CN105785024A - Aptamer-based enzyme-linked immunosorbent assay method for detecting iridovirus infection of groupers - Google Patents
Aptamer-based enzyme-linked immunosorbent assay method for detecting iridovirus infection of groupers Download PDFInfo
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Abstract
The invention discloses an aptamer-based enzyme-linked immunosorbent assay method for detecting iridovirus infection of groupers. The method comprises the following steps: incubating and combining a biotin-labeled aptamer and to-be-detected cells or tissues; cleaning a to-be-detected sample, adding horse radish peroxidase-labeled streptavidin, cleaning the to-be-detected sample after incubation completion, adding a TMB developing solution in a horse radish peroxidase developing kit for carrying out a developing reaction, and determining whether the to-be-detected cell or tissue sample is infected by grouper iridovirus according to light absorption value change. Compared with the existing antibody-based ELISA technology, the aptamer-based ELISA technology disclosed by the invention has the advantages that the method is high in specificity and sensitivity and convenient to operate, is suitable for large-scale rapid detection of iridovirus infection of cultured fishes in aquaculture farms and has good application prospects in the detection field of iridovirus infection.
Description
Technical field:
The invention belongs to grouper irido virus detection field, be specifically related to a kind of method that Enzyme-linked Immunosorbent Assay technology for detection grouper irido virus based on aptamer infects.
Background technology:
Cabrilla is one of coastal areas of southern China each province and the famousst and precious marine fish of Southeast Asian countries, and economic worth is high.But break out in cabrilla in recent years and seriously threaten the sea-farming of cabrilla with popular grouper irido virus (Singaporegrouperiridovirus, SGIV), cause huge economic loss.Cabrilla is after being infected by SGIV, it may appear that the histoorgan necrosis enlargements such as black matrix, liver spleen kidney, ultimately results in fish body dead.For the cabrilla of high-density breeding, it is possible to be used for quickly detecting grouper irido virus infects early stage and technical method easy and simple to handle, for controlling SGIV infection, minimizing is lost most important.Method for grouper irido virus diagnosis mainly includes based on virusology, histopathologic traditional method at present, based on the immunological detection method of antibody, and Protocols in Molecular Biology etc..Although the method that these detections SGIV infects possesses the feature of hypersensitivity, high accuracy, but owing to reagent and experimental apparatus are expensive, need professional to operate and detecting step is loaded down with trivial details, the shortcomings such as length consuming time and reagent preservation condition harshness, cause the detection that these methods are only applicable under laboratory condition.Therefore need the high specific of Development of Novel badly, high sensitivity, technology easy and simple to handle are monitored and are controlled grouper irido virus and infect.
Enzyme-linked Immunosorbent Assay technology (enzyme-linkedimmunosorbentassay, ELISA) is now widely used for harmful microbe detection.But traditional ELISA detection technique based on antibody has some shortcomings, such as antibody need length consuming time prepared by strict cryopreservation condition and antibody, costly, batch between quality there are differences, use first to resist with also want tape label after an anti-binding two during ELISA detection and hatch, cause complex operation length consuming time.Therefore, the substitute finding antibody the operating process simplifying detection technique become the main path optimizing elisa technique.Aglucon phyletic evolution technology (systematicevolutionofligandsbyexponentialenrichment, SELEX) of index concentration is the biological libraries technology of the extensively concerned novel detection of a class and treatment.Utilize the aptamer that SELEX technology screening obtains, i.e. single stranded DNA (ssDNA) or RNA, there is the high specific suitable with monoclonal antibody and affinity, facilitate storage and transport, the degeneration that variations in temperature causes is reversible, the short expense of screening time is low, it is possible to reuse, and the aptamer obtained is readily synthesized in the later stage and modifies.In work previously, we, based on index concentration aglucon evolution technology, screen with the cabrilla splenocyte infected by irido virus for target, it is thus achieved that can high specific identify iris SGIV infect aptamer.
Summary of the invention:
In order to overcome the existing deficiency based on antibody, detection viral infection Enzyme-linked Immunosorbent Assay technology, it is an object of the invention to provide a kind of high specific, high sensitivity, simple operation, detect, suitable in the Enzyme-linked Immunosorbent Assay technology (Aptamer-basedenzyme-linkedapta-sorbentassays, Apt-ELISA) based on grouper irido virus aptamer used plant, the method that grouper irido virus infects.
The method that the Enzyme-linked Immunosorbent Assay technology for detection grouper irido virus based on aptamer of the present invention infects, it is characterised in that comprise the following steps:
(1), biotin labeled for any one shown in SEQIDNO.1～3 grouper irido virus aptamer and testing sample are combined, then clean；
(2), the sample combining aptamer in step (1) is centrifuged, removes supernatant, the sample being enriched to is combined with horseradish peroxidase-Streptavidin (HRP-streptavidin) mixing；
(3), the sample combining horseradish peroxidase-Streptavidin that obtains of step (2) be carried out, then move in ELISA Plate, every hole adds the reaction of TMB nitrite ion lucifuge, is eventually adding stop buffer and terminates reaction；
(4), detect the testing sample light absorption value at 450nm place, come whether judgement sample room is infected by grouper irido virus according to the change of light absorption value.
Preferably, the biotin labeled grouper irido virus aptamer of step (1) first carries out renaturation before being combined with testing sample, particularly as follows: by biotin labeled aptamer at 94 DEG C of high-temperature denatured 10min, be then rapidly inserted into renaturation 10min in ice.
Aptamer described in step (1) is combined with testing sample, it is preferable that the aptamer of 200nM and the testing sample of 200 μ l combine.
Combination described in step (1), it is preferable that binding time is 1～30min, is 4～37 DEG C in conjunction with temperature.
Cleaning described in step (1), it is preferable that use PBS 3 times.
Horseradish peroxidase-Streptavidin described in step (2), it is preferable that horseradish peroxidase: the volume ratio of Streptavidin is 1:10000, addition is 200 μ l, and binding time is 15min.
Cleaning described in step (3), it is preferable that use PBS 5 times；Described ELISA Plate, it is preferred to 96 hole ELISA Plate；Described TMB nitrite ion lucifuge reaction, it is preferable that add 100 μ l lucifuge reaction 10min；Described addition stop buffer, it is preferable that add the H of 50 μ l2M2SO4。
The present invention adopts and quickly detects grouper irido virus based on three biotin labeled aptamers as the elisa technique of specific detection probe and infect.After cell or tissue sample clean to be measured, combination is hatched with biotin labeled aptamer, then after testing sample being cleaned, add the Streptavidin (HRP-streptavidin) of horseradish peroxidase-labeled, testing sample is cleaned after hatching combination, add the colour developing of TMB nitrite ion, the infection according to the change-detection grouper irido virus of light absorption value in horseradish peroxidase colour reagent box.Apt-ELISA stability is high, when temperature up to 37 DEG C when, still can be accurately detected the infection of irido virus；When Apt-ELISA amplifying nucleic acid aptamers and virus infected cell binding time are as short as 1min, what ELISA still can be sensitive detects the infection of cabrilla irisation virus.Apt-ELISA can be applicable to exploitation grouper irido virus and infects quick detection kit, it is adaptable to the high-volume whether infecting grouper irido virus plant Mesichthyes quickly detects, and has that short, easy and simple to handle, stability consuming time is strong, sensitivity advantages of higher.
Traditional elisa technique based on antibody has some shortcomings, length consuming time prepared by such as antibody, costly, batch between quality there are differences, the antibody prepared needs strict cryopreservation condition, use first to resist with also want tape label after an anti-binding two during ELISA detection and hatch, cause complex operation length consuming time.With existing based on compared with the elisa technique of antibody, in the present invention, the advantage of elisa technique based on aptamer is in that: high specificity, highly sensitive, simple operation, the high-volume that infects suitable in cultured fishes irido virus plant quickly detect, and the detection field infected at irido virus has good application prospect.
Accompanying drawing illustrates:
Fig. 1 is the specificity analyses of Apt-ELISA technology for detection SGIV infection cell in the embodiment of the present invention.Matched group arranges four groups: Con1, the Cell binding that biotin labeled aptamer infects with nervous necrosis virus；Con2, biotin labeled aptamer and the STIV Cell binding infected；Con3, biotin labeled aptamer is combined with normal cell；Con4, does not make the normal thin of any process；
In Fig. 2 embodiment of the present invention, Apt-ELISA detects the analysis hatching binding time needed for SGIV infects.Biotin labeled aptamer compares with Normocellular being combined as；
Fig. 3 is that in the embodiment of the present invention, Apt-ELISA detects the SGIV stability analysis infected, the SGIV cell infected is hatched in conjunction with 30min with biotin labeled aptamer in Apt-ELISA, is respectively set as 4 DEG C in conjunction with temperature, 28 DEG C, 37 DEG C, 45 DEG C and 55 DEG C；
Fig. 4 is the detection analysis of the Apt-ELISA cabrilla tissue to infecting SGIV in the embodiment of the present invention, and biotin labeled aptamer compares with being combined as of normal structure.
Detailed description of the invention:
Following example are further illustrating the present invention, rather than limitation of the present invention.Experimental technique in following example if no special instructions, is conventional method.
Embodiment 1:
Synthesizing three and detect the aptamer (being synthesized by ThermoFisherScientific) that grouper irido virus infects for ELISA, sequence is as follows:
Sequence 1:
5’-GACGCTTACTCAGGTGTGACTCGTATGTCCATGGCCGCATATTGGGAAGGTTGGTTTGGACTATGTGGAAGTTCGAAGGACGCAGATGAAGTCTC-3’
Sequence 2:
5’-GACGCTTACTCAGGTGTGACTCGTTTCGTGTTATGCTCCTCTTTATTGTCAGCTGAGTTTCTGCAGTGCGAAGGACGCAGATGAAGTCTC-3’
Sequence 3:
5’-GACGCTTACTCAGGTGTGACTCGTATTCGGGTTATTGCTCCTCTTTATTGTCACCTGGATGTATGATCGTGTAGCGAAGGACGCAGATGAAGTCTC-3’
Above-mentioned aptamer is at 5 ' ends or 3 ' terminal modified biotin labelings.
Based on the method that the Enzyme-linked Immunosorbent Assay technology for detection grouper irido virus of aptamer infects, comprise the following steps:
1, the renaturation of aptamer: biotin labeled aptamer, at 94 DEG C of high-temperature denatured 10min, is then rapidly inserted into renaturation 10min in ice；
2, by any one biotin labeled grouper irido virus aptamer 200nM of the sequence shown in above-mentioned with 200 μ l testing samples on ice in conjunction with 30min, then use PBS 3 times；
3, after the sample 1000 × g combining aptamer being centrifuged 3min, remove supernatant, the sample being enriched to is mixed in conjunction with 15min at the horseradish peroxidase-Streptavidin (streptavidin-HRP) (horseradish peroxidase: the volume ratio of Streptavidin is 1:10000) of 200 μ l；
4, will after combining the sample PBS 5 times of horseradish peroxidase-Streptavidin, being moved into by sample in 96 hole ELISA Plate, every hole adds 100 μ lTMB nitrite ion lucifuges reaction 10min, ultimately joins the H of 50 μ l2M2SO4Terminate reaction；
5, use microplate reader read the testing sample light absorption value at 450nm place and record result, come whether judgement sample is infected by grouper irido virus according to the change of light absorption value.
Apt-ELISA technology can recognition detection SGIV infection cell specifically
To infect the cabrilla spleen cell (GS cell) of SGIV with without the process of enzymic digestion liquid, 1000g is centrifuged off supernatant, with 5mLPBS centrifuge washing three times.Biotin labeled for 200nM aptamer is hatched in conjunction with 30min with the cell after above-mentioned process on ice, 1000g is centrifugal removes supernatant, with 5mLPBS centrifuge washing three times, after the centrifugal 3min of 1000 × g, remove supernatant, by streptavidin-HRP (1:10 at 200 μ l of the sample that is enriched to, 000) mixing is in conjunction with 15min, after PBS 5 times, is moved into by sample in 96 hole ELISA Plate, every hole adds 100 μ lTMB nitrite ion lucifuge reaction 10min, ultimately joins 50 μ l2MH2SO4Terminate reaction；Use microplate reader read the testing sample light absorption value at 450nm place and record result, come whether judgement sample is infected by grouper irido virus according to the change of light absorption value.Matched group arranges four groups: Con1, the Cell binding that biotin labeled aptamer infects with nervous necrosis virus；Con2, biotin labeled aptamer and the STIV Cell binding infected；Con3, biotin labeled aptamer is combined with normal cell；Con4, does not make the normal cell of any process.Testing result is as it is shown in figure 1, should as a result, it was confirmed that Apt-ELISA can the infection of specific recognition SGIV.
Apt-ELISA can be quickly detected the infection of SGIV
By the SGIV GS cell infected with biotin labeled aptamers (200nM) on ice respectively in connection with 30,20,10,5,2 and 1min；Follow-up concrete detection operation is referring to the operational approach of above-mentioned enzyme linked immunosorbent detection technology (Apt-ELISA)；Microplate reader is used to read the testing sample light absorption value at 450nm place and record result；Biotin labeled aptamer compares with Normocellular being combined as.Testing result as in figure 2 it is shown, should as a result, it was confirmed that when hatch be as short as 1min in conjunction with duration time, in Apt-ELISA, each sequence can detect that the infection of SGIV, and namely Apt-ELISA can be quickly detected the infection of SGIV.
Apt-ELISA detection technique has high stability
The SGIV GS cell infected is hatched in conjunction with 30min with biotin labeled aptamers (200nM), is respectively set as 4 DEG C in conjunction with temperature, 28 DEG C, 37 DEG C, 45 DEG C and 55 DEG C；Follow-up concrete detection operation is referring to the operational approach of above-mentioned enzyme linked immunosorbent detection technology (Apt-ELISA)；Microplate reader is used to read the testing sample light absorption value at 450nm place and record result；Biotin labeled aptamer and Normocellular being combined as and compare at the corresponding temperature.Testing result is as it is shown on figure 3, detection temperature is when 4 DEG C, and the Detection results of ELISA is best, and along with the rising of temperature, Detection results is gradually lowered, and when temperature is up to 37 DEG C, ELISA detection technique still may clearly detect the infection of irido virus.This result confirms that Apt-ELISA detection technique has high stability.
Apt-ELISA can detect the infection of SGIV in tissue
Filter with 100 mesh filter screens after the liver organization blade having infected SGIV is shredded and collect filtrate, with PBS 3 times；1000 × g is centrifuged 3min, collects cell；Cell and biotin labeled aptamers (200nM) are hatched in conjunction with 30min on ice；Follow-up concrete detection operation is referring to the operational approach of above-mentioned enzyme linked immunosorbent detection technology (Apt-ELISA)；Microplate reader is used to read the testing sample light absorption value at 450nm place and record result；Biotin labeled aptamer compares with being combined as of normal tissue cell.Testing result as shown in Figure 4, should as a result, it was confirmed that Apt-ELISA can detect the infection of SGIV in tissue sample.
Claims (7)
1. the method that the Enzyme-linked Immunosorbent Assay technology for detection grouper irido virus based on aptamer infects, it is characterised in that comprise the following steps:
(1), biotin labeled for any one shown in SEQIDNO.1～3 grouper irido virus aptamer and testing sample are combined, then clean；
(2), the sample combining aptamer in step (1) is centrifuged, removes supernatant, the sample being enriched to is combined with horseradish peroxidase-Streptavidin mixing；
(3), the sample combining horseradish peroxidase-Streptavidin that obtains of step (2) be carried out, then move in ELISA Plate, every hole adds the reaction of TMB nitrite ion lucifuge, is eventually adding stop buffer and terminates reaction；
(4), detect the testing sample light absorption value at 450nm place, come whether judgement sample room is infected by grouper irido virus according to the change of light absorption value.
2. the method that the Enzyme-linked Immunosorbent Assay technology for detection grouper irido virus based on aptamer according to claim 1 infects, it is characterized in that, the biotin labeled grouper irido virus aptamer of step (1) first carries out renaturation before being combined with testing sample, particularly as follows: by biotin labeled aptamer at 94 DEG C of high-temperature denatured 10min, be then rapidly inserted into renaturation 10min in ice.
3. the method that the Enzyme-linked Immunosorbent Assay technology for detection grouper irido virus based on aptamer according to claim 1 infects, it is characterized in that, aptamer described in step (1) is combined with testing sample, combines for the aptamer of 200nM and the testing sample of 200 μ l.
4. the method that the Enzyme-linked Immunosorbent Assay technology for detection grouper irido virus based on aptamer according to claim 1 infects, it is characterised in that the combination described in step (1), binding time is 1～30min, is 4～37 DEG C in conjunction with temperature.
5. the method that the Enzyme-linked Immunosorbent Assay technology for detection grouper irido virus based on aptamer according to claim 1 infects, it is characterised in that the cleaning described in step (1), is use PBS 3 times.
6. the method that the Enzyme-linked Immunosorbent Assay technology for detection grouper irido virus based on aptamer according to claim 1 infects, it is characterized in that, horseradish peroxidase described in step (2)-Streptavidin mixing combines, horseradish peroxidase: the volume ratio of Streptavidin is 1:10000, addition is 200 μ l, and binding time is 15min.
7. the method that the Enzyme-linked Immunosorbent Assay technology for detection grouper irido virus based on aptamer according to claim 1 infects, it is characterised in that the cleaning described in step (3), is use PBS 5 times；Described ELISA Plate is 96 hole ELISA Plate；Described TMB nitrite ion lucifuge reaction, for adding 100 μ l lucifuge reaction 10min；Described addition stop buffer, for adding the H of 50 μ l2M2SO4。
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| CN110618259A (en) * | 2019-09-17 | 2019-12-27 | 华南农业大学 | Colloidal gold test strip for detecting grouper iridovirus and preparation and detection methods thereof |
| CN111073892A (en) * | 2019-10-30 | 2020-04-28 | 广西科学院 | Aptamer for identifying grouper iridovirus infected cells and construction method and application thereof |
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| CN111363748A (en) * | 2020-02-07 | 2020-07-03 | 广西科学院 | Aptamer, construction method thereof and application thereof in detection of Chinese softshell turtle rainbow virus |
| CN111363748B (en) * | 2020-02-07 | 2022-12-20 | 广西科学院 | Aptamer, construction method thereof and application thereof in detection of Chinese softshell turtle rainbow virus |
| CN111363749B (en) * | 2020-02-07 | 2022-12-20 | 广西科学院 | Nucleic acid aptamer for detecting Chinese softshell turtle iridovirus as well as construction method and application thereof |
| CN114807149A (en) * | 2022-03-21 | 2022-07-29 | 华南农业大学 | Nucleic acid aptamer for specifically recognizing iridovirus of micropterus salmoides and application of nucleic acid aptamer |
| CN114807149B (en) * | 2022-03-21 | 2023-11-17 | 华南农业大学 | Nucleic acid aptamer for specifically recognizing largemouth black bass iridovirus and application thereof |
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Application publication date: 20160720 |