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Method for detecting and identifying variety of snake venom by utilizing adaptor technology

An aptamer and snake venom technology, applied in the field of biomedical testing, can solve the problems of blind treatment, missing the best time for treatment, and complex components of snake venom, and achieve rapid detection results

Active Publication Date: 2013-11-27
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, before using antivenom to treat snakebite, it is necessary to select the correct type of antivenom according to the type of venomous snake involved, in order to reduce the risk of allergic reactions, improve the efficiency of toxin antagonism and obtain ideal curative effect, otherwise it will lead to serious allergic reactions such as serum sickness. , missed the best time for treatment
At present, the clinical diagnosis of snakebite in my country mainly relies on medical history and clinical manifestations, and there is a lack of rapid and specific laboratory methods to identify snake venom types, which often leads to blindness in treatment.
The reason is that the components of snake venom are complex, and there are many homologous proteins and a large number of identical antigenic sites in snake venoms that are closely related, making it very difficult to find species-specific monoclonal antibodies

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A kind of Chinese cobra venom identification method based on aptamer technology, carries out as follows:

[0032]1) Screening of genus-specific aptamers of Chinese cobra venom, constructing a random single-stranded DNA (ssDNA) library and primers, synthesizing the single-stranded DNA library into a double-stranded DNA library by PCR, and amplifying the DNA by asymmetric PCR Library, PCR amplification products were purified and recovered by phenol-chloroform method to obtain a large number of purified single-stranded DNA libraries.

[0033] 2) Screening cycle of Chinese cobra venom: Dialyze the Chinese cobra venom and coat it on the enzyme-linked plate, react at 37°C for 3 hours, and set a blank control well at the same time. Sample wells and control wells were blocked with 3% BSA for 2 hours. Put 1ng of the purified single-stranded DNA library in the SELEX binding buffer to react with the blank control well at 37°C for 40 minutes, reverse-screen to remove the single-st...

Embodiment 2

[0042] A method for identifying Agkistrodon venom based on aptamer technology, which is carried out as follows:

[0043] 1) Screening of species-specific aptamers for Agkistrodon venom, constructing a random single-stranded DNA (ssDNA) library and primers, synthesizing the single-stranded DNA library into a double-stranded DNA library by PCR, and then using asymmetric PCR to amplify DNA library, PCR amplification products are purified and recovered by phenol-chloroform method to obtain a large number of purified single-stranded DNA libraries.

[0044] 2) Screening cycle of Agkistrodon venom: After dialysis, the venom of Agkistrodon was coated on the enzyme-linked plate, and reacted at 37°C for 3 hours, and a blank control well was set at the same time. Sample wells and control wells were blocked with 3% BSA for 2 hours. 1 ng of the purified single-stranded DNA library was first reacted with the blank control wells at 37°C for 40 minutes in SELEX binding buffer, reverse-screen...

Embodiment 3

[0053] A method for identifying the venom of Agkistrodon halys in Jiangsu and Zhejiang based on the aptamer technology is carried out as follows:

[0054] 1) Screening of species-specific aptamers for Agkistrodon venom in Jiangsu and Zhejiang, construction of random single-stranded DNA (ssDNA) library and primers, synthesis of double-stranded DNA library by PCR from single-stranded DNA library, and amplifying DNA by asymmetric PCR method Library, PCR amplification products were purified and recovered by phenol-chloroform method to obtain a large number of purified single-stranded DNA libraries.

[0055] 2), Jiangsu-Zhejiang Agkistrodon venom screening cycle: the Jiangsu-Zhejiang Agkistrodon venom was dialyzed and coated on an enzyme-linked plate, and reacted at 37°C for 3 hours, and a blank control well was set at the same time. Sample wells and control wells were blocked with 3% BSA for 2 hours. 1 ng of the purified single-stranded DNA library was first reacted with the blan...

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Abstract

The invention discloses a method for detecting and identifying the variety of snake venom by utilizing an adaptor technology, belonging to the field of biomedicine verification and relating to a method for detecting snake venom by utilizing the adaptor technology. Specific to the problems complex compositions of snake venom and poor specificity and sensitivity of the traditional snake bite detection method, the invention adopts a depletion SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology, and an adaptor with high-degree snake venom species specificity is obtained through screening and is converted into a report adaptor so as to establish a novel method for quickly detecting snake venom.

Description

technical field [0001] The invention belongs to the field of biomedical testing, and relates to a method for identifying and detecting snakebites of domestic common venomous snakes, in particular to a screening technology for specific aptamers of snake venom species by using the subtractive aptamer technology. Background technique [0002] Venomous snake injuries are a global public health problem that can lead to respiratory paralysis, fatal hemorrhagic sepsis, irreversible renal failure, and severe local tissue necrosis leading to permanent disability. According to statistics, there are more than 63 species of known venomous snakes in 4 families and 28 genera in my country. Common venomous snakes include Chinese cobra, king cobra, golden krait, corona snake, Agkistros chinensis, Viper rotundus, Agkistrodon halys, bamboo leaf green, sea snake, etc. . Antivenom is a specific drug commonly used in China to treat poisonous snake bites. At present, the common antivenoms in my c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 邓昆府伟灵
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA