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Primer, kit and detection method for rapid detection on ingredients of traditional Chinese medicinal material passer montanus

A technology for detection kits and detection methods, applied in biochemical equipment and methods, analytical materials, measuring devices, etc., can solve problems such as limiting on-site detection applications, and achieve the effects of simple operation, pollution avoidance, and high sensitivity

Pending Publication Date: 2022-01-14
山西省检验检测中心
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

However, no primers suitable for the PCR detection of finch brain components in Chinese medicinal materials have been found so far, and the common PCR detection technology has limitations, requiring electrophoresis detection to observe the detection results, and the price of real-time fluorescent PCR detection equipment is 5 times that of ordinary PCR detection equipment. to 10 times, these factors limit its application in on-site detection and grassroots areas where facilities are lacking

Method used

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  • Primer, kit and detection method for rapid detection on ingredients of traditional Chinese medicinal material passer montanus
  • Primer, kit and detection method for rapid detection on ingredients of traditional Chinese medicinal material passer montanus
  • Primer, kit and detection method for rapid detection on ingredients of traditional Chinese medicinal material passer montanus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] (1) Primer design

[0056] In the previous experiment, the upstream primer: LCO1490 (SEQ ID NO.3) 5'-GGTCAACAAATCATAAAGATATTGG-3', the downstream primer: HCO2198 (SEQ ID NO.4) 5'-TAAACTTCAGGGTGACCAAAAAATCA-3', (provided by Shanghai Sangon Biological Co., Ltd. Synthesized by the company), the amplification conditions are denaturation at 94°C for 5 minutes; 40 cycles of 94°C, 30s, 56°C, 30s, 72°C, 1min; 72°C extension for 10min. The sequencing results of the amplified products obtained from the samples (7 batches of samples collected in Example 5) and the results obtained after searching the database of NCBI obtained sparrows, quails, chickens, partridges, pigeons, geese, ducks, parrots, The sequence of the cytochrome oxidase 1 gene (CO1 sequence) of other common related species such as turkey, the homology comparison of these 7 sequencing samples and the sequence retrieved by NCBI to ensure that the sequence has no variation site at the primer, the obtained The target g...

Embodiment 3

[0094] In order to quickly extract the DNA in the sample, a reagent for rapid DNA extraction has been developed. The reagent for rapid DNA extraction includes a lysate and a cleaning solution. 100, 1.5% PVP, 150mmol / L NaCl, 0.5% polysorbate-20, 5mmol / L EDTA, 1.5% polyethylene glycol, the solvent is PBS solution (pH7.4). The wash formulation was PBS (pH 7.4) containing 0.15% polysorbate-20.

[0095] Use filter paper strips to adsorb DNA. The filter paper strips can be rectangular filter paper strips of 44mm×2mm, or filter paper strips soaked in paraffin at one end. The details are as follows: Use scissors to cut the filter paper into rectangular filter paper strips of 44mm×2mm Dip one end of the strip into heated and melted paraffin. After drying, a hydrophobic handle area of ​​40mm×2mm is formed for transferring the filter paper strip. The part of the front end that is not dipped in paraffin is the nucleic acid binding area, which is used for subsequent nucleic acid adsorptio...

Embodiment 4

[0111] Embodiment 4 Bird brain component nucleic acid detection test strip specificity experiment

[0112] Sample pretreatment: take 16 kinds of animal brain samples respectively (corresponding 16 kinds of animal brain tissue 1: sparrow 2: pigeon 3: goose 4, pig 5: donkey 6: fox 7: salmon 8: black snake 9: quail 10: Chicken 11: Rabbit 12: Sheep 13: Duck 14: Cattle 15: Horse 16: Mink) 20mg, grind the above sample into powder or paste, put it in a 2mL centrifuge tube, add 500μL lysate to lyse the sample for 7s, filter paper The strips were immersed in the lysate for 3s to absorb nucleic acids, and then the filter paper strips were transferred to 1750μL washing solution for 3s to wash away the impurities adsorbed on the surface. Finally, the filter paper strip was transferred to the prepared amplification reaction system for 3 seconds to elute the nucleic acid adsorbed on the surface, and the amplification system was prepared.

[0113] (2) Amplification reaction system, using th...

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Abstract

The invention relates to a primer, kit and detection method for rapid detection on ingredients of a traditional Chinese medicinal material passer montanus. Through nucleic acid thin-film chromatography detection test paper, visual observation of nucleic acid detection results can be realized. The ingredients of the traditional Chinese medicinal material passer montanus are detected through nucleic acid detection test paper, a detection result of a single sample can be obtained only in 65-70 minutes from pretreatment to detection completion, the detection test paper is free of non-specific reaction with other common animal-derived ingredients, and the detection test paper is easy and convenient to operate, high in specificity and sensitivity, low in cost of needed equipment, high in efficiency and especially suitable for on-site rapid detection and rapid market detection of basic supervision units.

Description

technical field [0001] The invention relates to a primer, a kit and a detection method for rapid detection of finch brain components of Chinese medicinal materials, and belongs to the technical field of biological detection. Background technique [0002] Finch brain is processed from the fresh brain tissue of the bird family sparrow (Passer montanus saturatus Stejneger). There are records in "Southern Yunnan Materia Medica", "Modern Health Care Traditional Chinese Medicine Dictionary", "Chinese Animal Chronicle", "Chinese Animal Medicine Chronicle", "Chinese Medicine Dictionary", "Meat, Poultry, Eggs and Milk Health", "Chinese Materia Medica", etc. Bird's brain is sweet in taste, flat in nature, and belongs to the kidney meridian. It has the effects of nourishing the kidney and rejuvenating yang, moisturizing the skin and promoting muscle growth. It should be taken orally. The application preparations are Guilingji Capsules and Yougui Pills. [0003] Finch brain is a cold...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6851C12N15/11G01N33/558G01N33/543
CPCC12Q1/6888C12Q1/6851G01N33/558G01N33/54313C12Q2531/113C12Q2563/107Y02A50/30
Inventor 朴晋华董培智杨宝李忠华连云岚马宁
Owner 山西省检验检测中心
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