DNA (deoxyribonucleic acid) barcoding based molecular identification method of leech

A molecular identification and barcoding technology, which is applied in the field of DNA barcoding molecular identification of wide-body golden thread leech, can solve the problems of difficulty in identification of leeches, poor stability of classification and description, and morphological damage.

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

At present, the traditional identification of traits lacks accurate internal anatomical features or the morphology is severely damaged during storage and processing. Therefore, it is urgent to find a new method to make up for the shortcomings of traditional classification methods

Method used

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  • DNA (deoxyribonucleic acid) barcoding based molecular identification method of leech
  • DNA (deoxyribonucleic acid) barcoding based molecular identification method of leech

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Embodiment Construction

[0023] The present invention will be further described below in conjunction with specific embodiment:

[0024] 1. Collection and preservation of leech specimens Collection strains: W1-stage I leeches for self-cultivated seedlings on the lake; W2-stage II of self-cultivated seedlings on the lake-leeches; W3-collected leech samples from the random market. The collected specimens were anesthetized with 10% alcohol for about 20 minutes, then soaked in 75% alcohol for 15 minutes and killed and preserved.

[0025] 2. Pretreatment of test samples: take out the leech specimens 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, take about 20 mg of the tail and place it in anatomical scissors 2ml EP tube, add magnetic beads, 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...

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Abstract

The invention discloses a DNA (deoxyribonucleic acid) barcoding based molecular identification method and CO I sequence of a leech (whitmania pigra). Polymerase chain reaction (PCR) is carried out by using the method to amplify a CO I gene of the leech, a PCR product is sent to a sequencing company to be sequenced after being verified by agarose gel, the sequencing results are subjected to manual proofreading and sequence splicing and are compared with public sequences, and the tissue to be tested can be judged to be the source of whitmania pigra if the homology of the sequencing results and a gene sequence SEQ ID NO.1 is above 99%. The whitmania pigra variety is quickly and accurately identified by adopting the reliable DNA molecular identification technology, thus improving the accuracy and the reliability and ensuring the safety of the leech as a medicine.

Description

technical field [0001] The invention relates to the field of species identification, in particular to a molecular identification method of wide-body leech DNA barcode. Background technique [0002] Wide-body golden thread leech (scientific name: Whitmania pigra) is a traditional precious Chinese medicinal material. Shuxuetong and many other traditional Chinese medicines for the treatment of cardiovascular diseases are the main raw materials. Therefore, the identification of the broad-bodied leech is the basis of medicine. In my country's medicinal material market, most animal medicines are precious and in short supply medicinal materials, which are usually used in the form of powder, etc. The market is chaotic and identification is very difficult. As for leeches, in the medicinal material market, the source is relatively clear, but there are still some counterfeit products circulating in the market. At present, the traditional identification of traits lacks accurate inter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2563/185
Inventor 李振国陈艳明务勇圣
Owner MUDANJIANG YOUBO PHARMA CO LTD
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