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Method for detecting bupleurum chinense and bupleurum scorzonerifolium by utilizing specific primer PCR (polymerase chain reaction)

A narrow-leaf Bupleurum and specificity technology, applied in the field of molecular biology detection of Bupleurum and narrow-leaved Bupleurum, can solve the problems of high operator requirements, high cost, cumbersome probe preparation, etc., and achieve high sensitivity and easy operation Simple and specific effect

Inactive Publication Date: 2014-02-12
SHANXI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RFLP technology is stable and reproducible, but the preparation of probes is cumbersome; RAPD method can explain the evolution relationship between relative species very well, but it is unstable and limited by experimental conditions; ISSR markers are fast, simple, and highly polymorphic. advantages, but its primers have poor versatility and amplification conditions require targeted design; AFLP technology perfectly combines RFLP and PCR technology, but its stability is poor, and it requires high operator requirements and costs a lot

Method used

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  • Method for detecting bupleurum chinense and bupleurum scorzonerifolium by utilizing specific primer PCR (polymerase chain reaction)
  • Method for detecting bupleurum chinense and bupleurum scorzonerifolium by utilizing specific primer PCR (polymerase chain reaction)
  • Method for detecting bupleurum chinense and bupleurum scorzonerifolium by utilizing specific primer PCR (polymerase chain reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 is used for detecting the synthesis of the PCR primer of Radix Bupleurum and Bupleurum angustifolia

[0029] A total of 12 ITS sequences of Bupleurum and Bupleurum angustifolia were obtained from GenBank, and the above sequences were compared with the biological software DNAMAN version4.0 to find out all the variation sites of Bupleurum and Bupleurum angustifolia that are different from other species . According to the position of the mutation site, primers of about 20 bases were designed respectively on the upstream and downstream of the ITS sequence; the designed primers were analyzed using the biological software Primer5. Primer F1: 5'-ATGCCTCCGCCCCGTTTGG-3' and downstream primer R1: 5'-TGCGTTTTCC GATCTCCGGT-3'; suitable primer pair for Bupleurum angustifolia, upstream primer F2: 5'-TGTCGTCGGCCTCGGCCTG-3' and downstream primer R2: 5 '-ACGACGAGGCACGGGAGGT-3'.

[0030] Primer synthesis was completed by Sangon Bioengineering (Shanghai) Co., Ltd.

Embodiment 2

[0031] Example 2 Utilize PCR primers to detect the specificity and sensitivity analysis of Radix Bupleuri

[0032] 1.1 Sample source

[0033] The samples of medicinal materials used in this example are: Bupleurum radix, collected from Hunyuan, Shanxi.

[0034] 1.2 DNA extraction

[0035] Take about 60 mg of the above samples, grind them into powder with liquid nitrogen, and use the kit method to extract DNA. The kit was purchased from Beijing Aidelai Biotechnology Co., Ltd., model: CTAB Plant Genomic DNA Rapid Extraction Kit (Cat. No.: DN14), 50 times.

[0036] 1.3 Serial dilution of sample DNA

[0037] The DNA concentration of the extracted Bupleuri herb was detected to be 181.1 ng / μl. The DNA samples were serially diluted, and the diluted concentrations were 1.811, 0.905, 0.603, 0.452, 0.362 ng / μl, and stored at -20°C for later use.

[0038] 1.4 PCR reaction

[0039] Reaction system (25μl):

[0040]

[0041] The PCR reaction conditions were: 93°C for 5 minutes; 35 cy...

Embodiment 3

[0045] Example 3 Utilize PCR primers to detect the specificity and sensitivity analysis of Bupleurum angustifolia

[0046] 1.1 Sample source

[0047] The medicinal samples used in this example are: Bupleurum angustifolia, collected from Lindian, Heilongjiang.

[0048] 1.2 DNA extraction

[0049] 1.2 with embodiment 2.

[0050] 1.3 Serial dilution of sample DNA

[0051] The DNA concentration of the above-mentioned extracted Bupleurum angustifolia medicinal material was detected to be 99.3 ng / μl. The DNA samples were serially diluted, and the diluted concentrations were 0.993, 0.496, 0.331, 0.248 ng / μl, and stored at -20°C for future use.

[0052] 1.4 PCR reaction

[0053] Except that the primers (F2-R2) used are different, the rest are the same as 1.4 in Example 2.

[0054] 1.5 Results

[0055] Detection method is the same as 1.5 of embodiment 2.

[0056] A band of about 407bp appeared, proving that the tested sample was Bupleurum angustifolia or contained Bupleurum ang...

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Abstract

The invention provides a method for detecting bupleurum chinense and bupleurum scorzonerifolium by utilizing a specific primer PCR (polymerase chain reaction). According to the method, a specific primer capable of rapidly detecting the bupleurum chinense and the bupleurum scorzonerifolium based on ITS (Internal Transcribed Spacer) sequences of the bupleurum chinense and the bupleurum scorzonerifolium is designed, and a PCR detection system is established based on the specific primer to respectively amplify PCR fragments of 336 bp and 407 bp, so that the molecular identification basis is provided for the medicinal material identification of the bupleurum chinense and the bupleurum scorzonerifolium and whether multiple Chinese patent medicines (honeyed pills and water-honeyed pills) and medicinal materials contain the bupleurum chinense and the bupleurum scorzonerifolium can be rapidly and accurately detected; a detection kit constructed according to the method has the advantages that the operation is simple and convenient, the specificity is good, the sensitivity is high, the sequencing is omitted and the like, and the detection to stems and leaves of the bupleurum chinense and the bupleurum scorzonerifolium or other parts of plants and to the medicinal materials and the Chinese patent medicines (honeyed pills and water-honeyed pills) can be realized.

Description

technical field [0001] The invention relates to molecular biology detection of Bupleurum and Bupleurum angustifolia, in particular to a method for detecting Bupleurum and Bupleurum angustifolia by using specific primer PCR. Background technique [0002] Bupleurum, a plant of the genus Bupleurum L. in the family Umbelliferae (Apioideae), was first recorded in "Shen Nong's Materia Medica", listed as top grade, and is a commonly used bulk Chinese herbal medicine in my country. Spicy, bitter, slightly cold in nature, it belongs to the liver, gallbladder, and lung meridians. It has the effects of dispersing and reducing fever, soothing the liver and concluding speech, and promoting Yang Qi. It is used to treat colds and fever, alternating cold and heat, chest and hypochondrium pain, and irregular menstruation. , Uterine prolapse, anal prolapse embolism. Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. are the dried roots of Bupleurum chinense DC. recorded in Chinese ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895
Inventor 张福生薛英李晓伟秦雪梅张丽增邢婕
Owner SHANXI UNIV
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