Primer and probe for detecting real-time fluorescence PCR of food allergen mustard components, kit and detection method
A technology for real-time fluorescence and primer detection, applied in biochemical equipment and methods, fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of pollution and low specificity, and achieve the effect of preventing pollution and rapid detection
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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
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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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