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Primer pair for identifying Scolopendra subspinipes multilans(L.)Koch and application of primer pair

A primer pair, centipede technology, applied in the field of molecular biology, can solve the problems of DNA barcoding method, such as time-consuming, inability to adapt to the needs of application, etc.

Active Publication Date: 2015-04-22
华润三九现代中药制药有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] With the development of molecular identification technology, DNA barcoding technology has been applied to PCR identification of spiny giant centipede and multi-thorny centipede (Zhang Hongyin, Chen Jun, Jia Jing, et al. Study on DNA barcoding identification of Chinese medicinal centipede and its counterfeit products, China Journal of Traditional Chinese Medicine, 2014,39(12):2208), but because the DNA barcoding method takes a long time and needs to be sequenced, it cannot meet the needs of the current on-site application of the molecular identification technology of traditional Chinese medicine
However, there has not been any report on the molecular specific identification between the less spiny giant centipede and the multi spiny centipede

Method used

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  • Primer pair for identifying Scolopendra subspinipes multilans(L.)Koch and application of primer pair
  • Primer pair for identifying Scolopendra subspinipes multilans(L.)Koch and application of primer pair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, the preparation and the use method of the kit for identification little spiny giant centipede

[0048] 1. Design and synthesis of primer pairs for identification of giant centipede

[0049] In this example, the universal primer COI (LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3'; HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAAATCA-3') was selected to amplify the samples of giant centipede less spiny and spiny centipede, and then sequence the obtained sequences. According to the source comparison, a pair of specific primers F1 and F2 were designed according to the variable region, as a specific primer pair for identifying the giant centipede. The sequence is as follows:

[0050] Primer F1: 5'-TCAATAATCGGAACAGCAT-3' (SEQ ID NO: 1);

[0051] Primer F2: 5'-ATACTGACCATACAAATAAAGGAG-3' (SEQ ID NO: 2).

[0052] 2. Assembly of kits for identification of giant centipedes

[0053] After the primers F1 and F2 designed and synthesized in step 1 are packaged separately, they are pac...

Embodiment 2

[0066] Embodiment 2, using the kit prepared in Example 1 to identify the giant centipede with few spines

[0067] Samples to be tested: 2 samples of Chinese herbal medicines shown in Table 1, giant centipede with few spines and centipede with many spines.

[0068] 1. Extract genomic DNA from the sample to be tested

[0069] Take the medicinal material sample, after surface disinfection with 75% (volume fraction) ethanol, use a sterilized scalpel to take 20 mg of the sample, then cut it into pieces with sterilized scissors, grind and pulverize it with a grinder, and use marine animal genomic DNA The extraction kit [Tiangen Biochemical Technology (Beijing) Co., Ltd.] was used to extract the total DNA. For details, please refer to the kit instruction manual, and store at -20°C.

[0070] 2. PCR amplification

[0071] Using the genomic DNA extracted from the sample to be tested in Step 1 as a template, the primer pair (primer F1 and primer F2) designed in Step 1 of Example 1 was ...

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Abstract

The invention discloses a primer pair for identifying Scolopendra subspinipes multilans(L.)Koch and an application of the primer pair. The primer pair disclosed by the invention for identifying or assistant-identifying Scolopendra subspinipes multilans(L.)Koch is specifically the primer pair of two single-chain DNA molecules shown in sequences 1 and 2 in a sequence table. Experiments verify that by adopting the primers disclosed by the invention, Scolopendra subspinipes multilans(L.)Koch and Scolopendra multidens Newport are quickly and accurately identified by virtue of a quick PCR method and a fluorescent staining method and a fluorescent dye method is introduced to detect a true and false identification result, so that a technical support for field utilization on medicinal material molecular identification is realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a pair of primers for identifying the giant centipede and its application. Background technique [0002] According to "Chinese Pharmacopoeia" (2010) edition one record, the source of centipede is the dried body of Scolopendra subspinipes mutilans L.Koch, a centipede animal with few spines. Centipede is a traditional precious Chinese medicinal material in my country. It has the effects of quenching wind and relieving spasm, dredging collaterals and relieving pain, and attacking toxins and dissipating stagnation. Headache, sores, scrofula, snake bites, etc., mainly produced in Hunan, Hubei, Guangxi, and Yunnan in my country (Wang Keqin, Fang Hong, Ye Maoxiang, etc. Investigation of Centipede Drug Sources and Product Identification, Chinese Medicinal Materials, 1997, 20(9):450). [0003] In recent years, centipede breeding has gradually emerged in my country. However, due to the uneve...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 黄璐琦袁媛崔占虎蒋超
Owner 华润三九现代中药制药有限公司
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