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Isothermal amplification detection primers, kits and methods for sunflower seed allergens

A technology of constant temperature amplification detection and constant temperature amplification, which is applied in biochemical equipment and methods, microbe determination/testing, DNA/RNA fragments, etc., to achieve high sensitivity, easy identification, and strong strain specificity

Inactive Publication Date: 2018-03-23
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no kit method that applies the LAMP method to the rapid detection of sunflower seeds

Method used

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  • Isothermal amplification detection primers, kits and methods for sunflower seed allergens
  • Isothermal amplification detection primers, kits and methods for sunflower seed allergens
  • Isothermal amplification detection primers, kits and methods for sunflower seed allergens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Design and Screening of Sunflower Seed Allergen-Specific Isothermal Amplification Primers

[0060] based on sunflower seeds HaG5 Three sets of constant temperature amplification primers were designed for the gene sequence, and each set of primers included outer primer 1 and outer primer 2, inner primer 1 and inner primer 2, loop primer 1 and loop primer 2. For primer screening, use the outer primers and inner primers in the 3 sets of primers for constant temperature amplification reaction. The 25 μL reaction system is as follows:

[0061] 5 μM each of outer primer 1 and outer primer 2, 40 μM each of inner primer 1 and inner primer 2, Bst 2.0 Warmstar DNA Polymerase 8U, Tris-HCl 20 mM, KCl 10 mM, pH 8.8, (NH4) 2 SO 4 10 mM, 0.1% Tween-20, dNTPs1.6mM, MgSO 4 0.8mM, betaine 0.8M, fluorescent dye SYTO-9 10μM, sample DNA to be tested 2μL, ddH 2 O to make up to 25 μL.

[0062] The reaction steps are as follows:

[0063] (1) Preparation of template DNA: t...

Embodiment 2

[0074] Example 2 Optimization of the constant temperature amplification reaction system for sunflower seed allergens

[0075] Using the above-mentioned specific constant temperature amplification primers SEQ ID NO: 1 ~ 6, with Mg 2+ The four conditions of concentration, betaine concentration, dNTPs concentration and reaction temperature are variable parameters to optimize the reaction conditions of constant temperature amplification:

[0076] (1) Mg 2+ Concentration optimization: set 4 Mg 2+ The concentration gradient is 0.4mM, 0.6mM, 0.8mM, 1.0mM in turn;

[0077] (2) Betaine concentration optimization: set 4 betaine concentration gradients as 0.6M, 0.8M, 1.0M, 1.2M;

[0078] (3) dNTPs concentration optimization: set 4 dNTPs concentration gradients to 1.2mM, 1.4mM, 1.6mM, 1.8mM in turn;

[0079] (4) Reaction temperature optimization: set 4 reaction temperature gradients in order of 63°C, 64°C, and 65°C.

[0080] When the reaction is in progress, the peak time of the reac...

Embodiment 3

[0086] Embodiment 3 Sensitivity detection

[0087] The sunflower seed standard was prepared into samples with mass concentrations of 100%, 10%, 5%, 1%, 0.5%, 0.1%, and 0.05%, respectively, for testing.

[0088] The DNA in the sample to be tested was extracted and purified by the CTAB method, and then detected by the method in Example 2. There are two methods for judging the results, curve method: the one with "S" type amplification curve is positive, and the one without "S" type amplification curve is negative, see image 3 ; Chromogenic method: if it appears green, it is positive, and orange is negative, see Figure 4 .

[0089] In this embodiment, both the curve method and the chromogenic method can detect 100%, 10%, 5%, 1%, 0.5% positive samples of sunflower seeds, but cannot detect 0.1% and 0.05% positive samples. The results are consistent.

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Abstract

The invention discloses an isothermal amplification detection primer of sunflower seed allergens, a kit and a method thereof. The method is based on a loop-mediated isothermal amplification technology, six specific primers are designed according to a HaG5 gene sequence of sunflower seed, under strand displacement activity-possessing Bst2.0WarmStarDNA polymerase effect, nucleic acid amplification is carried out for 45-60minutes at 63-65 DEG C, and the amplification efficiency can reach 109-1010 copy numbers. The method has the advantages of rapidity and high efficiency, simple operation, high specialty, high sensitivity and simple identification, and is suitable for on-site detection.

Description

technical field [0001] The invention belongs to the field of molecular biology technology and food safety detection, and relates to a detection method of allergen components in food, in particular to constant temperature amplification detection primers, a kit and a method for sunflower seed allergens. Background technique [0002] Sunflower seeds belong to the genus Helianthus (Asteraceae) Helianthus annuus ) The seeds of the herbaceous plant sunflower, used for food and oil. Sunflower seeds are common and widely eaten nuts. Mild allergic reactions caused by sunflower seeds can cause urticaria, edema of the lips and tongue, redness and swelling of the face, tearing, and runny nose. In severe cases, the respiratory system will be affected, causing asthma, shortness of breath, shortness of breath. Difficulty and other symptoms, some gastrointestinal symptoms such as diarrhea, vomiting, abdominal pain, severe and even death, so it belongs to the important category of food alle...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 高东微李志勇刘津武目涛陈源树凌莉席静刘青关丽军
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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