Specific probe for detecting expression of prophenoloxidase gene of Eriocheir sinensis and application of specific probe

A Chinese mitten crab, phenol oxidase technology, applied in the field of detection, can solve problems such as technical and time uncertainty

Pending Publication Date: 2022-04-29
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Antibody-antigen recognition is mainly used to detect specific proteins, but there are huge differences in technology and time to solve key problems such as wh

Method used

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  • Specific probe for detecting expression of prophenoloxidase gene of Eriocheir sinensis and application of specific probe
  • Specific probe for detecting expression of prophenoloxidase gene of Eriocheir sinensis and application of specific probe
  • Specific probe for detecting expression of prophenoloxidase gene of Eriocheir sinensis and application of specific probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Effects of Aeromonas hydrophila Infection on Hemocytes of Eriocheir sinensis

[0044] Materials and Methods

[0045] Experimental Materials

[0046] Eriocheir sinensis, body weight 50±20g. Aeromonas hydrophila strains were isolated and preserved in our laboratory. Reagent consumables and probes were purchased from relevant companies with reference to the instructions of the present invention. The probe sequence is: 5'-TAGACGAGGTGCCAGTGCCAGT -3', labeled with 5-terminal FAM fluorescence, and purified by HPLC. Anticoagulant components 338mmol / L sodium chloride, 115 mmol / L glucose, 30 mmol / L trisodium citrate, 10 mmol / L disodium ethylenediaminetetraacetic acid, pH=7.0, Carnoy's fixative component methanol The ratio with glacial acetic acid is 3:1, and it should be prepared before use.

[0047] experimental method

[0048] Select Chinese mitten crab individuals with no injury on the body surface and normal vitality, divide them into control group and experimental grou...

Embodiment 2

[0061] Rapid Analysis of Effects of Vibrio parahaemolyticus Infection on Hemocytes of Eriocheir sinensis

[0062] Materials and Methods

[0063] Experimental Materials

[0064] Chinese mitten crab, weighing 50±30g. The strains of Vibrio parahaemolyticus were isolated and preserved in this laboratory. Reagent consumables and fluorescent probes were purchased from related companies with reference to the instructions. The probe sequence is: 5'-TAGACGAGGTGCCAGTGCCAGT -3', labeled with 5-terminal FAM fluorescence, and purified by HPLC. Anticoagulant components 338 mmol / L sodium chloride, 115 mmol / L glucose, 30 mmol / L trisodium citrate, 10 mmol / L disodium ethylenediaminetetraacetic acid, pH=7.0, Carnos’s fixative component methanol The ratio with glacial acetic acid is 3:1, and it should be prepared before use.

[0065] experimental method

[0066] Select Chinese mitten crab individuals with no injury on the body surface and normal vitality, divide them into control group and ...

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Abstract

The invention discloses a specific probe for detecting Eriocheir sinensis phenol oxidase progene expression and application thereof. According to the method, a probe sequence is designed in a conserved region of a gene, a flow cytometry is introduced to carry out high-throughput and high-precision automatic measurement by utilizing a principle that a fluorescently-labeled probe is specifically combined with mRNA of a target gene, a single cell sample point can be accurately identified, and intracellular fluorescence intensity can be quantitatively analyzed, so that quantitative information of the mRNA expression level of the target gene is obtained. The accuracy, reliability and repeatability of results based on tens of thousands of sampling points are far better than those of conventional methods such as manual observation counting. The method does not need an antibody, is low in cost, is high in flow-based high-flux automatic analysis efficiency, is good in reliability, and has great application potential.

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 Applied Basic and Frontier Technology Research Program (19JCYBJC29700), the Open Fund of Tianjin Key Laboratory of Aquatic Ecology and Breeding (TJAE2015005) . technical field [0002] The invention belongs to the technical field of detection, and relates to an automation method for single-cell level gene expression measurement and cell classification based on the principle of specific and proportional combination of probe sequence and target sequence, combining fluorescence in situ hybridization and flow cytometry Detection method. This method does not rely on antibodies, and directly synthesizes probe sequences based on the conserved regions of mRNA of functional genes. The advanced flow measurement module further avoids the error of "different from person to person". High-throughput detect...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6816C12N15/11
CPCC12Q1/6888C12Q1/6816C12Q2600/124C12Q2563/107
Inventor 张亦陈刘逸尘孙金生
Owner TIANJIN NORMAL UNIVERSITY
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