Donkey-derived and horse-derived nest-type fluorescent PCR detection primer in donkey-hide gelatin, probe composition, kit, detection method and application

A technology for detecting primers and compositions, applied in the field of molecular biology, can solve problems such as linear genomic DNA breakage, failure to meet detection requirements, inaccurate false positive results, etc., achieve a high degree of DNA damage and improve detection accuracy and sensitivity, and the effect of improving detection sensitivity

Inactive Publication Date: 2016-07-27
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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Problems solved by technology

[0004] Because donkey-hide gelatin undergoes multiple processes of high-temperature decoction, the linear genomic DNA is broken into small fragments or even degraded. Although circular mitochondrial DNA is highly resistant to high temperatures, donkey-hide gelatin will damage DNA to varying degrees after deep processing , how to solve the degraded DNA has become a major bottleneck in the detection of the authenticity of donkey-hide gelatin based on DNA detection technology
Chinese patent (CN104988231.A) uses semi-nested PCR technology to identify the authenticity of donkey-hide gelatin, and the amplified fragment is about 700bp. For the deeply 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 is not only time-consuming, but the operation of opening the tube can easily cause cross-contamination and lead to false positives. The inaccuracy of the results can no longer meet the detection requirements.

Method used

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  • Donkey-derived and horse-derived nest-type fluorescent PCR detection primer in donkey-hide gelatin, probe composition, kit, detection method and application
  • Donkey-derived and horse-derived nest-type fluorescent PCR detection primer in donkey-hide gelatin, probe composition, kit, detection method and application
  • Donkey-derived and horse-derived nest-type fluorescent PCR detection primer in donkey-hide gelatin, probe composition, kit, detection method and application

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Experimental program
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Effect test

Embodiment 1

[0079] 1. DNA extraction from donkey-hide gelatin samples: use the method disclosed in the patent 201410317118.7 (a kit for quickly extracting DNA from donkey-hide gelatin and its extraction method), and the steps will not be repeated. The extracted genomic DNA is measured by an ultraviolet spectrophotometer. Purity and concentration. Determination of OD 260 / OD 280 The values ​​are all 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.

[0080] 2. 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 outer and inner primers are designed for donkeys and horses, and the amplified fragments are small, making it easier for the primers to bin...

Embodiment 2

[0085] Example 2 Kit Specificity Verification

[0086]Using the detection kit provided by the present invention, the genomic DNA is extracted from animal skins or fresh tissues such as cattle, pigs, donkeys, horses, camels, yaks, goats, sheep, rabbits, fish, chickens, ducks, dogs, minks and foxes. Template, multiple nested real-time fluorescent PCR detection was performed according to the above method to verify the specificity of this kit. The test results are shown in Table 3. Only the genomic DNA of donkey and horse was detected, and the rest of the animal-derived DNA was not detected, indicating that the detection method of this kit has good specificity.

[0087] Table 3 Kit specificity verification

[0088]

Embodiment 3

[0089] Embodiment 3 Sensitivity experiment

[0090] Quantify donkey and horse genomic DNA to 5×10 -2 ng / μl, 5pg / μl and 0.5pg / μl, 0.05pg / μl, 0.005pg / μl were added to each PCR reaction with different concentrations of donkey and horse DNA as templates, and the amount of addition was 2μl, that is, the DNA content was 0.1ng , 0.01ng, 1pg, 0.1pg, 0.01pg, amplified according to the above PCR system and detection method, the results are shown in Figure 4 and Figure 5 , 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 the common PCR method.

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Abstract

The invention provides a donkey-derived and horse-derived nest-type fluorescent PCR detection primer in donkey-hide gelatin, a probe composition, a kit, a detection method and an application, which relates to the technical field of the molecular biology. Donkey-derived and horse-derived components in the donkey-hide gelatin can be rapidly determined by utilizing the primer, the probe composition and the multiple nest-type fluorescent PCR detection kit. For trace DNA or degraded DNA in the donkey-hide gelatin, by adopting a nest-type PCR and small-fragment amplification technology, the probability of effectively identifying a target spot by the primer and a probe is greatly improved, so that the detection accuracy and sensitivity can be greatly improved; and the two-step PCR amplification is carried out in the same system, so that the beneficial effects of closed-tube operation, low pollution rate, high detection sensitivity, good specificity, high accuracy and high flux can be achieved.

Description

technical field [0001] The invention relates to animal-derived detection technology of gelatinous traditional Chinese medicine, in particular to a donkey-hide gelatin nest-type fluorescent PCR detection primer, probe composition, kit, detection method and application thereof, and belongs to the technical field of molecular biology. 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 "Compendium of Materia Medica" contains: "Eji...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6848C12Q1/6888C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2549/119C12Q2561/101C12Q2545/113
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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