Pure honeysuckle polymerase chain reaction-restriction map identification method

Abstract

The invention provides a polymerase chain reactino-restrictive enzyme-cut chart molecule (PCR-RFLP) method accurately identifying honeysuckle in genuine producing areas (Henan, Shandong) and nongenuine producing areas (other producing areas). The characteristic DNA alkali sequence of honeysuckle in genuine: 5'-GCCCCCCCGC CCCGCCTCCC ACAGGGTCGC G-3'; restrictive inner cut enayme EcoNi and its same cut-enzyme can identify and cut this sequence. The identifying course: extracting DNA of medical materials; using a pair of guide matters to make PCR increase; using restrictive inner cut enayme EcoNI and its same cut enayme to digest PCR product; analyzing the result by using gelation and electrophoresis of gelose; in contrast with the characteristic result of PCR-RFLP of genuine honeysuckle, judging if the tested product is genuine honeysuckle.

Description

1. Technical field:

[0001] The invention relates to the technical field of quality identification of germplasm of traditional Chinese medicinal materials. 2. Background technology:

[0002] Honeysuckle is the dry flower bud or the first flower of Lonicera japonica Thunb., a plant of Lonicera japonica Thunb. It has strong antibacterial effect and has good curative effect on various acute and chronic inflammations. The medicinal materials of honeysuckle are obviously authentic. For example, through our preliminary determination of the content of chlorogenic acid, the content of chlorogenic acid in Henan and Shandong honeysuckle in authentic areas is generally higher than that in other non-authentic areas, which shows that the quality of medicinal materials is related to the region, habitat, etc. closely related. However, honeysuckle from non-genuine areas and authentic areas cannot be distinguished by morphological and microscopic characteristics. Due to the isolation of geo...

Examples

Embodiment Construction

[0014] a. DNA extraction: proceed as usual, and adjust the DNA concentration of the test sample to 0.1-0.3 μg / μl with deionized water;

[0015] a. Polymerase chain reaction:

[0016] (1) For polymerase chain reaction, 30 μl is used as a reference, and the dosages of various items are as follows:

[0017] 10×PCR buffer 3μl

[0018] MgCl 2 (25mM) 2.4μl

[0019] dNTP (each 10mM) 0.6μl

[0020] Primers LjP1, LjP2 (30pmol / μl) 0.5μl each

[0021] 1~2 μl DNA template for test product

[0022] TaqDNA polymerase 1U (0.2μl)

[0023] Deionized water make up to 30μl

[0024] After mixing, centrifuge at high speed for a few seconds

[0025] (2) PCR reaction conditions: the reaction is carried out on a PCR instrument, and the reaction conditions are:

[0026] Pre-denature at 97°C for 4 minutes, and then carry out 30 cycles according to the following conditions:

[0027] Denaturation at 97°C for 15 seconds

[0028] Refolding at 53°C for ...

1. Technical field:

[0001] The invention relates to the technical field of quality identification of germplasm of traditional Chinese medicinal materials. 2. Background technology:

[0002] Honeysuckle is the dry flower bud or the first flower of Lonicera japonica Thunb., a plant of Lonicera japonica Thunb. It has strong antibacterial effect and has good curative effect on various acute and chronic inflammations. The medicinal materials of honeysuckle are obviously authentic. For example, through our preliminary determination of the content of chlorogenic acid, the content of chlorogenic acid in Henan and Shandong honeysuckle in authentic areas is generally higher than that in other non-authentic areas, which shows that the quality of medicinal materials is related to the region, habitat, etc. closely related. However, honeysuckle from non-genuine areas and authentic areas cannot be distinguished by morphological and microscopic characteristics. Due to the isolation of geo...

Examples

Embodiment Construction

[0014] a. DNA extraction: proceed as usual, and adjust the DNA concentration of the test sample to 0.1-0.3 μg / μl with deionized water;

[0015] a. Polymerase chain reaction:

[0016] (1) For polymerase chain reaction, 30 μl is used as a reference, and the dosages of various items are as follows:

[0017] 10×PCR buffer 3μl

[0018] MgCl 2 (25mM) 2.4μl

[0019] dNTP (each 10mM) 0.6μl

[0020] Primers LjP1, LjP2 (30pmol / μl) 0.5μl each

[0021] 1~2 μl DNA template for test product

[0022] TaqDNA polymerase 1U (0.2μl)

[0023] Deionized water make up to 30μl

[0024] After mixing, centrifuge at high speed for a few seconds

[0025] (2) PCR reaction conditions: the reaction is carried out on a PCR instrument, and the reaction conditions are:

[0026] Pre-denature at 97°C for 4 minutes, and then carry out 30 cycles according to the following conditions:

[0027] Denaturation at 97°C for 15 seconds

[0028] Refolding at 53°C for ...

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