Primer and probe for detecting real-time fluorescence PCR of food allergen mustard components, kit and detection method

A technology of real-time fluorescence and detection of primers, applied in biochemical equipment and methods, fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of low specificity and pollution, and achieve the effect of preventing pollution and rapid detection

Inactive Publication Date: 2012-08-22
郑秋月 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specificity of conventional PCR is lower than that of real-time fluorescent PCR, and there are pollution problems

Method used

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  • Primer and probe for detecting real-time fluorescence PCR of food allergen mustard components, kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1. Design of primers and probes for real-time fluorescent PCR detection of food allergenic mustard components

[0024] According to the results of homologous gene screening and comparison, the mustard housekeeping gene Sin gene (SEQ ID NO.4) with high specificity was selected as the target gene for detecting amplification. According to the nucleic acid sequences of the above-mentioned genes published on NCBI, and through comprehensive consideration of factors such as the consistency of the annealing temperature of the detection primers and probes and the similarity of GC content, the following specific primers and probes were designed:

[0025] Forward primer (SEQ ID NO.1): 5'-TGAGTTTGATTTTGAAGACGATATGG-3';

[0026] Reverse primer (SEQ ID NO.2): 5'-TGTRTAACSGCTTTGGATGCTC-3';

[0027] Probe (SEQ ID NO.3):

[0028] 5′-FAM-CAGGGHCCACACAGCAGAGGCCACC-Eclipse-3′;

[0029] 2. Construction of a real-time fluorescent PCR detection kit for food allergenic mustard components

...

Embodiment 2

[0075] Embodiment 2, specificity test

[0076] The detection kit and detection method containing specific primers and probes in Example 1 were used to detect mustard allergen samples, namely white mustard, brown mustard and black mustard. Simultaneous detection of white sesame, brown rice, mung bean, corn, black soybean, snow pea, Thai basmati rice, pinto bean, black adzuki bean, soybean, pea, rice bean, black rice, carrot, Chinese cabbage, leek, rape, peanut, sorghum , Barley and other non-mustard allergen samples. To assess the specificity of the method.

[0077] Test result: no fluorescent signal is detected in the FAM channel detected by the negative control, indicating that the reaction result is normal, and the reaction system is free from pollution; the FAM channel of the positive control test result has a fluorescent signal detected, and the reaction result is normal; the invention is used to detect mustard allergens The samples, that is, white mustard, brown mustard...

Embodiment 3

[0078] Embodiment 3, sensitivity test

[0079] 1. Detection sensitivity of mustard allergen in food of plant origin

[0080] Dextrose at concentrations of 50 mg / kg and 10 mg / kg were used as mustard allergen reference substances, and the sensitivity of the method was detected by the method of Example 1. The test results show that the negative control test results show that there is no fluorescent signal detected in the FAM channel, indicating that the reaction result is normal and the reaction system is free of pollution; the positive control test result shows that the FAM channel has a fluorescent signal detected, and the reaction result is normal; the sample test result shows that , the mustard allergen reference substances with concentrations of 50mg / kg and 10mg / kg respectively can be detected well.

[0081] The results showed that the standard method had a high sensitivity of 10 mg / kg for the detection of mustard allergens in food of plant origin.

[0082] 2. Detection se...

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Abstract

The invention relates to a primer and a probe for detecting real-time fluorescence PCR of food allergen mustard components, a kit and a detection method. A sequence of an upstream primer is SEQ ID NO.1, a sequence of a downstream primer is SEQ ID NO.2, and a sequence of the probe is SEQ ID NO.3. Based on the condition, the invention discloses the real-time fluorescence PCR detection kit containing the primer and the probe sequences and the corresponding detection method. The probe and the primer have strong specificity, and the detection kit and the method have simple and easy use, accurate result and high specificity and flexibility.

Description

technical field [0001] The invention relates to a gene detection method for food allergen components. Especially the real-time fluorescent PCR detection method of food allergen mustard components and the specific primers, probes and related kits involved therein. Background technique [0002] Mustard is a general term for several herbs in the Cruciferae family, or a condiment made from its spicy seeds. The leaves and fat stalks of mustard plants are also used as greens. The main varieties are: white mustard (Sinapis alba, or yellow mustard), native to the Mediterranean region; brown mustard (Brassica juncea, or mustard greens), native to the Himalayas. Brown mustard has almost completely replaced the former use of black mustard (Brassicanigra), which is now a much hated weed because it is not suitable for mechanical farming. Food allergy is a serious food safety problem. Food processors are responsible for controlling allergens and providing correct information. The maj...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64C12N15/11
Inventor 郑秋月曹际娟徐君怡徐静
Owner 郑秋月
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