Primers, probes, standards and applications for detection of Rutaceae plant allergen genes
A technology of allergens and standards, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problem of inaccurate detection of citrus allergen gene expression levels and quantitative evaluation of citrus Sensitivity and other issues, achieve high sensitivity, strong specificity, and improve food safety
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Embodiment 1
[0036] Example 1: Retrieval of sweet orange allergen gene information and mining of homologous genes
[0037] 1.1 Allergen protein retrieval
[0038] Information on known allergens in citrus can be obtained from the website of the Allergen Nomenclature Committee under the International Federation of Immunological Societies (http: / / www.allergen.org / ), as shown in Table 1.
[0039] Table 1 Citrus allergen information
[0040]
[0041] 1.2 Mining allergen genes based on sweet orange genome
[0042] Through the amino acid sequences of allergen proteins Cits 1.01 (GenBank Protein P84159), Cits2.01 (GenBank Protein CAI23765) and Cits 3.01 (GenBank Protein P84161) in published articles, through sequence homology in the sweet orange genome (http: http: / / citrus.hzau.edu.cn / orange / ) to search for the corresponding allergen gene and obtain the gene sequence, as shown in Table 2.
[0043] Table 2 Citrus allergen gene information
[0044]
[0045]
Embodiment 2
[0046] Example 2 Citrus Allergen Gene Quantitative Primer and Taqman Probe Design
[0047] According to the comparison of homologous sequences, the sequence-specific regions were searched, and the primers and probes were designed in this region to ensure the absolute quantification of the three genes. The primers and probes in the present invention are designed using Primer 5.0 software, and the nucleotide sequences of the primers and probes are shown in Table 3 or SEQ ID NO: 1 to SEQ ID NO: 9 in the sequence table.
[0048] Table 3 Primers and Taqman probes
[0049]
[0050] Note: F in the table is the forward primer, R is the reverse primer, and P is the probe.
Embodiment 3
[0051] Embodiment 3Realtime Taqman PCR standard product template preparation and the establishment of standard curve
[0052] 3.1Realtime Taqman PCR standard template preparation
[0053] 3.1.1 Subcloning into PMD18-T vector
[0054] use After routine PCR amplification by PCR System9700, the PCR product was electrophoresed on a 1.5% TAE agarose gel and detected in a UV imager ( figure 2 ). The PCR system is shown in Table 4, and the program is shown in Table 5. PCR Master Mix (2X) was purchased from Thermo Fisher Scientific (product number: K0171). Template cDNA is from Example 4.1. The primers used are shown in Table 3.
[0055] Table 4 PCR system
[0056]
[0057]
[0058] Table 5 PCR reaction conditions
[0059]
[0060] After electrophoresis of the PCR products on 1.5% TAE agarose gel, the corresponding strips were cut out on a UV imager. use The E.Z.N.A. TMGel Extraction kit was used to recover PCR products. The specific operations are as follows: (...
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