Specific probe for detecting hemocyanin gene expression of eriocheir sinensis and application of specific probe
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A technology of Chinese mitten crab and hemocyanin, which is applied in the field of detection and can solve problems affecting research and development
Pending Publication Date: 2022-07-01
TIANJIN NORMAL UNIVERSITY
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Problems solved by technology
For a long time, the research on immune cells of aquatic animals has mainly relied on manual methods such as staining and microscopic examination. The technical bottleneck of high-throughput automated classification and analysis has also greatly affected the development of related research.
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Embodiment 1
[0048] Effects of injection of Aeromonas hydrophila on blood cells of Eriocheir sinensis
[0049] Materials and Methods
[0050] Experimental Materials
[0051] Chinese mitten crab, weighing 50±20g. Aeromonas hydrophila was isolated and preserved by our laboratory. Reagent consumables and probes were purchased from related companies with reference to the instructions of the present invention. The probe sequence is: 5'-GAAGATGTTATCCATGTACTTGTGGAG-3', which is fluorescently labeled with 5-terminal FAM and purified by HPLC. The composition and concentration of anticoagulant were 338 mmol / L sodium chloride, 115 mmol / L glucose, 30 mmol / L trisodium citrate, 10 mmol / L disodium EDTA, pH=7.0, Carnot's fix The ratio of methanol to glacial acetic acid is 3:1, which should be prepared before use.
[0052] experimental method
[0053] Chinese mitten crab individuals with no injury on the body surface and normal vitality were selected and divided into control group and experimental gr...
Embodiment 2
[0064] Rapid Analysis of Effects of Ammonia Nitrogen Stress on Blood Cells of Eriocheir sinensis
[0065] Materials and Methods
[0066] Experimental Materials
[0067] Chinese mitten crab, weighing 50±20g. The ammonia nitrogen concentration is calculated according to the NH4+ concentration, and the stimulation concentration used in this example is 5 mg / L. Reagent consumables and fluorescent probes were purchased from related companies with reference to the instructions. The probe sequence was: 5'-GAAGATGTTATCCATGTACTTTGGGAG-3', which was labeled with 5-terminal FAM fluorescently and purified by HPLC. Anticoagulant component 338mmol / L sodium chloride, 115 mmol / L glucose, 30 mmol / L trisodium citrate, 10 mmol / L disodium EDTA, pH=7.0, Carnot's fixative component methanol The ratio to glacial acetic acid is 3:1, and it is prepared before use.
[0068] experimental method
[0069] The mitten crab individuals with no injury on the body surface and normal vitality were selected...
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Abstract
The invention discloses a specific probe for detecting hemocyanin gene expression of Eriocheir sinensis and application of the specific probe. Based on the principle that a probe sequence is specifically combined with mRNA of a target gene in proportion, a fluorescence in-situ hybridization technology is combined with image flow cytometry, different groups are distinguished according to whether fluorescence signals exist in cells or not, functional differences are analyzed according to signal strength, and the target gene can be identified according to the functional differences. And whether different genes are expressed in the same cell or not can be known by using multicolor fluorescence co-localization. Cell classification serves for diagnosis, function research and other requirements. Conventional methods mostly adopt antibody recognition to classify cells, but the current situation that aquatic animals lack commercially available antibodies limits the application. The method does not need an antibody, starts from a gene sequence, prepares a fluorescently-labeled short probe, is mild in hybridization condition, can carry out quantitative analysis at a complete single cell level, and has a good application prospect in cell classification and other aspects.
Description
[0001] This patent application was completed by the School of Life Sciences of Tianjin Normal University / Tianjin Key Laboratory of Animal and Plant Resistance, and was awarded the Tianjin Application Basic and Frontier Technology Research Program (19JCYBJC29700) and the Tianjin Key Laboratory of Aquatic Ecology and Aquaculture Open Fund (TJAE2015005). . technical field [0002] The invention belongs to the field of detection technology, and relates to a single-cell-level high-throughput gene based on the proportional specific combination of a probe sequence and a target gene target sequence, combining fluorescence in situ hybridization technology and flow cytometry detection technology Novel automated assays for expression measurement and cell classification. This method does not need to use antibodies to classify mitten crab blood cells, and only needs to obtain partial sequence information of the target gene, and then the probe sequence can be synthesized. The shorter probe ...
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Application Information
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