TaqMan probe primer mixture, kit and fluorescent quantitative PCR detection method for quickly identifying bungarus multicinctus blyth

A technology of the money snake and primer composition, which is applied in the direction of biochemical equipment and methods, microbe determination/testing, DNA/RNA fragments, etc., to achieve good accuracy, reduce the risk of interference, and high sensitivity

Inactive Publication Date: 2015-10-07
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is currently no disclosure or report of a method for rapidly identifying the origin of Sn

Method used

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  • TaqMan probe primer mixture, kit and fluorescent quantitative PCR detection method for quickly identifying bungarus multicinctus blyth
  • TaqMan probe primer mixture, kit and fluorescent quantitative PCR detection method for quickly identifying bungarus multicinctus blyth
  • TaqMan probe primer mixture, kit and fluorescent quantitative PCR detection method for quickly identifying bungarus multicinctus blyth

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] (1) Primer design: select the cytochrome C gene as the target gene based on DNA barcoding technology (as shown in SEQ ID NO.1), search and download the snake mitochondrial COI gene sequence in Genbank (GeneBank accession numbers are KF698926.1, JF700190.1, KF698937.1, KF698948.1, KF698944.1, JX233650.1, JN833612.1, JX233641.1, JX233637.1), by using the Mega5 analysis software to compare the money snake and common counterfeit red snake, Homology of COI gene sequence of golden krait, cobra, Elaphe japonica, yellow chain snake, lead-colored water snake, Chinese water snake and double whole white krait, design specific primers (as shown in SEQ ID NO.2 and SEQ ID NO.3 Shown) and TaqMan probe (as shown in SEQ ID NO.4), comparison results see figure 1 .

[0053] Design and construct a recombinant plasmid containing positively amplified internal standard DNA, and design the corresponding TaqMan probe (as shown in SEQ ID NO.5), the method is: use DNA random generation software ...

Embodiment 2

[0072] specificity test

[0073] Genomic DNA extracted from the confused Pinchia viper, golden krait, cobra, Elaphe japonica, yellow chain snake, lead-colored water snake, Chinese water snake, Shuangquan white ring snake and authentic money snake as templates, according to Example 1 The optimized reaction system and reaction conditions were used for fluorescent quantitative PCR to detect the specificity of primers and probes.

[0074]The results showed that only the DNA derived from the white snake had a CT value <35, while the other samples had no amplification signal. The results are shown in Table 4. It can be seen that the primers and probes in this kit have strong species specificity.

[0075] Table 4 Fluorescent quantitative PCR detection CT value

[0076]

Embodiment 3

[0077] Embodiment 3 sensitivity test

[0078] Genomic DNA Sensitivity Test

[0079] Extract target genomic DNA according to the kit used in Example 1, quantify to 50ng with Nanodrop, do 10× gradient dilution (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 ), 2 μl was taken as the template amount for each gradient, and 12 parallel samples were set for each gradient, and the DNA of Snake chrysalis was detected according to the above method to investigate the sensitivity of the kit.

[0080] The results show( Figure 4 , Figure 5 ), when the amount of fluorescent quantitative template DNA is 0.01ng, the probe still has an amplification curve and the CT value is 35, and there is basically no amplification curve, so the kit The detection limit of the method is 0.01ng;

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Abstract

The invention provides a TaqMan probe primer mixture, a kit and a qPCR detection method for quickly identifying bungarus multicinctus blyth and counterfeits thereof as well as application. Mitochondrion DNA (mtDNA) has the advantages of high sensitivity, good accuracy, high speed, low degradability, stability, easiness in operation and the like and thus serves as a target gene; a competitive positive internal amplification control is artificially synthesized, a specific probe is added into the system and shares a pair of primers with the target gene, thus false negative can be effectively indicated; double multicolor fluorescent quantitative PCR refers to detection in the same tube, has the beneficial effects of accuracy and stability, easiness in operation, remarkably high sensitivity, high specificity, large flux and the like, and is especially applicable to the identification of traditional Chinese medicines with low DNA content and high degradability caused by processing, so that a new way is explored for identifying the animal-derived ingredients in food and drugs.

Description

technical field [0001] The invention relates to the technical field of identification of famous and precious Chinese herbal medicines, in particular to a method for identifying white snakes, specifically a TaqMan probe primer mixture, a kit and a fluorescent quantitative PCR detection method for quickly identifying the authenticity of white snakes. Background technique [0002] Bungarus multicinctus Blyth is a commonly used traditional Chinese medicine recorded in the 2010 edition of "Chinese Pharmacopoeia". It is derived from the dried body of young snakes of the Cobra family animal Bungarus multicinctus Blyth, which is disc-shaped with a diameter of 3-6cm. The diameter of the snake body is 0.2-0.4cm, the gas is slightly fishy, ​​and the taste is slightly salty. The golden snake has the effects of expelling wind, dredging collaterals, and relieving spasm. It is mainly used to treat rheumatism obstinate numbness, numbness and spasm, apoplexy, crooked mouth and eyes, hemipleg...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q1/6851C12Q2600/16C12Q2600/166
Inventor 步迅刘艳艳张全芳卞如如陈杰
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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