Nest-type fluorescence PCR detection primers, probe composition and kit for donkey and pig-sourced ingredients in colla corii asini and detection method and application

Abstract

The invention provides nest-type fluorescence PCR detection primers, a probe composition and a kit for donkey and pig-sourced ingredients in colla corii asini and a detection method and application, and relates to the technical field of molecular biology. By means of the primers, the probe composition and the multiple nest-type fluorescence PCR detection kit, the donkey and pig-sourced ingredients in colla corii asini can be rapidly identified. For trace DNA or degraded DNA in colla corii asini, a nest-type PCR and small fragment amplification technology is adopted, the probability for effectively identifying target points by means of the primers and a probe is greatly increased, and therefore the detection accuracy and sensitivity are greatly improved; two steps of PCR amplification are conducted in a same system, and the advantages that closed tube operation is achieved, the contamination rate is low, the detection sensitivity is high, the specificity is good, the accuracy is high, and the flux is large are achieved.

Description

technical field [0001] The present invention relates to the technical field of animal-derived detection 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 pig-derived 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 Ma...

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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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