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Isothermal amplification detection primer of Macadamia allergens, kit and method thereof

A technology of constant temperature amplification detection and constant temperature amplification, which is applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the problems of rapid detection of macadamia nuts, etc., and achieve primer-dimer The effect of low probability, mild conditions and easy operation

Active Publication Date: 2015-05-20
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 macadamia nuts

Method used

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  • Isothermal amplification detection primer of Macadamia allergens, kit and method thereof
  • Isothermal amplification detection primer of Macadamia allergens, kit and method thereof
  • Isothermal amplification detection primer of Macadamia allergens, kit and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Design and screening of Australian Nuts Allergen Specific Hanger -Specific Estaber

[0060] According to the Australian Nuts Special Gene Sequence AMP2 Design 3 sets of constant temperature amplification primers. Each set of primers includes outer primers 1 and outer primers, inner primers, and inner primers 2, ring quotes 1 and ring quotes.The primer screening adopts the outer and internal primers of the 3 sets of quotes for a constant temperature amplification reaction. The 25 μL reaction system is as follows:

[0061] Outer primer 1 and outer primer 2 each 5 μm each, inner primer 1 and inner primer 2 each, 40 μm each, and Bst 2.0 Warmstar DNA polymerase 8u, pH8.8 Tris-HCL 20 mm, KCL 10 mm, (NH4) 2 So 4 10 mm, 0.1% Tween-20, DNTPS 1.6mm, MGSO 4 0.8mm, 0.8m of beet-alkali, fluorescent dye SYTO-910 μm, DNA 2 μL for samples to be checked, DDH 2 O complement to 25 μL.

[0062] The response steps are as follows:

[0063] (1) Preparation of Template DNA: Use the CTAB...

Embodiment 2

[0074] Example 2 Optimization of Australian Nuts Allergen's constant temperature amplification reaction system

[0075] Use the above -mentioned specific constant temperature amplification primers SEQ ID NO: 1 ~ 6, MG 2+ The concentration, beet -alkali concentration, DNTPS concentration and reaction temperature 4 conditions are variable parameters, and the reaction conditions for constant temperature amplification are optimized:

[0076] (1) MG 2+ Concentration optimization: set 4 mg 2+ The concentration gradient is 0.4mm, 0.6mm, 0.8mm, 1.0mm;

[0077] (2) Optimization of beet -alkaline concentration: Set 4 beet -alkaline concentration gradients 0.6m, 0.8m, 1.0m, 1.2m;

[0078] (3) DNTPS concentration optimization: Set 4 DNTPS concentration gradients 1.2mm, 1.4mm, 1.6mm, 1.8mm;

[0079] (4) Optimization of reaction temperature: Set 4 reaction temperature gradients at 63 ° C, 64 ° C, 65 ° C.

[0080] When the reaction is in progress, the peak time of the reaction curve is used as a...

Embodiment 3

[0086] Example 3 sensitivity detection

[0087] The Australian Nut Standard products are prepared to test the quality concentrations of 100%, 10%, 5%, 1%, 0.5%, 0.1%, and 0.05%for testing samples for testing.

[0088] The CTAB method extracts the DNA in the sample of the purified sample, and then uses the method of Example 2 to detect it.As a result, there are two types of judgment methods. The curve method: the "S" type amplification curve is positive, and the "S" type amplification curve is negative. See image 3 ; Color rendering method: If green is appeared, it is positive, and orange is negative. See Figure 4 Essence

[0089] In this embodiment, the curve method and color rendering method can be detected by 100%, 10%, 5%, 1%, and 0.5%of Australian nut positive samples.The results are consistent.

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Abstract

The invention discloses an isothermal amplification detection primer of Macadamia 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 specific target gene sequence AMP2 of macadimia nut, 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 present invention is the field of molecular biology technology and food safety testing. It involves the detection method of allergen components in food. LAMP detection of primer groups, detection kits and detection methods that specialize in Australian nuts. Background technique [0002] Macadamia( Macadamia Ternifolia ), Also known as Hawaii, Australian Ho Tao, Queensland, are plants in Australian Nuts in Shanlong Ophthalmology.Australian nuts are common and widely edible tree -born nuts, and are important categories of food allergen identification management.The International Standard CODEX Stan 1-1985 "CODEX STAN 1-1985" Codex Stan of the International Food Code clearly stipulates that Australian nuts and their products must be declared on the pre-packaged food label.On the basis of international standards, countries around the world have promulgated their respective regulations and provisions on the management of allergen components such as Australi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2531/119C12Q2545/101
Inventor 刘津张隽李志勇何日荣李婷凌莉刘青席静关丽军
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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