Method for detecting leopard bone component in mixture and primer applied

A technology of mixture and leopard bone, which is applied in the detection of medicinal materials, or leopard bone components in traditional Chinese medicine preparations using leopard bone or substitutes, and in the field of specific primers, which can solve the problem of high subjectivity

Active Publication Date: 2013-08-28
BEIJING TONGRENTANG CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the traditional morphological method is mainly used for the identification of leopard bones, which relies on the experience of the identification personnel and is highly subjective. Therefore, an accurate and effective identification method is needed

Method used

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  • Method for detecting leopard bone component in mixture and primer applied
  • Method for detecting leopard bone component in mixture and primer applied
  • Method for detecting leopard bone component in mixture and primer applied

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Identification of Leopard Bone Components in Zaizao Pills (Leopard Bone Prescription) in Example 1

[0057] 1. Extraction of total DNA from leopard bone

[0058] This extraction step can be performed according to conventional operations in the field. The specific operations of the present embodiment are as follows: leopard bone is ground into fritters with a mortar, and can be used for DNA extraction after 48 hours of EDTA decalcification (10% EDTA solution (pH 7.0) earlier. 200 μ g sample (leopard bone after decalcification, initial Add 1ml of lysate A (10mM Tris-HCl pH8.0, 0.1MEDTApH8.0, 0.5% SDS, 200μg / ml proteinase K) to heavy) and digest overnight at 55°C. The digested sample was centrifuged at 12000rpm for 15min and the supernatant was extracted with phenolform Extract. Then use 2 times the volume of absolute ethanol to precipitate DNA, then wash the precipitate twice with 75% ethanol, and dissolve in an appropriate amount of 50 μl double-distilled water. The sam...

Embodiment 2

[0089] Identification of Leopard Bone Components in Tongren Dahuoluo Pills (Leopard Bone Prescription) in Example 2

[0090] Select 3 batches (10 pills / batch) of Tongren Dahuoluo Pills (Leopard Bone Prescription)—a total of 50 medicines, in which the Leopard Bone component accounts for about 0.8% (mass percentage) as the sample to be tested, and extract the total DNA. And carry out single-plex PCR amplification respectively with described primer pair SEQID Nos.1 and 2, SEQ ID Nos.3 and 4, and SEQ ID Nos.5 and 6 respectively. The extraction process of the total DNA of the sample to be tested, the PCR amplification and the analysis process of the amplification result are the same as in Example 1.

[0091] See results Figure 5 (Take the experimental results of one sample as an example, the rest are the same), using the DNA extraction method of the present invention and the designed leopard species-specific primers can effectively amplify the target fragment from the correspondi...

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Abstract

The invention provides a method for detecting a leopard bone component in a mixture and a primer applied. The method mainly comprises steps of: extracting a total DNA from a mixture to be measured; carrying out PCR amplification by a specific primer, the extracted total DNA serving as a template, wherein the specific primer is one pair, two pairs or three pairs of primer selected from the group of SEQ ID Nos.1 and 2, SEQ ID Nos.3 and 4 and SEQ ID Nos.5 and 6; and carrying out analysis and determination on an amplification result. The invention further provides a kit containing the above primer for detecting leopard bone component in the mixture and especially for detecting whether a mixture is leopard bone medicinal material or detecting leopard bone component in a traditional Chinese medicine preparation using a drug of leopard bone or a substitute. The primer of the present invention has high singularity and sensitivity.

Description

technical field [0001] The invention relates to a method for detecting leopard bone components in mixtures, especially medicinal materials, using molecular biology techniques, in particular to a method for detecting leopard bone components in medicinal materials, or traditional Chinese medicine preparations that use leopard bone or substitutes for medicine, and the method used in the method specific primers. Background technique [0002] Leopard bones, also known as Chuansi legs and leopard bones, are derived from the bones of Panthera pardus L. and other leopards. Leopard bone has been used in traditional Chinese medicine for more than a thousand years. It has the effects of dispelling wind and dredging collaterals, strengthening tendons and bones, and is used to treat rheumatic arthralgia, soreness of waist and knees and other diseases. However, at present, the traditional morphological method is mainly used for the identification of leopard bones, which relies on the exp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 赵兴波彭鹏姚璐杜菁张薇刘莹
Owner BEIJING TONGRENTANG CO LTD
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