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Method for extracting trace agkistrodon halys venom genes, specific detection method and kit

An extraction method and specific technology, applied in the field of trace gene extraction from snake venom, can solve problems such as no specific primers and methods, difficulty in identification by non-professionals, missed treatment opportunities, etc., to achieve rapid identification, specific identification, and high sensitivity Effect

Inactive Publication Date: 2020-07-28
SHANGHAI SERUM BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the wide variety of venomous snakes, it is often difficult for non-professionals to identify
The treatment of venomous snakebite patients needs to correctly identify the species of venomous snake that caused the accident, so that the appropriate antivenom can be selected correctly, the efficiency of toxin antagonism can be improved to obtain the ideal curative effect, and at the same time, the chance of injection of unmatched antivenom can be reduced, thereby reducing the seriousness of serum sickness and the like. Risk of anaphylaxis, missed opportunity for treatment
However, at present, the clinical diagnosis of snakebite in my country mainly relies on the patient's oral medical history and clinical manifestations, which may cause blindness in treatment. Due to the lack of rapid and specific laboratory methods for identifying snake venom species, it is difficult to carry out targeted treatment
The complete genome sequence of Agkistrodon halys has not been published before, and there are no specific primers and methods for extracting DNA from samples, especially snake venom, and amplifying target sequences

Method used

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  • Method for extracting trace agkistrodon halys venom genes, specific detection method and kit
  • Method for extracting trace agkistrodon halys venom genes, specific detection method and kit
  • Method for extracting trace agkistrodon halys venom genes, specific detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Design of Viper gene-specific primers

[0055] According to the nucleotide sequence of Agkistrodon halys gene in the Gen Bank database, the online software (http: / / www.ncbi.nlm.nih.gov) was used for homology analysis and comparison, and the primer design software was used to determine the composition and composition of the base sequence. Analyze stability and other aspects, and refer to the principles of primer design, and design multiple pairs of primers with comprehensive consideration. The sequences of the primer pairs are as follows:

[0056]

Embodiment 2

[0057] Embodiment 2: Minor Genomic DNA Extraction

[0058] Take an appropriate amount of Agkistrodon halys tissue or toxin, put it in a centrifuge tube, add an appropriate amount of pure water with a final concentration of 100 μg / ml PK, incubate at 37°C, centrifuge for 10 minutes, suck the supernatant into another centrifuge tube, then add an equal volume of balanced phenol, Shake or invert to mix thoroughly, centrifuge at 12000g for 10min, absorb the supernatant water phase and place it in another centrifuge tube, add an equal volume of 1:1 mixed equilibrium phenol: chloroform, then shake or invert thoroughly, centrifuge at 12000g for 10min, absorb the supernatant water phase Place in another centrifuge tube, add an equal volume of chloroform, oscillate or invert to mix thoroughly, centrifuge at 12000g for 10min, absorb the supernatant water phase into another tube, add 2.5 times the volume of cold absolute ethanol to freeze for more than 10min, centrifuge at 12000g for 10min,...

Embodiment 3

[0060] Embodiment 3: PCR amplification reaction

[0061] 1. PCR reaction conditions

[0062] The PCR reaction system includes: 20 μl total volume, 2.0 μl 10X PCR buffer; 2.0 μl 10X dNTPs, 0.2-1.5 μl upstream primer (final concentration: 0.2 μmol / l-1.5 μmol / l); 0.2-1.5 μl downstream primer (final concentration: 0.2μmol / l-1.5μmol / l); 0.4μl-2μl Taq enzyme (final concentration: 0.1 U / μl–0.5U / μl); DNA template 0.5μl; double distilled water to make up 20μl.

[0063] The PCR reaction parameters are:

[0064]

[0065] 2. Optimization of PCR reaction parameters

[0066] The preferred PCR reaction system is: total volume 20 μl, 10X PCR buffer 2.0 μl; 10X dNTPs 2.0 μl, upstream primer final concentration 0.5 μmol / l; downstream primer final concentration 0.5 μmol / l, Taq enzyme final concentration according to the reagent instruction manual; DNA Template 0.5μl; make up to 20μl with double distilled water.

[0067] The preferred PCR reaction temperature and time for primer pair 1 are...

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Abstract

The invention discloses a method for extracting trace agkistrodon halys venom genes, a specific detection method and a kit, and belongs to the field of biotechnology. By extracting the trace genes invenom, different primer pairs are used as specific primers separately, products are obtained through PCR amplification, and it is verified that obtained sequence is agkistrodon specific gene sequenceafter agarose gel electrophoresis and sequencing are carried out. The detection method has the advantages of high specificity, good stability, rapid detection and easy operation, is beneficial to large-scale and automated detection and analysis, and is a method for quickly and specifically identifying agkistrodon and venom thereof.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method for extracting trace genes in Agkistrodon venom, one of the domestic common poisonous snakes; in addition, the invention also relates to a detection method and a detection kit for trace genes in Agkistrodon venom. Background technique [0002] Pit vipers belong to the order Snakes and the family Viperidae. The body length of Agkistrodon halys is 60-70 cm, and the head is slightly triangular. Also known as earth snake, big male snake, and land snake, it is named for its earth-like color. It is found in my country except Tibet, Qinghai, and Yunnan provinces, and it is the most widely distributed kind of venomous snake in my country. The bites of Agkistrodon halys are more common in summer and autumn. Agkistrodon halys bites are one of the sudden acute poisoning events. Agkistrodon halys toxin is a mixed toxin, which can cause heart, liver, lung, kidney and other organ ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12N15/10C12N15/11C12Q1/6888
CPCC12Q1/6806C12Q1/6888C12Q2521/537
Inventor 陈则易应磊罗湘初陈成杨智吴琼卞聪华王德慧范铁炯
Owner SHANGHAI SERUM BIOTECH
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