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A group of specific primers for identifying turtle shells and its identification method

A specific, turtle-shell technology, applied in the field of traditional Chinese medicine identification, can solve the problems such as the difficulty in determining the threshold of intra-species variation, the inability to identify decoction pieces and adulterated products, and the doubtful accuracy of test results, so as to reduce the probability of false negative amplification, The effect of flexible concentration and low cost

Active Publication Date: 2020-09-25
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the study of using molecular marker technology to identify turtle shells, the existing technology can only be applied to the medicinal materials and basic samples of turtle shells, and it is still unable to identify decoction pieces and adulterated products, and the scope of application is relatively limited
In addition, some researchers have used DNA barcoding technology based on COI sequences to carry out molecular identification of soft-shelled turtles and common counterfeit products. It is difficult to determine the threshold of intraspecific variation, and the accuracy of the test results is doubtful
In addition, there are inherent defects such as cumbersome steps, high time and cost
[0005] PCR has the advantages of good specificity, high sensitivity (exponential increase), good repeatability, easy operation, and high throughput (each instrument can amplify 96 samples at a time); however, in conventional PCR technology, DNA sequences are used for species In the process of identification, due to the use of general primers in COI, ITS and other regions, after the amplification is completed, the product needs to be sequenced and compared with the gene database, and then the species can be determined. There are many steps, time-consuming, and special equipment; in addition, the detection of adulterated products cannot be achieved, or the identification results are affected by adulterated phenomena

Method used

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  • A group of specific primers for identifying turtle shells and its identification method
  • A group of specific primers for identifying turtle shells and its identification method
  • A group of specific primers for identifying turtle shells and its identification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Design, screening, optimization and verification of specific primers for the turtle shell kit

[0065] (1) Reagent

[0066] Sodium Lauryl Sulfate, Sodium Chloride, Tris, Hydrochloric Acid, Disodium EDTA, Proteinase K (20mg / mL, Sigma-Aldrich, P6556), Tris Balanced Phenol ( LIFE SCIENCES, T0250), chloroform, isoamyl alcohol, absolute ethanol, Ezup Column Animal Genomic DNA Extraction Kit (Shenggong, B518251), DreamTaqGreen DNA Polymerase (Thermo Scientific, #EP0712), dNTP Mix (Thermo Scientific, #R0192), Agarose, Ethidium Bromide (SunShineBio, SN314-1), boric acid, all reagents were of analytical grade.

[0067] (2) Method

[0068] First design specific primers:

[0069] According to the gene difference of soft-shelled turtle Pelodiscus sinensis (Accession No.: NC_006132.1, AY687385.1, AY962573.1) and Florida soft-shelled turtle Apalone ferox (Accession No.: NC_014054.1, FJ890514.1), specific primers were designed, and according to Requirement modification...

Embodiment 2

[0081] Embodiment 2: turtle shell kit detection method

[0082] Crush the turtle shell product, weigh 50-200 mg of powder below 40 mesh, add 995 μL of SDS lysate and 5 μL of proteinase K, vortex and mix well, extract in a constant temperature mixer at 55°C-65°C for 1-8 hours, and take it by centrifugation Supernatant solution; Tris equilibrated phenol, phenol-chloroform-isoamyl alcohol (25:24:1), chloroform-isoamyl alcohol (24:1) with the supernatant in turn Mix and centrifuge to take the supernatant; add 5M potassium acetate solution 0.1 times the volume of the supernatant and pre-cooled 96% ethanol twice the volume of the supernatant to the supernatant, mix, place at -20°C overnight, and centrifuge Then discard the supernatant; add 500 μL of pre-cooled 70% ethanol to wash the precipitate, centrifuge and discard the supernatant; dry or freeze-dry at room temperature, dissolve with 25 μL TE buffer to obtain the genomic DNA test solution, quality inspection , Store at -20°C or...

Embodiment 3

[0087] Example 3: Application of the turtle shell kit to the detection of self-made adulterated products

[0088] Detect according to the method of embodiment 2:

[0089] The PCR program is: 95°C for 3 minutes; 35 cycles of 95°C for 30s, 66°C for 30s, 72°C for 1min; 72°C for 7min.

[0090] The adulterated turtle shells were prepared by mixing pre-crushed soft-shelled turtles and soft-shelled turtle shell powders in five different ratios (7:1, 3:1, 1:1, 1:3, 1:7). The final mass of the sample was 50 mg. Then it was extracted and purified by SDS method, and analyzed under optimized conditions.

[0091] Prefabricated reaction system for preparing primers, including: 1×PCR buffer, 2.0mmol / L MgCl 2 , 0.2mmol / LdNTPs, primer pair, 0.625U Taq DNA polymerase, and add sterilized double distilled water to make up to 25μL; then, put it into a sterile EP tube, add 1μL DNA test solution, mix well, that is get a reaction system. Thereafter, the reaction system was placed in a PCR instru...

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Abstract

The invention relates to a group of specific primers for identifying turtle shells and an identification method thereof, and belongs to the technical field of identification of traditional Chinese medicines. Firstly, a DNA test sample solution is prepared, and the sample is used as a template, designed and screened specific nucleotide sequences are selected as primers for PCR amplification, then products are subjected to agarose gel electrophoresis analysis and gel imaging system detection. Finally, authenticity and adulteration of turtle shell products are judged based on test results. The method can determine whether the sample is adulterated. The method is easy to operate, does not require sequencing, has strong specificity, strong anti-interference ability, good reproducibility, has awide range of application, and can be used for accurate identification of turtle shell origins, medicinal materials and decoction pieces, and a practical method can be provided for judging the authenticity and adulteration of the turtle shell products.

Description

technical field [0001] The invention relates to a group of specific primers for identifying turtle shells and an identification method thereof, belonging to the technical field of traditional Chinese medicine identification. Background technique [0002] Trionycis Carapax, the carapace of the soft-shelled turtle (Pelodiscus sinensis), is a traditional Chinese medicine, first recorded in "Shen Nong's Materia Medica", and listed as a middle grade. It has been recorded in successive dynasties of herbal medicine, and is now listed as a dual-purpose traditional Chinese medicine by the National Health Bureau. Turtle shell is slightly cold in nature, salty in taste, belongs to the liver and kidney meridian, can nourish yin and subdue yang, reduce fever and steam, soften hard and resolve stagnation. Indications for fever due to yin deficiency, bone steaming and labor-heat, yin deficiency and yang hyperactivity, dizziness, internal movement of deficiency wind, cramps in hands and fe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/166C12Q2565/125
Inventor 杨欢虞平添沈玉萍蔡宝昌焦兆群陈立群陈宇菲鹿蓓蓓
Owner JIANGSU UNIV
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