LAMP (Loop-Mediated Isothermal Amplification) primer composition for assaying peanut allergen Ara h6 gene and kit thereof
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A peanut allergen and primer combination technology, applied in DNA/RNA fragments, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of unused peanut allergen detection, complicated operation process, expensive instruments, etc. The effect of simple result determination, wide application range and simple operation
Inactive Publication Date: 2017-11-03
SHANGHAI IGENETEC DIAGNOSTICS CO LTD +2
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[0006] 2) Detection of allergen gene residues, such as PCR detection technology, pyrosequencing technology, etc. Among them, PCR detection method is the most important and most accurate method, but the operation process of this type of method is relatively complicated and requires professionals; the instruments used are relatively Expensive and time consuming to test
[0008] Loop-Mediated Isothermal Amplification (Loop-Mediated Isothermal Amplification, hereinafter referred to as LAMP method) is a fast, efficient and specific nucleic acid isothermal amplification technology established by Notomi et al. High, easy to popularize, safe and reliable, etc. At present, this technology has not been used to detect peanut allergens
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Embodiment 1
[0056] This embodiment is a peanut allergen fluorescence detection method:
[0057] 1. LAMP detection kit for peanut allergen Ara h6 gene, which includes LAMP working solution, primer solution, BstDNA polymerase, and control, as follows:
[0069] 4) Bst DNA polymerase: the concentration is 8 U / μL;
[0070] 5)...
Embodiment 2
[0085] In this example, the detection method of implementation 1 is used to detect the fluorescence of peanuts from different origins, and the detection results are as follows: figure 2 shown.
[0086] Through experiments on peanut samples from different origins (purchased from Suzhou, Qingyang, Tongzhou, and Zhengyang), it was found that the peanut samples from four different origins all had amplification signals ( figure 2 ), other negative controls have no amplification signal (negative controls are soybean, pistachio, almond, hazelnut, sesame, walnut, mustard, water).
Embodiment 3
[0088] In this example, the detection method of Implementation 1 is used to detect DNA samples with different concentrations to analyze the sensitivity of peanuts. The DNA concentrations are 10, 1, 1×10 -1 , 1×10 -2 , 1×10 -3 , 1×10 -4 , 1×10 -5 , unit ng / μL, the fluorescence detection results are shown in the figure image 3 shown.
[0089] according to image 3 As shown in the peanut sensitivity fluorescence detection results, it was found that 10ng / μL and 1ng / μL had amplification signals ( image 3 ), while there is no amplification signal at other concentrations, the amplification signal in the range from 1ng / μL to 0.1ng / μL can be further explored to further determine the detection sensitivity.
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Abstract
The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) primer composition for assaying a peanut allergen Ara h6 gene, an assay kit and an assay method. The assay primer composition consists of sequences shown as SEQ ID NO: 1 to SEQ ID NO: 4. The assay kit consists of an LAMP working solution, a fluorescence indicator and / or developer, Bst DNApolymerase, the LAMP primer composition, a positive control, and a negative control. The assay method includes the following steps: a CTAB (Cetyl Trimethyl AmmoniumBromide) method is used for extracting the DNA of a sample to be assayed, the sample DNA is then added into a reaction system, and is amplified under an isothermal condition of 60 DEG C to 65 DEG C for 45 to 90 minutes, and a fluorescencedetector is used for detecting the fluorescence curve of an amplification result and / or the color change in a reaction tube filled with the developer is directly observed to judge whether peanut allergen is contained in the sample. The primer composition, the assay kit and the assay method have the advantages of quickness, simplicity in operation, high specificity and high sensitivity, and is applicable to real-time field test.
Description
technical field [0001] The invention relates to the technical field of gene detection, in particular to a LAMP primer composition, a detection kit and a detection method for detecting the peanut allergen Ara h6 gene. Background technique [0002] Food allergy is an abnormal immune response produced by the body's immune system to certain foods. The corresponding immunoglobulins will combine with these foods and release many chemical substances, causing allergic symptoms (such as nausea, vomiting, eczema, etc.), and severe cases can cause Anaphylactic shock occurs. The World Food and Agriculture Organization reported in 1995 that more than 90% of food allergies were caused by peanuts, soybeans, milk, eggs, fish, shellfish, and nuts. According to reports, peanut is an important food allergen, accounting for 10% to 47% of food allergies, which can cause allergic diseases such as allergic enteritis, allergic gastritis, and even anaphylactic shock and anaphylactic death in severe...
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