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One group of specific primers for identifying leech as well as identification method thereof

A specific and leech technology, applied in the field of traditional Chinese medicine identification, can solve the problems of difficult application, inability to detect adulterated products, cumbersome and time-consuming operation, etc., and achieves the effects of simple operation, high application value, and convenient and fast operation.

Inactive Publication Date: 2018-04-20
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, a researcher uses the COI sequence of leeches to carry out DNA molecular identification research on leeches, that is, extracts the DNA of leech samples, uses COI universal primers to perform PCR amplification, and after sequencing the amplified products, constructs an N-J clustering analysis tree, which realizes The classification of leech samples is cumbersome, time-consuming and laborious. It can only be used to test samples of known leech species. For unknown species of leech, it needs to be compared with the sequence in the database, and adulterants cannot be detected. , it is difficult to apply

Method used

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  • One group of specific primers for identifying leech as well as identification method thereof
  • One group of specific primers for identifying leech as well as identification method thereof
  • One group of specific primers for identifying leech as well as identification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051]Example 1: Design and verification of leech kit-specific primers

[0052] (1) Reagent

[0053] 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, isopropanol, absolute ethanol, Ezup Column Animal Genomic DNA Extraction Kit (Shenggong, B518251), DreamTaq Green DNA Polymerase (Thermo Scientific, #EP0712) , dNTP Mix (ThermoScientific, #R0192), Agarose, Ethidium Bromide (SunShineBio, SN314-1), boric acid, all reagents are of analytical grade.

[0054] (2) Method

[0055] A. Design of heterosexual primers

[0056] Design specific primers according to the genetic differences between genuine leeches (Whitmania pigra Whitman), willow leaf leeches (Whitmania acronulata Whitman, and Hirudonip ponica Whitman) and counterfeit leeches published in GENEBANK, as shown in the above table Shown, synthesized by Shanghai Sangong Co., Ltd...

Embodiment 2

[0061] Embodiment 2: the detection of specific primers of the present invention to commercially available leech samples

[0062] Different batches of commercially available leech samples (including the counterfeit Hirudo phenanthae) were pulverized, and 10 mg of powder below 40 mesh was weighed, extracted, purified and quality checked by the SDS method to obtain a DNA test solution. The specific operations were as follows:

[0063] (1) Crush the leech product, sieve it with 40 mesh, collect the fine powder, add appropriate amount of SDS lysate and proteinase K (material-to-liquid ratio: 50-200: 1mg / mL), mix well, and incubate at 56°C for extraction 6h, take the supernatant after centrifugation;

[0064] (2) Add an equal volume of Tris-balanced phenol to the supernatant, fully mix, get the supernatant after centrifugation, add Tris-balanced phenol-chloroform-isoamyl alcohol (25:24: 1), mix well, get the supernatant after centrifugation;

[0065] (3) Add the same amount of chl...

Embodiment 3

[0069] Example 3: Detection of self-made adulterated products by leeches kit

[0070] Use pre-crushed genuine leech and fake leeches samples to prepare leech adulterants by mixing 5 different ratios (9:1, 7:3, 1:1, 3:7, 1:9), each sample The final mass of 20mg. Then extract and purify by SDS method, analyze according to the method of embodiment 2.

[0071] Such as Figure 7 As shown, 5 different ratios of mixed leech adulterants were amplified and analyzed by the specific primers of the corresponding species, and the band positions were consistent with the length of the expected amplified target fragment, indicating that the specific primers could identify adulterants corresponding species in the product.

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Abstract

The invention relates to a method for identifying leech by one group of specific primers and belongs to the technical field of traditional Chinese medicine identification. The method comprises the following steps: firstly, preparing a DNA test sample solution; secondly, taking the DNA test sample solution as a template, selecting specific nucleotide sequence obtained through design and screening as primers to perform PCR amplification; thirdly, performing agarose gel electrophoresis analysis and gel imaging system detection on products; and finally, judging the true and false and the adulteration condition of the leech according to the detection result. The specific primer amplification method is simple in operation, sequencing is not needed, the result is intuitive, and the adulteration product can be detected and identified; meanwhile, the method is high in anti-interference ability and high in repeatability, can be used for accurately identifying leech foundation and medicinal materials, provides an efficient and rapid method for detecting the leech products, and has a relatively good application value.

Description

technical field [0001] The invention relates to a group of specific primers for identifying leeches and an identification method thereof, belonging to the field of traditional Chinese medicine identification. Background technique [0002] Leeches, commonly known as leeches, are commonly used animal medicinal materials. The 2015 edition of the "Chinese Pharmacopoeia" stipulates that leeches are the dried whole body of leeches (Whitmania pigra Whitman), leeches (Hirudonip ponica Whitman) and willow leaf leeches (Whitmania acronulata Whitman). This traditional Chinese medicine was first recorded in "Shen Nong's Materia Medica". It tastes salty and flat, and has the effects of breaking blood, dredging menstrual flow, and removing stasis. In the market of Chinese herbal medicines, the main source of leeches is leeches, and there are a small amount of leeches. With the continuous development of traditional Chinese medicine preparations of leeches in recent years, the market deman...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11C12N15/10
Inventor 杨欢郑阳沈玉萍蔡挺严辉夏国华杨娅娅徐利丽鹿蓓蓓陈宇菲
Owner JIANGSU UNIV
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