Molecular identification method for DNA bar code of lumbricus

A technology of molecular identification and barcoding, which is applied in the field of species identification, can solve the problems of non-specific tissue characteristics, difficulty in identifying earthworm stems, and destruction of microscopic features.

Inactive Publication Date: 2017-06-13
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, it is difficult to see the shape and color of the species itself, and the microscopic characteristics are also damaged to varying degrees, resulting in the dry product of Dilong. It is difficult to identify

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  • Molecular identification method for DNA bar code of lumbricus
  • Molecular identification method for DNA bar code of lumbricus
  • Molecular identification method for DNA bar code of lumbricus

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Embodiment Construction

[0026] The present invention will be further described below in conjunction with specific embodiment:

[0027] 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.

[0028] 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 molecular identification method for a DNA bar code of lumbricus and a CO I sequence thereof. The method comprises the following steps: amplifying a sample CO I gene through polymerase chain reaction (PCR); verifying the PCR product through agarose gel and then sending to a biological sequencing company for sequencing; manually correcting and splicing sequence for the sequencing result; comparing with a public sequence; and if the homology of the gene sequence SEQ ID NO.1-NO.4 is above 99%, confirming a to-be-detected tissue as a source of pheretima aspergillum or Shanghai lumbricus. According to the invention, a reliable DNA molecular identification technique is adopted; the morphological identification for lumbricus varieties is not required; the lumbricus varieties and non-drug lumbricus varieties specified in Chinese Pharmacopoeia can be quickly and accurately identified; besides, the pheretima aspergillum and the Shanghai lumbricus can be further distinguished; the accuracy and the reliability can be enhanced; the safety of the lumbricus used as a medicinal material can be guaranteed.

Description

[0001] This application is a divisional application for the invention patent with the patent number 201410162028.5. The filing date of the original application is April 21, 2014, and the application number is 201410162028.5. The invention and creation name is a DNA barcode molecular identification method of Earthworm. technical field [0002] The invention relates to the field of species identification, in particular to a DNA barcode molecular identification method of earthworm. Background technique [0003] 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 "Guan...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
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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