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Detection kit and detection method of honeysuckle

A detection method and detection reagent technology, applied in DNA/RNA fragments, recombinant DNA technology and other directions, can solve problems such as difficulty in mastering, and achieve the effects of simple operation, ensuring food safety, and good application prospects.

Inactive Publication Date: 2016-04-13
LUZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of counterfeit honeysuckle is usually based on the color, shape and other traits as the standard, which is difficult to grasp and has certain limitations

Method used

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  • Detection kit and detection method of honeysuckle
  • Detection kit and detection method of honeysuckle
  • Detection kit and detection method of honeysuckle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 RAPD technology amplification of honeysuckle-specific SCAR markers

[0024] 1. Extraction of plant DNA by CTAB method

[0025] 1. Collect samples of honeysuckle and other plant species from Guangdong, Guangxi, Sichuan Emei, Sichuan Leshan and Hunan, and collect honeysuckle from Houston, USA. Store in a 4°C refrigerator for later use;

[0026] 2. Take about 2g of plant leaves or flower buds, crush them and put them in a mortar, add quartz sand and quickly grind them into powder, and put the powder into a 5ml centrifuge tube, the amount of which is about 1 / 3 of the centrifuge tube;

[0027] 3. Add 2ml of CTAB extraction buffer (Hexadecyltrimethyl-ammoniumbromide) preheated at 65°C and 4µl of β-mercaptoethanol, mix thoroughly, put in a water bath at 65°C for 40min, and shake once every 10min to avoid agglomeration;

[0028] 4. Add an equal volume of chloroform:isoamyl alcohol (24:1) to each tube, oscillate fully for 5 minutes to make it milky white, centrifuge ...

Embodiment 2

[0140] Embodiment 2 uses the method of the present invention to specifically amplify the honeysuckle gene

[0141] 1. Experimental method

[0142] (1) According to the sequences shown in SEQ ID NO.1-4 obtained in Example 1, 4 pairs of primers were designed and synthesized.

[0143]

[0144] (2) PCR amplification

[0145] 1. Take a number of samples of honeysuckle and other species (18 samples) and one DNA variety of Lilynia japonica in Xiushan County, Chongqing, and dilute it to 10ng / μl for later use.

[0146] 2. PCR amplification:

[0147] (1) PCR system (10μl)

[0148]

[0149] After fully mixing, centrifuge at 12000rpm for 15s in a centrifuge, and place in a PCR instrument (Applied Biosystems 96-Well Thermal Cycler, Life Technology, USA).

[0150] (2) PCR amplification conditions

[0151]

[0152]

[0153] 3. Agarose gel electrophoresis (the concentration of the gel depends on the size of the target fragment).

[0154] For details, please refer to "Refined...

Embodiment 3

[0167] Embodiment 3 honeysuckle detection kit of the present invention

[0168] 1. Kit composition

[0169] This kit contains:

[0170] CTAB extraction buffer (200ml);

[0171] 2×TaqMasterMix, wherein the primers can be selected from a pair of primers or multiple pairs of primers (500 μl) in the primers shown in SEQ ID NO.5-6, SEQ ID NO.7-8, and SEQ ID NO.9-10;

[0172] One tube (50 μl) of negative control template DNA (Mountain Silver Flower DNA);

[0173] One tube (50 μl) of positive control template DNA (Honeysuckle DNA);

[0174] Sterilized ddH 2 O (1500 μl).

[0175] (1) CTAB extraction buffer

[0176] CTAB extraction buffer (100ml)

[0177]

[0178] Adjust to pH 5.0 with HCL, add H 2 O to 100ml.

[0179] (2) Formula of PCR system 2×TaqMasterMix

[0180]

[0181] 2. Detection method

[0182] (1) PCR amplification

[0183] 1. CTAB extraction buffer is used to extract the DNA of the sample to be tested.

[0184] 2. Perform PCR amplification:

[0185] det...

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PUM

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Abstract

The invention discloses a honeysuckle nucleotide sequence and further discloses application of a reagent for detection the sequence in preparation of a honeysuckle detection reagent, a honeysuckle detection kit and a detection method. The detection kit and the detection method can accurately and effectively distinguish honeysuckles, are good in specificity, consume short time and have good application prospect.

Description

technical field [0001] The invention relates to a detection kit and detection method of honeysuckle. Background technique [0002] Honeysuckle has another name called honeysuckle (scientific name: Lonicera japonica). The medicinal material honeysuckle is the dried flower buds or flowers of the same genus Lonicera Lonicera and the same genus. Honeysuckle has been known as a good medicine for clearing away heat and detoxification since ancient times. It is sweet and cold in nature and fragrant, sweet and cold to clear away heat without hurting the stomach, and the fragrance is thorough and can dispel evil. Honeysuckle can not only disperse wind-heat, but also clear blood toxin. It is used for various febrile diseases, such as body heat, rash, spots, heat-toxin sores, sore throat, etc., and the effect is remarkable. Modern studies have proved that honeysuckle contains chlorogenic acid, luteolin and other pharmacologically active ingredients, which have strong inhibitory effe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895
Inventor 傅俊江
Owner LUZHOU MEDICAL COLLEGE