Nested fluorescent PCR detection primer, probe, kit and detection method of donkey-origin, horse-origin, swine-origin and bovine-origin in donkey-hide gelatin, and applications thereof

A technology for detecting primers and porcine origin, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., and can solve problems such as DNA damage, linear genomic DNA fragmentation, inaccuracy

Inactive Publication Date: 2016-08-31
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The test results require electrophoresis and gel imaging systems to see the results. It takes about 5 hours from PCR to test results, and there is a possibility of cross-contamination
Because donkey-hide gelatin undergoes multiple processes of high-temperature decoction, the linear genomic DNA is broken into small fragments or even degraded. Although the tolerance of circular mitochondrial DNA to high temperatures is stronger than that of linear Different degrees of damage to DNA, how to solve the problem of degrading DNA has become a major bottleneck in detecting the authenticity of Ejiao based on DNA detection technology
Chinese patent (CN 104988231.A) uses semi-nested PCR technology to identify the authenticity of donkey-hide gelatin, and the amplified fragment is about 700bp. For the highly processed gelatinous traditional Chinese medicine, the DNA has been highly damaged, and it is difficult to successfully amplify large fragments. Two rounds of PCR amplification are required, which is not only time-consuming, but the operation of opening the tube can easily cause cross-contamination, resulting in false positives and inaccurate results, so it cannot meet the requirements of authenticity detection of donkey-hide gelatin

Method used

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  • Nested fluorescent PCR detection primer, probe, kit and detection method of donkey-origin, horse-origin, swine-origin and bovine-origin in donkey-hide gelatin, and applications thereof
  • Nested fluorescent PCR detection primer, probe, kit and detection method of donkey-origin, horse-origin, swine-origin and bovine-origin in donkey-hide gelatin, and applications thereof
  • Nested fluorescent PCR detection primer, probe, kit and detection method of donkey-origin, horse-origin, swine-origin and bovine-origin in donkey-hide gelatin, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] DNA extraction based on donkey-hide gelatin samples: using the method disclosed in the patent 201410317118.7 (a kit for quickly extracting DNA from donkey-hide gelatin and its extraction method), the steps will not be repeated, and the purity of the extracted genomic DNA is determined by ultraviolet spectrophotometer and concentration. The measured OD260 / OD280 values ​​are both about 1.8-1.9, and the concentration is above 10ng / μl, indicating that the DNA is of high purity and moderate concentration, which meets the requirements of PCR amplification.

[0090]1. Selection of target genes and design of primers: Compared with the genome, the copy number of mitochondria in tissues is higher, and the degree of damage of donkey-hide gelatin after deep processing is relatively small, so the mitochondrial 16SrDNA gene is preferred. The external primers and internal primers are designed for donkeys and pigs, and the amplified fragments are small, making it easier for the primers...

Embodiment 2

[0095] Example 2 Kit specificity verification

[0096] Using the detection kit provided by the present invention, the genomic DNA is extracted from animal skins or fresh tissues such as cattle, pigs, horses, donkeys, camels, yaks, goats, sheep, rabbits, fish, chickens, ducks, minks and foxes as templates, Perform multiple nested real-time fluorescent PCR detection according to the above method to verify the specificity of this kit. The test results are shown in Table 5. Only the genomic DNA of donkey, pig, horse and cow was detected, and the rest of the animal-derived DNA was not detected, indicating that the detection method of this kit has good specificity.

[0097] Table 3 Specificity Verification

[0098]

[0099]

Embodiment 3

[0100] Embodiment 3 Sensitivity experiment

[0101] Quantify donkey, pig, horse and bovine genomic DNA to 5×10 -2 ng / μl, 5pg / μl and 0.5pg / μl, 0.05pg / μl, 0.005pg / μl Each PCR reaction was added with different concentrations of donkey, horse, cow and pig DNA as a template, and the amount added was 2μl, that is, the DNA content 0.1ng, 0.01ng, 1pg, 0.1pg, 0.01pg respectively, amplified according to the above PCR system and detection method, the results are shown in Figure 8 to Figure 11 , as can be seen from the figure, the detection limit of the present invention is 0.1pg, and the detection sensitivity reaches the picogram level, which is 1000 times higher than that of ordinary nested PCR amplification.

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Abstract

The invention provides a nested fluorescent PCR detection primer, a probe, a kit and a detection method of donkey-origin, horse-origin, swine-origin and bovine-origin in donkey-hide gelatin, and applications thereof, and relates to the technical field of molecular biology. Using combinations of the primer and probe of the invention as well as multiple nested fluorescent PCR detection kits can quickly detection the donkey-origin, horse-origin, swine-origin and bovine-origin components in donkey-hide gelatin. Aiming at microscale DNA or degradation DNA in donkey-hide gelatin, the nested fluorescent PCR detection primer, the probe, the kit and the detection method greatly increase the effective target spot identification rate of the primer and the probe by nested PCR and short PCR fragment technologies, thereby greatly increasing the detection accuracy and sensitivity; two steps of PCR amplifications are conducted within one system, thereby providing advantages such as closed pipe operation, low contamination possibility, high detection sensitivity, good specificity, high accuracy, great flux, etc.

Description

technical field [0001] The present invention relates to the technical field of animal-derived detection of jelly traditional Chinese medicine, in particular to a nested fluorescent PCR detection primer, probe, kit, detection method and application of donkey-hide gelatin derived from donkey, horse, cattle and pig, belonging to molecular The field of biological technology. Background technique [0002] Donkey-hide gelatin is a solid gelatin made from the donkey skin of the equine animal, which is boiled and concentrated. It is originally produced in the Pandong-E region of Shandong Province. It was first recorded in "Shen Nong's Materia Medica". , has a history of nearly three thousand years. Donkey-hide gelatin blood-tonifying holy medicine, tastes sweet and flat, enters the lung, liver, and kidney meridian, has the functions of nourishing blood and stopping bleeding, nourishing yin and moistening dryness, etc. Power, applicable to a wide range of people. Li Shizhen's "Com...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2600/16C12Q2561/113C12Q2563/107C12Q2545/114C12Q2549/119C12Q2537/143C12Q2531/113C12Q2545/113
Inventor 张全芳刘艳艳范阳阳步迅
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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