. The results are shown in Table 4, where the coefficient of variation for the intra-batch repeat was 4.80% maximum and the coefficient of variation for the inter-batch repeat was 7.85%, both less than 10%, indicating that the test has high reproducibility.
TABLE 4 repeatability tests
Example 6 sensitivity test
The sensitivity of the ELISA and the liquid phase chip is compared by using the ELISA kit and the established PRV single heavy liquid phase chip detection method to detect the positive serum with the dilution of 1:1 to 1: 1000.
The ELISA kit is used for detecting negative and positive serum with different dilution ratios, and the positive serum can be judged to be positive by calculating the detection result to be less than or equal to 0.7891. FIG. 4 shows the ELISA detection results of different dilutions of serum, and it can be seen from FIG. 4 that the undiluted positive serum and 10-fold diluted positive serum can be detected by using the ELISA kit as positive results, and the positive sera in the rest dilution ratios are not detected as positive.
The coupled microspheres are used for detecting the positive and negative serum with the same dilution ratio by using a liquid phase chip, and the positive serum can be judged by calculating that the detection result is greater than a critical value 167. FIG. 5 shows the detection results of liquid-phase chip under different dilutions of serum, and it can be seen from FIG. 5 that the positive serum diluted 1000 times can still be detected by the liquid-phase chip detection method, which proves that the liquid-phase chip detection method is more sensitive than ELISA.
Example 7 specificity test of PRV antibody liquid phase chip detection method
In order to verify the specificity of the PRV antibody liquid phase chip detection method established by the invention, the detection method is used for detecting the positive serum of PRV, PRRSV and VSV, the fluorescence intensity among the three is plotted and compared, and the result is shown in figure 6. As can be seen from FIG. 6, the PRV detection result is positive, the PRRSV and VSV detection results are negative, and the difference of fluorescence values is significant, which indicates that the kit has no cross reaction with the serum of PRSRV and VSV and has better specificity.
Example 8 consistency test of PRV antibody liquid phase chip detection method
In order to verify the consistency and the conformity of the detection samples of the established PRV antibody liquid chip detection method and the commercial PRV ELISA kit, 64 serum samples are detected by using the ELISA kit and the liquid chip detection method respectively, so that the consistency and the conformity of the detection results of the two are verified. As shown in Table 5, the number of positive samples accounted for 76.56% of the total samples, and the number of negative samples accounted for 14.06% of the total samples. Therefore, the number of samples with consistent results of the two detection methods is 58, which accounts for 90.63% of the total samples, namely the samples are 90.63% consistent, which indicates that the PRV antibody liquid phase chip detection method and the ELISA detection method established by the test have good consistency.
TABLE 6 comparison of the identity of xMAP and ELISA
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (7)
1. A liquid phase chip for detecting PRV antibody is characterized in that the liquid phase chip contains coupled magnetic beads, biotin-labeled detection antibody and reporter molecule; the coupled magnetic beads are coupled with PRV-gB protein.
2. The liquid phase chip of claim 1, wherein: the biotin-labeled detection antibody is a biotin-labeled secondary antibody.
3. The liquid phase chip of claim 1, wherein: the reporter molecule is streptavidin marked by fluorescein.
4. The liquid phase chip of claim 3, wherein: the streptavidin marked by the fluorescein is SA-PE.
5. The liquid phase chip of claim 1, wherein the coupled magnetic beads are prepared by: and adding PRV-gB protein after the blank magnetic beads are activated, and incubating overnight at 4 ℃ in a dark place to obtain the coupled magnetic beads.
6. The liquid phase chip of claim 5, wherein the blank magnetic beads are activated by the following specific steps: suspending blank magnetic beads by using microsphere cleaning fluid, activating in microsphere activating solution, adding carboxyl activating agent and protective agent for carboxyl activation, and oscillating and incubating at room temperature in a dark place.
7. The liquid phase chip of claim 6, wherein: the carboxyl activating agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the protective agent is N-hydroxy thiosuccinimide.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106290867A (en) * | 2016-08-17 | 2017-01-04 | 广东省农业科学院动物卫生研究所 | The liquid-phase chip of five kinds of fowl vertical transmission disease antibodies of a kind of detection simultaneously and method |
| CN108303530A (en) * | 2017-01-11 | 2018-07-20 | 上海鸣捷生物科技有限公司 | A kind of pseudorabies gB antibody assay kits and its detection method |
| US20180256705A1 (en) * | 2015-06-29 | 2018-09-13 | Pulike Biological Engineering, Inc. | Subunit vaccine of porcine pseudorabies virus and preparation method |
| CN109307772A (en) * | 2018-10-12 | 2019-02-05 | 华南农业大学 | A kind of Pseudorabies virus gE and gB IgG antibody double fluorescent microballoon immunological detection method |
| AU2020102093A4 (en) * | 2020-09-01 | 2020-10-08 | Chinese Academy Of Inspection And Quarantine | A test kit for simultaneously detecting ten insect-borne disease pathogens and application thereof |
-
2020
- 2020-12-01 CN CN202011385385.XA patent/CN112666349A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180256705A1 (en) * | 2015-06-29 | 2018-09-13 | Pulike Biological Engineering, Inc. | Subunit vaccine of porcine pseudorabies virus and preparation method |
| CN106290867A (en) * | 2016-08-17 | 2017-01-04 | 广东省农业科学院动物卫生研究所 | The liquid-phase chip of five kinds of fowl vertical transmission disease antibodies of a kind of detection simultaneously and method |
| CN108303530A (en) * | 2017-01-11 | 2018-07-20 | 上海鸣捷生物科技有限公司 | A kind of pseudorabies gB antibody assay kits and its detection method |
| CN109307772A (en) * | 2018-10-12 | 2019-02-05 | 华南农业大学 | A kind of Pseudorabies virus gE and gB IgG antibody double fluorescent microballoon immunological detection method |
| AU2020102093A4 (en) * | 2020-09-01 | 2020-10-08 | Chinese Academy Of Inspection And Quarantine | A test kit for simultaneously detecting ten insect-borne disease pathogens and application thereof |
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