Method for identifying gynostemma pentaphylla and making distinction between gynostemma pentaphylla and cayratia japonica at deoxyribonucleic acid (DNA) level

A technology of Gynostemma pentaphyllum and black twig, applied in the field of identifying Gynostemma pentaphyllum at the DNA level and distinguishing it from black twig

Inactive Publication Date: 2011-09-21
GUANGXI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Purpose of the present invention: in order to overcome the traditional identification of the medicinal plant Gynostemma cucurbitaceae mainly based on the limitations and complexity of methods such as character or/and tissue microscopic identification...

Method used

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  • Method for identifying gynostemma pentaphylla and making distinction between gynostemma pentaphylla and cayratia japonica at deoxyribonucleic acid (DNA) level
  • Method for identifying gynostemma pentaphylla and making distinction between gynostemma pentaphylla and cayratia japonica at deoxyribonucleic acid (DNA) level
  • Method for identifying gynostemma pentaphylla and making distinction between gynostemma pentaphylla and cayratia japonica at deoxyribonucleic acid (DNA) level

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Experimental program
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Embodiment

[0057] 1. Extract DNA:

[0058] DNAs of Gynostemma cucurbitaceae and Ussica berry of grape family were extracted respectively by the CTAB method improved by the inventor.

[0059] (1) DNA extraction from fresh samples of Cucurbitaceae Gynostemma

[0060] ① Weigh 0.5g leaves and place them in a pre-cooled mortar, add liquid nitrogen and grind them into fine powder, quickly transfer them to a 7ml centrifuge tube, add 3ml preheated 1.5×CTAB extraction buffer, mix well, and place in a 65°C water bath Keep warm for 20min.

[0061] ② Cool the extract from step ① to room temperature, and centrifuge at 7500rpm for 10min. Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (24 / 1) for extraction, mix well, and centrifuge at 7500rpm for 10min.

[0062] ③ Take the centrifuged supernatant in step ②, add 1 / 10 volume of 1×CTAB extraction buffer and an equal volume of chloroform / isoamyl alcohol (24 / 1), mix well, and centrifuge at 7500rpm for 10min.

[0063] ④ Take the ...

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Abstract

The invention discloses a method for identifying gynostemma pentaphylla and making a distinction between gynostemma pentaphylla and cayratia japonica at deoxyribonucleic acid (DNA) level. The method is characterized by designing gynostemma pentaphylla SCAR marked specific primer pair WJS-T1 and WJS-T2 by using the result of medicinal plant cucurbitaceae gynostemma pentaphylla DNA characteristic segment SCAR marked clone sequencing, designing internal reference primer pair WHL-18S-C1 and WHL-18S-C2 according to a cucurbitaceae gynostemma pentaphylla 18S rRNA conserved region, forming a polymerase chain reaction (PCR) composite system 1, and amplifying the cucurbitaceae gynostemma pentaphylla DNA by using a touchdown PCR program to obtain two zones, namely a 359bp zone and a 293bp zone; anddesigning the internal reference primer pair WPT-18S-C1 and WPT-18S-C2 according to a vitaceae 18S rRNA conserved region, combining the internal reference primer pair WPT-18S-C1 and WPT-18S-C2 and the gynostemma pentaphylla SCAR marked specific primer pair WJS-T1 and WJS-T2 to form a PCR composite system 2, and amplifying a vitaceae cayratia japonica DNA by using the touchdown PCR program to obtain an 18S rRNA 177bp zone. Therefore, the DNA level of medicinal plant cucurbitaceae gynostemma pentaphylla is identified, and the distinction between the gynostemma pentaphylla and the vitaceae cayratia japonica which is easy to confound with the cucurbitaceae gynostemma pentaphylla in shape is made.

Description

technical field [0001] The invention relates to a method for identifying Gynostemma pentaphyllum at the DNA level and differentiating it from black currant. Background technique [0002] my country is rich in medicinal plant resources. Due to the wide variety, the phenomenon of homonyms or different names in different places is common; the difference between authentic and non-authentic medicinal materials of the same species is not very obvious in terms of morphology, tissue structure, etc., but The quality sometimes varies so much that it is hard to identify. Even for the same species, the content of active ingredients may vary in different places of origin. In the sales of Chinese herbal medicines, adulteration and falsehoods due to profit-driven situations often occur, so the variety and quality identification of Chinese herbal medicines is particularly important. [0003] Traditional identification methods of Chinese herbal medicines are mainly character and / or tissue m...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 吴耀生周娟罗育
Owner GUANGXI MEDICAL UNIVERSITY
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