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Molecular identification kit and identification method for rabdosia serra

A technology of molecular identification and yellow grass, applied in the molecular identification kit and identification field of yellow grass, can solve the problems of time and cost increase, and achieve the effects of rapid identification, good stability and good specificity

Active Publication Date: 2021-12-07
GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent CN 104404129A discloses a method of DNA barcode identification of Xihuangcao and its close genus. The patent provides a gene bank including IST2 gene sequence fragments of Labiatae plants Xihuangcao and C. Amplify the ITS2 sequence by PCR and then compare it with the gene bank to identify whether the tested plant is Xihuangcao or Cinnamon chinensis. However, this identification method needs to sequence the PCR amplification products, which increases the time and cost.
Chinese patent CN 104404629A discloses a DNA identification method of Xihuangcao and Xianhuaxiangchacai. After the rbcl sequence is amplified by PCR and compared with the sequence of the gene bank, the tested plants are identified, but this method also requires sequencing of the PCR products

Method used

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  • Molecular identification kit and identification method for rabdosia serra
  • Molecular identification kit and identification method for rabdosia serra
  • Molecular identification kit and identification method for rabdosia serra

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1I

[0032] Example 1ISSR screening marker universal primers embodiment

[0033] Was screened with primers specific detection Xihuangcao ISSR markers from the universal primers, the present invention is firstly selected and Xihuangcao similar known samples, and the extracted genomic DNA. The present invention is a known sample populations of wild and cultivated plants, medicinal Xihuangcao germplasm collection of information selected with reference to different environments shown in Table 1. Table 1 sample identified Professor Liu Junmin Guangzhou University of Traditional tea is lined dish, vegetable fiber floral tea, tea leaves vegetables, tea Xihuangcao and significant pulse vegetables are grown in the Guangzhou University of Traditional Chinese Medicine when Jane mountain in the yellow grass germplasm nursery, specimen samples stored in the Department of Guangzhou University of Traditional Chinese medicine resources.

[0034] Table Creek to collect information in different populati...

Embodiment 2

[0044] Optimization of the reaction conditions in Example 2

[0045] The present invention is screened in the embodiment 1 based primer 3 (UBC-815) on, respectively, the PCR reaction the amount of template and primer concentrations set different gradient optimization. The amount of template and 100ng were set up 10,20,25,40,60,80 7 gradient, provided the primer concentration and 1.0μmol·L 0.2,0.4,0.6,0.8 -1 Gradient of 5. Experiments were repeated at least three times until stable results.

[0046] In a PCR machine for processing different gradient amplification, amplification program was: 94 ℃ denaturation 7min; 94 ℃ denaturation 2min, 52 ℃ 45s annealing, extension 72 ℃ 2min, 40 cycles total; extending 10min 72 ℃; extender yield was stored 4 ℃.

[0047] Optimization results, such as the amount of template image 3 Shown, wherein lanes 1 to 7 corresponding to the amount of template and 100 ng 10,20,25,40,60,80 respectively, by the image 3 Found, Xihuangcao ISSR reaction system is n...

Embodiment 3I

[0051] Design and Optimization of the annealing temperature Example 3ISSR-SCAR embodiment labeled primer

[0052] Extraction kit agarose gel with a large number of DNA (DP210) the figure 1 Located in 750 ~ 1000bp band range for specific amplification Gel Extraction, referring to specific steps Gel Extraction kit instructions. The recovered fragment was then connected by pClone007 Vector Kit (TSV-007) with a cloning vector instructions, as follows:

[0053] 1, connection

[0054] Suction pClone007 Vector 1μL in 0.2mL sterile centrifuge tube at the bottom, suction 1μL 10 × Topo Mix gently added to the centrifuge tube mixed by pipetting, then DNA fragment was added 8μL recovered rt connected 5min.

[0055] 2, transformation

[0056] The ligation product was added to 100μL E. coli DH5α competent cells ice bath was 30min. After the hot water bath 42 ℃ shock 90s, the ice bath was quickly transferred to ice 5min. After an ice bath was added in a centrifuge tube 400μL of LB liquid medium,...

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Abstract

The invention discloses a molecular identification kit and an identification method for rabdosia serra. A pair of ISSR-SCAR marker primers capable of specifically detecting rabdosia serra is designed and screened by utilizing an ISSR marker and a sequencing result obtained by amplification, and the molecular identification kit and the identification method of the rabdosia serra are provided on the basis of the marker primers, so that false positive caused by diversity among populations can be avoided, and the identification method and the kit are suitable for quickly and accurately identifying the rabdosia serra.

Description

Technical field [0001] The present invention belongs to the field of biotechnology. More particularly, to a Xihuangcao molecular identification method and identification kit. Background technique [0002] Hara (Rabdosia serra (Maxim.) Hara) is a perennial herb of the mint family Isodon, with heat and dampness, the efficacy of jaundice dampness, cooling blood stasis, for the treatment of acute jaundice hepatitis, acute cholecystitis, dysentery, enteritis, bone-bruising pain and other symptoms. Meanwhile, the component having a significant anticancer activity Xihuangcao contained Xihuangcao A and A Xihuangcao factors such great potential for development. The Xihuangcao medicinal herbs as common characteristics of the Lingnan region, "Guangdong Chinese Materia Medica Standards" for inclusion (Rabdosia lophanthoides var Labiatae (Labiatae) lined plant Plectranthus (Rabdosia lophanthoides) and its variants fiber floral tea dishes .graciliflora) or Xihuangcao (Rabdosia serra) of the dr...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/156C12Q2600/13C12Q2565/125Y02A50/30
Inventor 詹若挺刘军民孟爽爽王德勤
Owner GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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