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Method for identifying dried Pheretima aspergillum based on PCR-RFLP technology

A technology of PCR-RFLP and technical identification, which is applied in the field of identification of Chinese herbal medicine resources, can solve problems such as no reports of earthworm species, and achieve simple and reliable operation

Inactive Publication Date: 2019-02-01
INST OF ZOOLOGY GUANGDONG ACAD OF SCI
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Problems solved by technology

But there is no report about using PCR-RFLP technology to identify earthworm species

Method used

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  • Method for identifying dried Pheretima aspergillum based on PCR-RFLP technology
  • Method for identifying dried Pheretima aspergillum based on PCR-RFLP technology
  • Method for identifying dried Pheretima aspergillum based on PCR-RFLP technology

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Embodiment 1

[0014] 1. Extraction of DNA:

[0015] Take 30 mg of different kinds of earthworms (Guangdilong, Eisenia foetida, Amynthasobscurito porus, Pheretima guillelmi and Metaphire magna) tissues, and use animal Tissue DNA Extraction Kit (Tiangen) was used for DNA extraction to obtain genomic DNA as a template.

[0016] 2. COI fragment amplification, purification and sequencing:

[0017] PCR amplification was performed with COI gene universal primers HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' (as shown in SEQ ID NO.1) and LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3' (as shown in SEQ ID NO.2), to establish PCR Reaction system, PCR reaction is carried out in a 25 μL system, including 1 μL of DNA template, 2.5 μL of 10×PCR Buffer, MgCl 2 (25mmol / L) 2μL, dNTPs (10mmol / L) 0.5μL, Taq DNA polymerase (2.5U / μL) 0.4μL, HCO2198 and LCO1490 primers (10μmol / L) each 0.5μL, add sterilized ultrapure water to 25μL; The reaction program was: pre-denaturation at 95°C for 4 min; denaturation at 95°C for 45 ...

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Abstract

The invention discloses a method for identifying dried Pheretima aspergillum based on a PCR-RFLP technology. Universal primers of a COI gene are used to carry out PCR amplification to obtain mitochondrial cytochrome oxidase (COI) gene fragments of all kinds of earthworms, the sequences of the fragments are sequenced and compared, the complete sequences of the COI fragments of the different kinds of earthworms undergo restriction enzyme map analysis by DNAMAN software to finally screen a restriction enzyme RsaI, the restriction enzyme RsaI is used to carry out enzyme digestion on the above obtained PCR product, the enzyme digestion result of the PCR product is detected by 2% agarose gel, and the enzyme digestion maps are used to identify the dried Pheretima aspergillum and other varieties.The method is simple and reliable to operate, and provides a new technical way for the identification of dried Pheretima aspergillum products.

Description

Technical field: [0001] The invention belongs to the technical field of resource identification of traditional Chinese medicinal materials, and in particular relates to a method for identifying A. guangdilong based on PCR-RFLP technology. Background technique: [0002] Guangdilong is the dried body of Pheretima aspergillum, an animal of the family Pheretima aspergillum. It is mainly produced in Guangxi, Guangdong. It is a traditional Chinese medicinal material for animals. Guangdilong preparations are widely used in clinical practice. Guangdilong has a variety of pharmacological effects, mainly including lowering blood pressure, relieving asthma, antipyretic and anti-inflammatory, antithrombotic, and antitumor. Dilong has a history of thousands of years of folk medicine in China. According to the Chinese Pharmacopoeia, Dilong includes Guangdilong and Hudilong. The quality of Dilong is considered to be the best in the industry. There are many varieties of earthworms in China...

Claims

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Application Information

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IPC IPC(8): C12Q1/683C12Q1/6888
CPCC12Q1/683C12Q1/6888C12Q2600/156C12Q2531/113
Inventor 黄庆毛润乾李志武
Owner INST OF ZOOLOGY GUANGDONG ACAD OF SCI
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