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Polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for distinguishing honeysuckle and lonicerae flos

A technology of PCR-RFLP and syringae, which is applied in the field of PCR-RFLP for identifying honeysuckle and syringae, can solve the problems of complex composition analysis experiment operation, strong subjectivity of morphological identification, poor stability of results, etc., and achieves reliable performance. Identification method, protection of rights and interests, effect of high demand

Inactive Publication Date: 2013-09-11
TONGJI UNIV
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AI Technical Summary

Problems solved by technology

Traditional morphological identification and component analysis are difficult to identify the mountain silver flower doped in honeysuckle, the morphological identification is highly subjective, the experimental operation of component analysis is complicated, and the stability of the results is not good

Method used

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  • Polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for distinguishing honeysuckle and lonicerae flos

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] 1. Extraction of DNA:

[0018] references [1] , and make a small change. Collect samples of honeysuckle and mountain silver-flower medicinal materials.

[0019] 1) Take 0.3g of the sample in a mortar, add liquid nitrogen to grind it into a fine powder, and carefully transfer it to a 5ml centrifuge tube.

[0020] 2) Add 3000 μl of refrigerated extraction buffer (0.25M NaCl, 0.25M Tris-HCl, 50 mM EDTA) to the centrifuge tube, mix well, and place on ice for 10 min.

[0021] 3) Centrifuge at 10000 r / min for 10 min, pour off the supernatant; repeat again.

[0022] 4) Add 3000μl 65℃ preheated 2×CTAB extract solution (2% CTAB, 100mM Tris-HCl, pH=8.0, 20 mM EDTA pH = 8.0, 1.4 M NaCl), 80μl β-mercaptoethanol, mix well , keep warm in a 65°C water bath for 2 hours, and shake gently from time to time.

[0023] 5) Take out the centrifuge tube, centrifuge at 12000r / min for 10min, suck out the supernatant with a micropipette, and put it into a clean centrifuge tube; add an equal...

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Abstract

The invention belongs to the technical field of distinguishing traditional Chinese medicinal materials, and in particular relates to a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for distinguishing honeysuckle and lonicerae flos. The feature DNA base sequence of the honeysuckle is 5'-Y GGCCR-3' and can be identified and cut by restriction enzyme CfrI or EaeI. The distinguishing process comprises the following steps of: extracting medicinal material DNA; performing PCR amplification by a pair of primers; digesting a PCR product by the restriction enzyme CfrI or EaeI; performing agarose gel electrophoresis analysis on the result; and comparing the result with a PCR-RFLP feature result of the lonicerae flos, and judging whether the test piece is the honeysuckle or the lonicerae flos. The quick, convenient and reliable PCR-RFLP method is realized according to the difference between the DNA sequences of the honeysuckle and the lonicerae flos; and the PCR-RFLP method has an important significance for guaranteeing the quality of a honeysuckle medicinal material, cracking down the ill behavior that sellers sell the lonicerae flos as the honeysuckle and protecting the rights and interests of customers.

Description

technical field [0001] The invention belongs to the technical field of germplasm identification of traditional Chinese medicinal materials, and in particular relates to a PCR-RFLP method for identifying honeysuckle and mountain silver flowers. Background technique [0002] The 2010 edition of "The Pharmacopoeia of the People's Republic of China" records that honeysuckle is the plant Lonicera Lonicera ( Lonicera japonica Thunb.) dry flower buds or with first-opened flowers, mountain silver flower is the Lonicera chinensis Lonicerae Lonicerae ( Lonicera macranthoides Hand. -Mazz.), red gland honeysuckle ( Lonicera hypoglauca Miq.), South China honeysuckle ( Lonicera confusa DC.) and Tawny Honeysuckle ( Lonicera fulvotomentosa Hsu et S.C.Cheng.) dry buds or with first flowers. Honeysuckle and mountain silver flower are very similar in plant morphology and medicinal material shape, so it is difficult to distinguish them. However, there are obvious differences in the e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 朱云国王庆霞程舟杨晓伶李珊
Owner TONGJI UNIV
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