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Extracting micro viper gene and specific detection method thereof

A gene and trace technology, applied in the fields of biochemical equipment and methods, microbial determination/inspection, DNA preparation, etc., can solve the problems of missing the best time for treatment, blind treatment, unpublished whole genome sequence of Viper, etc. Achieve high sensitivity, good stability and good specificity

Inactive Publication Date: 2019-10-29
SHANGHAI SERUM BIOTECH
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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 whole genome sequence of viper has not been published at home and abroad before, and there are no specific primers and methods for extracting DNA from samples, especially snake venom, and amplifying the target sequence

Method used

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  • Extracting micro viper gene and specific detection method thereof
  • Extracting micro viper gene and specific detection method thereof
  • Extracting micro viper gene and specific detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Viper gene-specific primer design

[0056] According to the nucleotide sequence of the viper gene in the Gen Bank database, the online software (http: / / www.ncbi.nlm.nih.gov) was used to perform Blast homology analysis and comparison, and the conserved and specific nucleotide regions were screened out. Use the primer design software to analyze the composition and stability of the base sequence, and comprehensively consider the principles of primer design, design multiple pairs of primers, and submit them to Shanghai Sangong for synthesis. The sequence of the primer pairs is as follows:

[0057] Table 1

[0058]

[0059]

Embodiment 2

[0060] Embodiment 2: Minor Genomic DNA Extraction

[0061] Take an appropriate amount of viper tissue or viper venom, put it in a centrifuge tube, add an appropriate amount of pure water with a final concentration of 100 μg / ml PK (i.e. proteinase K), incubate at 37°C, centrifuge at 12000g for 10min, suck the supernatant and put it in another centrifuge Then add an equal volume of balanced phenol, shake or invert to mix thoroughly, centrifuge at 12,000g for 10 minutes, absorb the supernatant aqueous phase and put it in another centrifuge tube, add an equal volume of 1:1 to mix and balance phenol:chloroform, then shake or invert to mix thoroughly , centrifuge at 12000g for 10min, absorb the supernatant water phase and put it 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 Store for ...

Embodiment 3

[0063] Embodiment 3: PCR amplification reaction

[0064] 1. PCR reaction conditions

[0065] PCR reaction system includes: total volume 20μl, 10XPCR buffer 2.0μl; 10XdNTPs 2.0μl, 0.2-1.5μl primer P-F (final concentration: 0.2μmol / l-1.5μmol / l); 0.2-1.5μl primer P-R (final concentration: 0.2μmol / l-1.5μmol / l); 0.4μl-2μl Taq enzyme (5U / μl, final concentration: 0.1 U / μl-0.5U / μl); DNA template 0.5μl; double distilled water 10.5μl-14.7μl.

[0066] The PCR reaction conditions are:

[0067]

[0068] 2. Optimization of PCR reaction conditions

[0069] The preferred PCR reaction system is: total volume 20μl, 10X PCR buffer 2.0μl; 10XdNTPs 2.0μl, primer P-F final concentration 0.5μmol / l; primer P-R final concentration 0.5μmol / l, Taq enzyme final concentration 0.25U / μl; DNA template 0.5μl; make up to 20μl with double distilled water.

[0070] Primer pair 1: The preferred PCR reaction temperature and time are:

[0071]

[0072] The preferred PCR reaction temperature and time for ...

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Abstract

The invention discloses an extracting micro viper gene and a specific detection method thereof, and belongs to the technical field of biology. According to extracting micro viper gene and the specificdetection method thereof, by extracting genes from viper tissue and toxin, different primer pairs are used as specific primers to obtain PCR amplification products, and then through agarose gel electrophoresis detection, the PCR products are used as specific gene sequences of a viper after being sequenced. The detection method has the advantages of high specificity, good stability, rapid detection, easy operation and the like, is conducive to large-scale and automatic detection and analysis, and is a fast and specific method for identifying the viper and the toxin of the viper.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to viper, one of domestic common venomous snakes, and its toxin trace gene extraction and detection method. 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. [0003] The round-spotted viper belongs to the genus Viperiae and is mainly distributed in Fujian, Guangdong, Guangxi and other places in my country (Zhao Ermi. Chinese snakes [M]. Hefei: Anhui Science and Technology Press, 2006.148-149.), It is a blood poisonous snake and is one of the top ten poisonous snakes in China. Severe coagulation dysfunction can occur in the early stage after the bite of the viper, which is prone to complications such as disseminated intravascular coagulation, intracranial or visceral hemorrhage (Zhang ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806C12Q1/686C12Q1/6888C12N15/11
CPCC12N15/1003C12Q1/6806C12Q1/686C12Q1/6888C12Q2521/537C12Q2523/32C12Q2565/125
Inventor 陈则朱崇日易应磊王德慧罗敏李鑫卞聪华范铁炯
Owner SHANGHAI SERUM BIOTECH
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