Rapid detection kit for snake source components and application of rapid detection kit

A snake-derived, rapid technology, applied in the field of biological species identification, can solve the problems of difficult identification and achieve the effect of low copy number, easy observation and high sensitivity

Active Publication Date: 2021-03-16
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Xiong Xuemin et al. used thin-layer chromatography to screen the components of black snake in Shefen capsules to provide a basis for qualitative identification, but

Method used

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  • Rapid detection kit for snake source components and application of rapid detection kit
  • Rapid detection kit for snake source components and application of rapid detection kit
  • Rapid detection kit for snake source components and application of rapid detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Snake source universal internal standard gene JUN screening

[0050] The JUN (Gen bank No. EF144048.1 ) gene was screened out by searching the gene information about snakes in Gen Bank, and using BLAST software for homology and specificity analysis.

[0051] The internal standard gene screening process is as follows:

[0052] Step 1. Query the genome information of the target species in the NCBI database, analyze its integrity, select and download the species genome with complete sequencing information, and store it in the ".FASTA" format. Species that need to be screened and distinguished from the target species are limited, that is, "compared species", which are generally common species that are closely related to the target species. The selection of comparison species will directly affect the speed and accuracy of screening comparison, that is, the number of comparison species is large and the comparison speed is slow, so it is necessary to choose flexib...

Embodiment 2

[0064] Example 2 Establishment of LAMP detection method for snake source components

[0065] against JUN Gene (Genbank No. EF144048.1), using the Japanese Rongyan Co., Ltd. ring-mediated primer online design software, a total of 4 pairs of primers were designed, named JUN-1, JUN-2, JUN-3, JUN-4 (Table 1). Utilize these 4 pairs of primers to carry out LAMP amplification to snake source genomic DNA, and use 2% agarose gel to carry out electrophoresis analysis to the amplified product ( figure 2 ), the results showed that the band of primer JUN-1 was the clearest and brightest, and JUN-1 was selected for subsequent experiments.

[0066] Table 1 LAMP primer sequence

[0067]

[0068] Use LAMP to quickly detect snake samples, the reaction system is 25 μL, including 1×Bst Thermal buffer, 0.6mM dNTP, 3.6 mM MgSO 4 , 0.6 M betaine, 1.6 μM primers JUN - 1-FIP, 1.6 μM primer JUN - 1-BIP, 0.2 μM primer JUN -1-F3, 0.2 μM primer JUN - 1-B3, 8 U Bst DNA polymerase large fra...

Embodiment 3

[0070] Example 3 Universal identification of the standard gene LAMP in snakes

[0071]This application uses the designed primers JUN-F / R to carry out LAMP amplification on three kinds of snakes and use 2% agarose gel to perform electrophoresis analysis on the amplified products. The results showed that the snake meat of the three species had bright amplification bands ( Figure 4 ). This indicates that the gene and corresponding primers can be used to detect snake-derived products universally.

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Abstract

The invention provides a rapid detection kit for snake source components and application of the rapid detection kit, and relates to the technical field of biological species identification. Accordingto the rapid detection kit, a snake source general internal standard gene JUN (Gen bank No. EF144048.1) is screened out firstly, the nucleotide sequence of the snake source general internal standard gene JUN is shown as SEQ ID NO.1, the snake source general internal standard gene JUN is located on a chromosome, the copy number of the snake source general internal standard gene JUN in snake speciesis constant, allele variation does not exist, and the snake source general internal standard gene JUN can serve as a target gene for identifying a snake source. A constant-temperature amplification primer is designed with the snake source general internal standard gene JUN as a target sequence, and a constant-temperature amplification reaction product is used for colloidal gold nucleic acid teststrip detection. The rapid detection kit is formed by combining constant-temperature amplification reaction with colloidal gold nucleic acid test strip detection, the snake source components can be rapidly and sensitively detected, and the detection sensitivity can reach 0.8% (w/w). The rapid detection kit is simple in using method, low in cost, good in specificity and quite suitable for on-site real-time detection, and a reaction result is easy to observe.

Description

technical field [0001] The invention relates to the technical field of biological species identification, in particular to a loop-mediated isothermal amplification technique (LAMP) combined with a colloidal gold nucleic acid test strip detection kit for detecting snake-derived components. Background technique [0002] my country has a long history of using snakes as medicine. As early as the Ming Dynasty medical scientist Li Shizhen’s "Compendium of Materia Medica" wrote about the medicinal knowledge of snakes. So far, there are as many as More than 70 species. With the development of economy and society, its medicinal and commercial value has become increasingly prominent, and the social demand and application of snake resources are increasing year by year. In order to obtain huge profits, some unscrupulous traders mix counterfeit and confused products with snake medicinal materials, preparations, leather and other products. It is difficult to make a correct judgment only b...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11C12N15/12
CPCC12Q1/6844C12Q1/6888C12Q2600/166C12Q2531/119C12Q2565/625
Inventor 许文涛黄昆仑朱龙佼杜再慧邱申
Owner CHINA AGRI UNIV
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