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Method for detecting Osteon Myospalacem Baileyi in mixture and primers used thereby

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

Active Publication Date: 2013-04-17
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 Theron 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 Osteon Myospalacem Baileyi in mixture and primers used thereby
  • Method for detecting Osteon Myospalacem Baileyi in mixture and primers used thereby
  • Method for detecting Osteon Myospalacem Baileyi in mixture and primers used thereby

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Identification of bone components in Sailong in Example 1 Zaizao Pill (Sailong Fang)

[0060] 1. Extraction of total DNA from Theron bone

[0061]This extraction step can be performed according to conventional operations in the field. The specific operations of the present embodiment are as follows: the Sialon bone is ground into small pieces with a mortar, and can be used for DNA extraction after 48 hours of EDTA decalcification (10% EDTA solution (pH7.0). 200 μg sample (Sialon after decalcification) Bone, initial weight) was added to 1ml lysate A (10mM Tris-HCl pH8.0, 0.1MEDTA pH8.0, 0.5% SDS, 200μg / ml proteinase K), and digested overnight at 55°C. The digested sample was centrifuged at 12000rpm for 15min to take the supernatant Carry out phenolic extraction. Then use 2 times of volume dehydrated alcohol precipitation DNA, then wash precipitation twice with 75% ethanol, appropriate 50 μ l double-distilled water dissolves. Sample DNA is through PCR purification kit (K...

Embodiment 2

[0094] Example 2 Identification of Theron Bone Components in Sailong Fengshi Capsules

[0095] Select 3 batches (10 capsules / batch) of Silong Fengshi Capsules finished medicine (a total of one medicine) as the samples to be tested, take 4g of the capsule content for each sample, extract DNA, and use the primers to pair SEQ ID Nos. .1 and 2, SEQ ID Nos.3 and 4, and SEQ ID Nos.5 and 6 were subjected to single-plex PCR amplification, respectively.

[0096] see results Image 6 (Take the experimental results of one sample as an example, and the rest are the same). The Theron species-specific primer can amplify the target fragment from the corresponding finished drug.

[0097] Image 6 Among them, swimming lane 1 represents the detection result taking reconstituted pill (Leopard bone prescription) as the test sample; Swimming lane 2 represents the detection result taking Sailong Fengshi Capsule (Leopard bone prescription) as the test sample; Swimming lane 3 represents the reconst...

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Abstract

The invention provides a method for detecting Osteon Myospalacem Baileyi in a mixture and primers used thereby. The method mainly comprises the following steps: 1, extracting total DNA from a mixture to be detected; 2, carrying out PCR amplification through specific primers with the extracted total DNA as a template, wherein the specific primers are selected from one, two or three of SEQ ID Nos.1and SEQ ID Nos.2, SEQ ID Nos.3 and SEQ ID Nos.4, and SEQ ID Nos.5 and SEQ ID Nos.6; and 3, analyzing and determining amplification results. The invention further provides a kit which contains the primers and is used for discriminating whether a mixture to be detected is an Osteon Myospalacem Baileyi medicine or detecting Osteon Myospalacem Baileyi in a traditional Chinese medicinal preparation containing Osteon Myospalacem Baileyi or a substitute thereof. The primers of the invention have high specificity and sensitivity.

Description

technical field [0001] The present invention relates to a method of applying molecular biology technology to detect the components of the bone in the mixture, especially the bone in the medicinal material, in particular to a method for detecting the bone in the bone in the medicinal material or the traditional Chinese medicine preparation with the bone in the medicine or its substitute, and the method The specific primers used in . Background technique [0002] Theron, whose scientific name is Myospalax Baileyi, belongs to the family Rodentia Hamsteridae, and is a mammalian pest endemic to the Qinghai-Tibet Plateau. Sailong dry whole bone belongs to the national first-class animal new medicinal material. It has the functions of expelling wind and cold, dehumidification, menstrual flow and pain relief, and nourishing liver and kidney. However, at present, the traditional morphological method is mainly used for the identification of Theron bones, which relies on the experienc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 解素花姚璐曹梦杜菁王海刘名张薇张晓兰
Owner BEIJING TONGRENTANG CO LTD
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