Sea snake venom specificity detection method and kit

A technology for detection kits and detection methods, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve problems such as difficult to treat, difficult to identify by non-professionals, and poor results, and achieve Good stability, high sensitivity, rapid identification effect

Pending Publication Date: 2021-12-17
SHANGHAI SERUM BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Injection of antivenom is currently the most effective first aid measure for venomous snake bites worldwide. Few countries abroad have developed anti-sea snake venom products, and some of them have been used in clinical treatment. Sea snake bites are currently not available in China Special drugs, patients are often sent to local small hospitals, medical units at all levels carry out symptomatic treatment according to their own experience, there is no standardized emergency plan, and the effect is not good
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 entire genome sequence of sea snakes 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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  • Sea snake venom specificity detection method and kit
  • Sea snake venom specificity detection method and kit
  • Sea snake venom specificity detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Sea snake gene-specific primer design

[0052] According to the nucleotide sequence of the sea snake gene in the Gen Bank database, use online software for homology analysis and comparison, use primer design software to analyze the composition and stability of the base sequence, and conduct homology and sequence specificity analysis , refer to the primer design principles, and design multiple pairs of primers with comprehensive consideration. The sequences of the primer pairs are as follows:

[0053]

Embodiment 2

[0054] Embodiment 2: Minor Genomic DNA Extraction

[0055] Take an appropriate amount of sea snake tissue or toxin, put it in a centrifuge tube, add an appropriate amount of pure water and protease at a final concentration of 100 μg / ml, incubate at 37°C, centrifuge for 10 minutes, suck the supernatant into another centrifuge tube, then add an equal volume of equilibrated phenol, shake Or invert and mix thoroughly, centrifuge at 12000×g for 10min, absorb the supernatant aqueous phase and put it in another centrifuge tube, add an equal volume of 1:1 mixed equilibrium phenol: chloroform, then shake or invert thoroughly, centrifuge at 12000×g for 10min, draw Put the supernatant aqueous phase in another centrifuge tube, add an equal volume of chloroform, oscillate or invert to mix thoroughly, centrifuge at 12000×g for 10 min, absorb the supernatant aqueous phase into another tube, add 2.5 times the volume of cold absolute ethanol and freeze for more than 10 min. Centrifuge at 12000...

Embodiment 3

[0057] Embodiment 3: PCR amplification reaction

[0058] 1. PCR reaction conditions

[0059] PCR reaction system includes: total volume 20μl, 10×PCR buffer 2.0μl; 10×dNTPs 2.0μl, 0.2-1.5μl upstream primer (final concentration: 0.2μmol / l-1.5μmol / l); 0.2-1.5μl 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.

[0060] The PCR reaction parameters are:

[0061]

[0062] 2. Optimization of PCR reaction parameters

[0063] The preferred PCR reaction system is: total volume 20 μl, 10× PCR buffer 2.0 μl; 10× dNTPs 2.0 μl, final concentration of upstream primers 0.5 μmol / l; final concentration of downstream primers 0.5 μmol / l, final concentration of Taq enzyme recommended by reagent instructions Concentration; DNA template 0.5μl; double distilled water to make up to 20μl.

[0064] The preferred PCR reaction temperature and time for primer pair 1 a...

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Abstract

The invention discloses a trace sea snake venom specificity detection method and a kit, and belongs to the technical field of biology. According to the invention, trace genes in toxins are extracted, different primer pairs are respectively used as specific primers, a product is obtained through PCR amplification, and the product is verified to be a sea snake specific gene sequence through agarose gel electrophoresis and sequencing. The detection method and the kit provided by the invention have the advantages of high specificity, good stability, rapid detection, easy operation and the like, are beneficial to large-scale and automatic detection analysis, and are a method for rapidly and specifically identifying the sea snakes and the toxins of the sea snakes.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a detection method and a detection kit for sea snake venom in domestic common venomous snakes; in particular, it relates to a specific detection method and detection kit for sea snake venom. Background technique [0002] According to the "Chinese Snake Atlas", there are 9 genera and 16 species of sea snakes inhabiting the coast of my country, which are the main marine poisonous creatures. Sea snakes and cobras are closely related, both highly venomous snakes. Sea snake venom is among the strongest animal poisons. Sea snakes are widely distributed in warm waters along the South China Sea, Beibu Gulf, Hainan, Taiwan, Guangxi, Guangdong and the East China Sea. The southeast coastal area of ​​my country is the key area for the development of China's marine industry. In recent years, with the continuous increase in the development and utilization of marine resources, marine worker...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2565/125
Inventor 陈则张黎明易应磊柳国艳周东王博范铁炯王蓓蕾
Owner SHANGHAI SERUM BIOTECH
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