Method for identifying DNA bar code molecule of earthworm

A molecular identification and barcoding technology, applied in the field of species identification, can solve the problems of microscopic feature destruction, difficulty in seeing species shape and color, and non-specific tissue features.

Inactive Publication Date: 2014-07-02
MUDANJIANG YOUBO PHARMA CO LTD
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Problems solved by technology

However, since most of the animal medicinal materials have similar external shapes and non-specific tissue characteristics, and after rough processing in the place of origin and processing of decoction pieces

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  • Method for identifying DNA bar code molecule of earthworm
  • Method for identifying DNA bar code molecule of earthworm
  • Method for identifying DNA bar code molecule of earthworm

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Embodiment Construction

[0025] The present invention will be further described below in conjunction with specific embodiment:

[0026] 1. Collection and preservation of Earthworm specimens: The collected samples come from Guangxi, Shanghai and other places. The collected specimens were anesthetized with 10% alcohol for about 20 minutes, then soaked in 75% alcohol for 15 minutes and killed and preserved.

[0027] 2. Pretreatment of test samples: take out the specimens of Earthworm preserved in 75% alcohol, remove the viscera to avoid contamination of visceral contents, soak and wash the remaining part in distilled water for 3 times, wash away the alcohol, and take about 20 mg of the tail with anatomical scissors. Put it into a 2ml EP tube, add magnetic beads, and shake to crush. Proteinase K was incubated at 56°C for 1 hour until the tissue was completely dissolved. Add 350 μl of chloroform:isoamyl alcohol (24:1) and mix well, centrifuge at 10,000×g for 5 minutes at room temperature, and carefully p...

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Abstract

The invention discloses a method for identifying a DNA bar code molecule of an earthworm and a COI sequence of the DNA bar code molecule of the earthworm. The method comprises the steps of amplifying the gene of a sample COI through polymerase chain reaction (PCR), verifying the PCR product by agarose gel, then sending to a biological sequencing company for sequencing, according to the sequencing result, sequence splicing through manual correcting, comparing with a public sequence, then confirming the tissue to be tested as the source of pheretima aspergillum or pheretima vulgaris if the homology of the spliced sequence and the gene sequence SEQ ID NO.1-NO.4 is over 99%. By adopting the reliable DNA molecular identification technology, the method can rapidly and accurately identify the earthworm varieties specified by the 'pharmacopoeia of China' and non-medicinal material earthworm varieties without conducting morphological identification on the earthworm varieties, can further distinguish the pheretima aspergillum varieties from pheretima vulgaris varieties, improves the accuracy and reliability, and guarantees the safety of the earthworm as the medicinal material.

Description

technical field [0001] The invention relates to the field of species identification, in particular to a DNA barcode molecular identification method of earthworm. Background technique [0002] Earthworm, that is, earthworm, is cold in nature and slightly salty in taste. It is a traditional Chinese medicinal material. However, only the annelid phylum Megaterridae is recorded under the item "Earth Dragon" in the Pharmacopoeia of the People's Republic of China 2010 Edition (Part One). Dried body of Pheretima aspergillum (E. Perrier), Pheretima vulgaris Chen, Pheretima guillelmi (Michaelsen) or Pheretima pectinifera Michaelsen. The former is commonly called "Guangdilong", and the latter three are commonly called "Shanghaidilong". Earthworm is listed as inferior in "Shen Nong's Materia Medica", which has the effects of clearing away heat and calming convulsions, dredging collaterals, relieving asthma, and diuresis. It is often fried and used for high fever, coma, convulsions, ar...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6869C12Q1/6888C12Q2563/185C12Q2531/113
Inventor 李振国陈艳明务勇圣
Owner MUDANJIANG YOUBO PHARMA CO LTD
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