Semi-nested-multiple PCR (Polymerase Chain Reaction) method for discerning authenticity of donkey-hide gelatin

A semi-nested and donkey-hide gelatin technology, which is applied in the field of semi-nested-multiple PCR method for identifying the authenticity of donkey-hide gelatin, can solve the problems of detection sensitivity and detection time that cannot meet expectations, and detection specificity is not good, so as to shorten the detection time, Effect of Improving Detection Sensitivity

Inactive Publication Date: 2015-10-21
山东世通检测评价技术服务有限公司
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Problems solved by technology

[0004] Chinese patent (CN201510018058.3) discloses a primer-probe composition, a kit and a multiplex real-time fluorescent quantitative PCR detection method for identifying multiple animal origins in donkey-hide gelatin. The detection specificity is not good, and the detection sensitivity and detection time cannot meet expectations.

Method used

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  • Semi-nested-multiple PCR (Polymerase Chain Reaction) method for discerning authenticity of donkey-hide gelatin
  • Semi-nested-multiple PCR (Polymerase Chain Reaction) method for discerning authenticity of donkey-hide gelatin
  • Semi-nested-multiple PCR (Polymerase Chain Reaction) method for discerning authenticity of donkey-hide gelatin

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Experimental program
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Embodiment 1

[0048] 1. Materials and Reagents

[0049] Liquid nitrogen, mortar, 1.5ml EP centrifuge tube, TE lysate, 10% SDS solution, proteinase K, RNase, potassium acetate solution, phenol: chloroform: isoamyl alcohol, isopropanol, ethanol, primers, PCR enzyme, PCR Buffer

[0050] 2. Main instruments and equipment

[0051] PCR instrument: Hangzhou Bioer Technology Co., Ltd.; gel electrophoresis system: Beijing Liuyi Machinery Factory; gel imaging system: American Alpha.

[0052] 3. Detection method

[0053] (1) Extraction of 4 kinds of animal (donkey, horse, pig and cow) skin and donkey-hide gelatin DNA

[0054]Weigh 0.2g skins of 4 kinds of animals (donkey, horse, pig and cow) and donkey-hide gelatin successively, put them into the mortar pre-cooled with liquid nitrogen respectively, then slowly add liquid nitrogen to the mortar, grind rapidly until the sample into a fine powder and placed in a 1.5ml centrifuge tube; add 600 μL of pre-cooled TE lysate to the treated sample, then add...

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Abstract

The invention relates to the field of food and drug detection technologies, in particular to a semi-nested-multiple PCR (Polymerase Chain Reaction) method for discerning the authenticity of donkey-hide gelatin, and the semi-nested-multiple PCR method is used for discerning whether components of horse, pig and cattle are doped in the donkey-hide gelatin, comprises the steps of adopting a modified SDS (Sodium Dodecyl Sulfonate) method to extract high quality DNA from the donkey-hide gelatin treated by liquid nitrogen grinding, and then designing universal primers P1 and P2 to perform the first round of amplification, wherein an amplification product is taken as a template of the second round of PRC; performing the second round of multiple PCR amplification by using mixed primers including the universal primer P1 and specific primers A, B, C and D of four species (donkey, horse, pig and cattle), detecting an amplification product of the second round by cataphoresis to discern whether the donkey-hide gelatin is adulterated according to the size of an electrophoretic band. By adopting the technical scheme of the invention, the detection sensitivity of the authenticity detection method of the donkey-hide gelatin on the basis of the existing PCR technology is improved in premise of ensuring the detection specificity, meanwhile, the detection time is shortened.

Description

technical field [0001] The invention relates to the technical field of food and drug detection, in particular to a semi-nested-multiple PCR method for identifying the authenticity of donkey-hide gelatin, which is used to identify whether donkey-hide gelatin is mixed with components derived from horses, pigs and cattle. Background technique [0002] Ejiao has a history of thousands of years of consumption in China. It has the effects of nourishing blood and stopping bleeding, nourishing yin and moistening lungs. It is a special Chinese medicinal material formed by boiling donkey skin, and its main component is collagen. Pure donkey-hide gelatin must come from 100% donkey skin in order to ensure the nutritional efficacy of the product. Due to the unique efficacy of donkey-hide gelatin, the supply is in short supply, and counterfeit and shoddy products have appeared in many places. Most of these low-quality glues are made of animal skin, bones, and connective tissues such as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6848C12Q1/686C12Q2549/119C12Q2537/143C12Q2565/125
Inventor 张慧孙海新黄金发许娜孙丕春
Owner 山东世通检测评价技术服务有限公司
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