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A kind of specific primer pair and method for identifying Tibetan calamus and Shichangpu

A technology of Tibetan calamus and Shichangpu, which is applied to the specific primer pair of Shichangpu to identify the field of Tibetan calamus respectively, which can solve the problems of carcinogenicity, name confusion, and many common names, and achieve the effect of ensuring the quality of medicinal materials

Active Publication Date: 2019-01-22
HUNAN INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the toxicity of α-asarone and β-asarone, the active ingredients of Tibetan calamus, to human beings, the FDA (Food and Drug Administration) has banned the consumption of Tibetan calamus and its Extract
Historically, the use of Tibetan calamus and stone calamus has not been clearly separated, and due to the wide range of regions, there are too many common names, which leads to confusion in names

Method used

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  • A kind of specific primer pair and method for identifying Tibetan calamus and Shichangpu
  • A kind of specific primer pair and method for identifying Tibetan calamus and Shichangpu
  • A kind of specific primer pair and method for identifying Tibetan calamus and Shichangpu

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The method for the identification of Tibetan calamus and calamus calamus comprises the following steps:

[0044] 1. Extraction of medicinal material DNA: proceed as usual, and adjust the DNA concentration of the test sample to 0.2-0.5 μg / μL with deionized water;

[0045] 2. Polymerase chain reaction:

[0046] (1) Polymerase chain reaction takes 25 μL as a reference, and the dosages of various items are as follows:

[0047]

[0048]

[0049] (2) PCR reaction conditions: The reaction was carried out on a PCR instrument, and the reaction conditions were: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 62°C for 30 seconds, extension at 72°C for 45 seconds, and after 35 cycles, extension at 72°C for 10 minutes.

[0050] (3) Electrophoresis detection of PCR products: Take 5 μL of the above reaction solution, mix it with 1.0 μL sample loading buffer, and use 2% agarose gel (containing 0.5 μg / μGelRed) to detect the amplification ...

Embodiment 2

[0059] 为了保证分子鉴定结果的可靠性,本实验收集了藏菖蒲和石菖蒲的多种同属伪品和市面上流通的50种常用中药材,以对本实验方法进行适用性验证,确保本方法准确、可靠。

[0060] 1材料(见表1)

[0061] 表1本发明所有样品来源表

[0062]

[0063]

[0064] 2 DNA提取

[0065] 取本品0.1g,依次用75%乙醇1ml、灭菌超纯水1ml清洗,吸干表面水分,置乳钵中研磨成极细粉。取25mg,置1.5ml离心管中,用新型广谱植物基因组DNA快速提取试剂盒提取DNA,加入0.7mL65℃预热缓冲液GP1,加入巯基乙醇,使其浓度为0.1%,轻轻混匀,将离心管放于65℃水浴20min,并不时摇动。加入0.7mL三氯甲烷 / 异戊醇(24:1),充分混匀,12000rpm离心5分钟。吸取上清至预先装有0.7mLGP2缓冲液的新离心管中,充分混匀。将混匀液加入到吸附柱中,12000rpm离心30sec,弃掉废液;将吸附柱放回收集管中,加入500uL缓冲液GD,12000rpm离心30sec,弃去废液;将吸附柱放回收集管中,加入600uL漂洗液PW,12000rpm离心30sec,弃去废液,重复洗脱一次;将柱放回收集管中,12000rpm离心2分钟后,弃去废液,开盖室温放置以除去残留漂洗液;将吸附柱放入新的洁净1.5mL离心管中,在柱中央加入50uL洗脱液TE,室温放置5min,12000rpm室温离心2min,离心管中的液体即为基因组DNA,4℃保存备用。另取藏菖蒲对照药材0.1g,同法制成对照药材模板DNA溶液。

[0066] 3 PCR-RFLP反应

[0067] 鉴别引物:5'CGGATGCGGATGTTGGC 3'(SEQ ID NO.1)和5'TGGGCTCTTCCCGTTTCG 3'(SEQ ID NO.2)。PCR反应体系:在200uL离心管中进行,反应总体积为25uL,反应体系包括10×PCR缓冲液2.7uL,dNTP(2.5mmol·L -1 )1.8uL,非高保真TaqDAN聚合酶(2.5U·L -1 )0.5uL,模板1uL,无菌超纯水18.5uL。将离心管置PCR仪,PCR反应参数:94℃预变性5分钟,循环反应35次(94℃30秒,62℃30秒,72℃45秒)...

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Abstract

The invention discloses a specific primer pair and a method thereof for identifying acorus calamus L. and acorus tatarinowii schott. The primer pair includes a primer P1 and a primer P2, wherein the sequence of the primer P1 is as shown in SEQ ID NO.1 and the sequence of the primer P2 is as shown in SEQ ID NO.2. The method can be used for respectively identifying acorus calamus L. and acorus tatarinowii schott by carrying out simple DNA extraction, PCR specific amplification, enzyme digestion and electrophoresis detection on samples; the method has the advantages of simple operation, strong specificity, good repeatability and the like; and the method is applicable to rapid identification of acorus calamus L. and acorus tatarinowii schott medicinal materials.

Description

technical field [0001] The invention relates to the technical field of quality identification of germplasm of traditional Chinese medicinal materials, in particular to a pair of specific primers and a method for identifying Tibetan calamus and calamus calamus respectively. Background technique [0002] Both Tibetan calamus and Shichangpu are included in the 2010 edition of the Chinese Pharmacopoeia. Tibetan calamus is the dried rhizome of Acorus calamus L., and Shichangpu is the dried rhizome of Acorus tatarinowii Schott. Traditional Tibetan medicinal materials. Tibetan calamus and Shichangpu medicinal materials are widely used in my country, and they are important species with the same source of medicine and food. They were first recorded in "Shen Nong's Herbal Classic" and are still used today. In Tibetan areas, the use and history of Tibetan calamus are of great value. It is listed as a first-class endangered Tibetan medicinal material, and the medicinal material resourc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/683C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2521/301C12Q2563/185
Inventor 聂平肖炳燚罗晖明李文莉刘丽丁野舒毕琼孙辉
Owner HUNAN INST FOR FOOD & DRUG CONTROL
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