Specific primers for identifying tortoise shells and identification method thereof

A specific, tortoise-shell technology, applied in the field of traditional Chinese medicine identification, can solve the problems of difficulty in determining the threshold of intra-species variation, inability to identify decoction pieces and adulterated products, and inability to detect adulterated products, so as to reduce the probability of false negative amplification , the concentration is flexible and variable, and the effect of a wide range of applications

Inactive Publication Date: 2018-05-11
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Some researchers also designed 2 pairs of specific primers for the Cyt B and COI sequences, and established a composite PCR technique to identify the authenticity of tortoise shell medicinal materials; Counterfeit products, the scope of application is relatively limited
In addition, the DNA barcoding technology based on the COI sequence is used for molecular identification of tortoises and common counterfeit products. The threshold is difficult to determine, 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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  • Specific primers for identifying tortoise shells and identification method thereof
  • Specific primers for identifying tortoise shells and identification method thereof
  • Specific primers for identifying tortoise shells and identification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Design, screening, optimization and verification of specific primers for tortoiseshell kit

[0058] (1) Reagent

[0059] Sodium lauryl sulfate, sodium chloride, tris(hydroxymethyl)aminomethane, hydrochloric acid, disodium edetate, 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, reagents are all analytical pure.

[0060] (2) Method

[0061] Design specific primers:

[0062] The genetic differences between turtles and their common fakes compared by DNAMAN software, Chinemys reevesii (Accession No.: NC_006082.1, AY676201.1, FJ469674.1, KJ700438.1), Trachemysscripta (Accession No.: NC_011573.1, FJ392294) .1, KM216749.1), Mauremys sinensis (Accession No.: NC_...

Embodiment 2

[0075] Example 2: Application of tortoiseshell kit primers for detection of homemade adulterants

[0076] The tortoise shell adulterated products are prepared by mixing the pre-crushed tortoise, Brazilian tortoise, and the original carapace powder of the tortoise shell. The two tortoise shell adulterated products (CR:TS, CR:MS) are in 5 different ratios (7:1, 3) :1, 1:1, 1:3, 1:7) to mix, the final mass of each sample is 50mg. Then it was extracted and purified by SDS method, and analyzed under optimized conditions.

[0077] First prepare the test solution, and then use PCR and agarose gel electrophoresis to quickly detect the target fragment.

[0078] The preparation method of the test solution is carried out according to the following steps:

[0079] (1) Crush the tortoise shell product, pass a 40-mesh sieve, collect the fine powder, add an appropriate amount of SDS lysis solution and proteinase K (the ratio of material to liquid is 10-200:1 mg / mL), mix well, and incubate at 56°C f...

Embodiment 3

[0088] Example 3: Detection of different batches of commercially available tortoiseshell samples

[0089] The method is the same as in Example 2.

[0090] Different batches of commercially available tortoiseshell samples (primitives, medicinal materials, and decoction pieces) were crushed, and 50 mg of the powder below 40 mesh was weighed, and extracted, purified and quality checked by SDS method. After that, the PCR reaction system was prepared and placed in a PCR machine for amplification under the above-mentioned optimized conditions. Subsequently, 8μL of the amplified product was analyzed using 2% agarose gel containing EB and TBE buffer. After the electrophoresis, the gel imaging system was used to detect the tortoise shell according to the presence or absence of electrophoresis bands and molecular weight. Authenticity. In addition, the positive control substances of tortoise, Brazilian tortoise and terrapins were prepared by weighing 50 mg of the original sample powder belo...

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Abstract

The invention relates to a group of specific primers for identifying tortoise shells and an identification method thereof, and belongs to the technical field of identification of traditional Chinese medicines. Firstly, genomic DNA is extracted and purified after tortoise shell products are crushed, 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 tortoise shell productsare judged based on test results. The identification method is easy to operate, does not require sequencing, has strong specificity, strong anti-interference ability, good reproducibility, has a widerange of application, and can be used for accurate identification of tortoise shell origins, medicinal materials and decoction pieces, and a practical method is provided for judging the authenticityand adulteration of the tortoise shell products.

Description

Technical field [0001] The invention relates to a set of specific primers for identifying tortoise shells and an identification method thereof, and belongs to the technical field of traditional Chinese medicine identification. Background technique [0002] The tortoise shell (Testudinis Carapax ET Plastrum), the carapace and plastron of the tortoise (Chinemysreevesii) of the tortoise family, is a traditional animal medicine. It was first recorded in the "Shen Nong's Herbal Classic" and was listed as the top grade. Materia Medica has been recorded in the past dynasties, and it is now listed as a dual-purpose Chinese medicine by the National Health Bureau. Tortoise shell is slightly cold, tastes salty and sweet, returns to the liver, kidneys and heart meridian, can nourish yin and yang, nourish the kidney and strengthen the bone, nourish blood and nourish the heart, strengthen the meridian and stop collapse, etc. Indications of yin deficiency hot flashes, bone steaming, night sweat...

Claims

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Application Information

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Patent Type & Authority Applications(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陈立群焦兆群鹿蓓蓓夏国华
Owner JIANGSU UNIV
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