Primer, kit and method for isothermal amplification detection of chestnut allergen

A technology of constant temperature amplification detection and constant temperature amplification, which is applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve problems such as chestnuts, and achieve low probability of primer dimers, Strong strain specificity, high specificity effect

Active Publication Date: 2018-05-01
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 for applying the LAMP method to the rapid detection of chestnut

Method used

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  • Primer, kit and method for isothermal amplification detection of chestnut allergen
  • Primer, kit and method for isothermal amplification detection of chestnut allergen
  • Primer, kit and method for isothermal amplification detection of chestnut allergen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0060] Three sets of constant temperature amplification primers were designed according to the specific sequence of chestnut leafy-like protein mRNA, 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 temp...

Embodiment 2

[0074] Example 2 Optimization of the constant temperature amplification reaction system for chestnut 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 reaction c...

Embodiment 3

[0086] Embodiment 3 Sensitivity detection

[0087] Chestnut standard samples were prepared into samples to be tested with mass concentrations of 100%, 10%, 5%, 1%, 0.5%, 0.1%, and 0.05%, respectively, for detection.

[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% chestnut positive samples, but cannot detect 0.1% and 0.05% positive samples. The results of the two methods consistent.

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Abstract

The invention discloses an isothermal amplification detection primer of Chinese chestnut 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 Leafy-likeproteinmRNA specific sequence of Chinese chestnut, 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 fields 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 chestnut allergens. Background technique [0002] Chestnut( Castanea mollissima ), also known as chestnut, Chinese chestnut, is a plant of the genus Chestnut in the family Fagaceae. Chestnut is a common and widely edible tree nut. The allergic symptoms caused by chestnut include facial redness, eye pain, redness, itching, hives, asthma, red and swollen lips, nausea, abdominal pain, low blood pressure, dizziness and other symptoms, so it belongs to Important categories of food allergen labeling management. The international standard CODEX STAN 1-1985 "General Principles for the Labeling of Prepackaged Foods" formulated by the Codex Alimentarius Commission clearly stipulates that tree nuts and their pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2600/158C12Q2531/119
Inventor 李志勇陈源树刘津凌莉席静武目涛刘青关丽军
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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