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Real-time fluorescent PCR (polymerase chain reaction) detection method of opium poppy component

A detection method and real-time fluorescence technology, applied in the field of bioengineering, can solve the problems of inability to accurately identify the source of opium poppy species, cannot fully meet the law enforcement inspection and regulation of opium poppy contraband ingredients, and achieve high sensitivity and good specificity

Inactive Publication Date: 2016-03-02
PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have a wide range of applications, but they can only conduct indirect detection of alkaloids remaining in poppy shell food, and cannot accurately characterize the source of poppy species, and cannot fully meet the needs of law enforcement, inspection and regulation of opium poppy's prohibited ingredients

Method used

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  • Real-time fluorescent PCR (polymerase chain reaction) detection method of opium poppy component
  • Real-time fluorescent PCR (polymerase chain reaction) detection method of opium poppy component
  • Real-time fluorescent PCR (polymerase chain reaction) detection method of opium poppy component

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The extraction of the DNA of embodiment 1 poppy and spices, medical material

[0038] (1) Solid sample: Take 25g of poppy seeds, grind and pulverize them with a liquid nitrogen freezing grinder, weigh 200mg of poppy seed powder into a centrifuge tube.

[0039] (2) Liquid sample: Weigh 250g of poppy seeds, pour it into a 500mL separatory filter, and let it stand for 1-2 hours. The sample will be divided into 3 layers, the upper layer is oil, the middle is the water phase, and the lower layer is solid residue. Residue treatment: separate the residue into a 100ml beaker, take part of the residue into a mortar or grinder, grind and pulverize it, and put 1mL into a 2ml centrifuge tube. Water phase treatment: After the residue is separated, add 100mL petroleum ether to the separatory filter funnel, shake for 5 minutes and let it stand still, collect the lower aqueous phase, and concentrate it to 1 -5mL, take 1mL to a 2mL centrifuge tube.

[0040] (3) Using TianGenDP-305 Pla...

Embodiment 2

[0043] The design of embodiment 2 primer pair and probe

[0044] (1) The sequences of the primer pairs and probes of this embodiment are shown in Table 1:

[0045] Table 1 Primer and probe sequences

[0046]

[0047] (2) The real-time fluorescent PCR reaction system of the present embodiment is shown in Table 2:

[0048] Among them, the TaqMan real-time fluorescent PCR reaction program is: 50°C, 2min; 95°C, 10min, 90°C, 10s, 60°C, 1min, 45 cycles.

[0049] Table 2 SYBRGreen real-time fluorescent PCR reaction system

[0050]

Embodiment 3

[0051] Example 3 Primer and Probe Specific Detection of Poppy Components Real-time Fluorescent PCR Amplification

[0052] Choose common spicy seasonings used to prepare compound seasonings, including cinnamon, star anise, bay leaves, cumin, pepper, pepper, cumin, a total of 7 kinds, and commonly used medicinal materials include Houttuynia cordata, honeysuckle, purple flower, etc. A total of 36 species were extracted and purified together with poppy seeds. The extraction conditions are shown in Table 3 and Table 4. Poppy seed DNA was used as a positive control and ultrapure water was used as a negative control. The amplification system selected in Table 2 was used for Primers were tested for specificity. Amplification results such as figure 1 .

[0053] DNA extraction situation in table 37 kinds of spices

[0054]

[0055] DNA extraction situation in table 436 medicinal materials

[0056]

[0057]

[0058] Conclusion: The primers and probes are only positive for DNA ...

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Abstract

The present invention relates to a real-time fluorescent PCR (polymerase chain reaction) detection method of opium poppy component, and the detection method comprises the following steps: 1, opium poppy seed DNA is used as a template for fluorescent quantitative PCR amplification to obtain a PCR amplification product; and 2, the fluorescence signal of the amplification product is detected, wherein the reaction system for the PCR amplification comprises an opium poppy component amplification specific primer pair and an opium poppy component amplification specific probe. The opium poppy component real-time fluorescent PCR specific primer pair and the opium poppy component real-time fluorescent PCR specific probe have good specificity and high sensitivity, a very good method for quickly and accurately detecting whether a food additive or a flavoring ingredient is added illegally with the opium poppy component is provided, and the method has good application prospects.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a real-time fluorescent PCR detection method for poppy ingredients in food or condiments. Background technique [0002] Poppy husk is the dried and mature husk of poppy, a plant of the family Papaveraceae. It has the effects of astringing the lungs, relieving cough, astringent intestines, and relieving pain. It contains more than 20 alkaloids such as morphine, codeine, and papaverine. Smoking opium poppy shells and its products will make the smokers addicted psychologically and physically, and have toxic effects on the liver, heart and other organs of the human body. Therefore, opium poppy husks are included in the scope of narcotic drugs for control in our country. As non-edible substances, it is strictly forbidden to add them to food and condiments. [0003] At present, the detection methods of poppy components mainly focus on the detection of alkaloid components deri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2545/101C12Q2561/113C12Q2563/107
Inventor 杨捷琳高琴宗凯
Owner PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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