Nest-type fluorescent PCR detecting primer, probe composition and kit for donkey material and bovine-derived material in donkey-hide gelatin, detecting method and application

Abstract

The invention provides a nest-type fluorescent PCR detecting primer, a probe composition and a kit for a donkey material and a bovine-derived material in donkey-hide gelatin, a detecting method and application and relates to the technical field of molecular biology.By the utilization of the primer, the probe composition and the multi-nested fluorescent PCR detecting kit, the donkey material and the bovine-derived material in the donkey-hide gelatin can be rapidly identified.For trace DNA or degraded DNA in the donkey-hide gelatin, the probability of effectively recognizing a target point is greatly increased for the primer and probe through nest-type PCR and a small-fragment amplification technology, and therefore detecting accuracy and sensitivity are greatly improved; two-step PCR amplification is performed in the same system, and the detecting method has the advantages that tube-closed operation is achieved, the pollution rate is low, detecting sensitivity is high, specificity is good, accuracy is high, and the flux is large.

Description

technical field [0001] The invention relates to the technical field of detection of animal origin of gelatinous traditional Chinese medicine, in particular to a nested fluorescent PCR detection primer, probe composition, kit, detection method and application of donkey-hide gelatin and bovine origin, belonging to molecular biology technology field. 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 Medic...

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Problems solved by technology

However, there are also disadvantages such as long time consumption, easy contamination and false positives, that is, the whole process takes about 5 hours from the beginning of PCR amplification, and the test results require electrophoresis and gel imaging systems to see the results, multiple tube opening operations, and the possibility of cross-contamination Big

[0005] In addition, because donkey-hide gelatin undergoes multiple processes of high-temperature decoction, the linear genomic DNA is broken into small fragments and even degraded. Although the tolerance of circular mitochondrial DNA to high temperature is stronger than that of linear genomic DNA, after intensive processing DNA will be damaged to varying degrees. How to solve the problem of degrading DNA has become a major bottleneck in detecting 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, resulting in false positives and low accuracy of the results. Therefore, the traditional PCR detection method can no longer meet the current requirements for authenticity detection of donkey-hide gelatin.

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