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Loop-mediated isothermal amplification primer and kit for detecting apium graveolens ingredients and application of loop-mediated isothermal amplification primer

A loop-mediated isothermal, amplification primer technology is used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to save time, reduce detection costs, and simple result judgment.

Inactive Publication Date: 2014-01-01
曹际娟 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are large differences in the detection limits of different methods, so a standard method with more accurate and repeatable test results should be adopted between institutions and countries

Method used

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  • Loop-mediated isothermal amplification primer and kit for detecting apium graveolens ingredients and application of loop-mediated isothermal amplification primer
  • Loop-mediated isothermal amplification primer and kit for detecting apium graveolens ingredients and application of loop-mediated isothermal amplification primer
  • Loop-mediated isothermal amplification primer and kit for detecting apium graveolens ingredients and application of loop-mediated isothermal amplification primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] (1) LAMP primer design steps are as follows:

[0052] ①Refer to the relevant literature of celery to understand the specific genes in different strains of celery, and select the MTD gene as the specific gene of celery;

[0053] ② Search the selected gene sequence on the NCBI website;

[0054] ③Using DNAMAN software to compare the found sequences, the comparison results show that the MTD gene has a certain degree of conservation;

[0055] ① Design two sets of specific primers, namely celery LAMP primer MTD-1 and celery LAMP primer MTD-2, the base sequences are as follows:

[0056] Celery LAMP Primer Mtd-1

[0057] F3cacaagattt gtttaatagg aact (SEQ ID NO. 1)

[0058] B3accaaacata taaaaacaga taacg (SEQ ID NO. 2)

[0059] FIP(F1c+F2)tacgggaaac aggctgtaag tattcaatat cttagtcgga tcacc (SEQ ID NO. 3)

[0060] BIP(B1c+B2) acatgctgag tcacgatgag cctgactcat cacaccgtaa (SEQ ID NO. 4)

[0061] Celery LAMP Primer Mtd-2

[0062] F3cacaagattt gtttaatagg aact (SEQ ID NO.5)

[006...

Embodiment 2

[0079] Embodiment 2 MTD-1 primer sensitivity experiment

[0080] According to the method described in Example 1, the DNA of samples celery and mustard (purchased from farmers’ markets) were respectively refined:

[0081] Dilute celery DNA with mustard DNA to a mass ratio of 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, and 0.01% gradients, and take 2 μl of each dilution for LAMP experiments. 2 O was used as a negative control instead of the DNA template, and MTD-1 was used as a LAMP primer for LAMP amplification. According to the instruction manual of the fluorescent PCR instrument, the reaction program was set as follows: the incubation stage was 63°C for 30s, 1 cycle; the cycle stage was 63°C for 15s , 63°C for 45s, 45 cycles, and collect fluorescence signals at 63°C for 45s to determine the sensitivity of the LAMP reaction.

[0082] To determine the sensitivity of the LAMP reaction, and at the same time determine the reaction time based on the peak time of the lowest sensitivity and the...

Embodiment 3

[0089] Example 3 LAMP primer specificity experiment

[0090] According to the method described in Example 1, the DNA of the following samples (purchased from farmers' markets) were refined and extracted respectively:

[0091]Celery, celery, mustard, peanuts, soybeans, black kidney beans, black sesame, white sesame, wheat, rice, panda beans, mung beans, yellow kidney beans, corn, red kidney beans, white kidney beans, buckwheat kernels, milk kidney beans, flaxseed , red bean, large white kidney bean, pea, purple kidney bean, black rice, white pea, coix seed, sorghum, oats, Arabidopsis, Chinese cabbage, cauliflower, Shanghai green, carrot, white radish, leek, Chinese cabbage, rape, milk Chinese cabbage.

[0092] The DNA of each of the samples above was extracted (after extraction, the concentration of the sample DNA was 100ng / μL) as the template for the LAMP reaction, and MTD-1 was used as the LAMP primer to carry out the LAMP reaction. The reaction system was the same as in Exa...

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Abstract

The invention discloses a loop-mediated isothermal amplification primer and kit for detecting apium graveolens ingredients and application of the loop-mediated isothermal amplification (LAMP) primer. The base sequence of the loop-mediated isothermal amplification primer is as shown as SEQ ID NO.1-4; an LAMP detection technology is adopted, and a method suitable for detection of specificity of the apium graveolens ingredients in food is created. The method provided by the invention is simple to operate, and does not rely on an expensive instrument, the detection time is about one hour, the sensitivity of the method can reach 0.5 percent, the detection efficiency of the method can reach that of a traditional PCR method or fluorogenic quantitative PCR method, and the method can be applicable to practical work of inspection and quarantine, and especially applicable to rapid preliminary screening of a large number of samples and food quality monitoring in various base laboratories, and suitable for application and promotion as an industrial recommended standard.

Description

technical field [0001] The invention belongs to the field of food inspection techniques and methods, and in particular relates to a celery component loop-mediated isothermal amplification (LAMP) primer, a kit and a detection method. Background technique [0002] Food allergy, also known as allergic reaction in medicine, refers to the production of corresponding antibodies or sensitized lymphocytes after the body is stimulated by antigens (including haptens). Cause tissue damage or physiological dysfunction of the body. It is a disease with a high recurrence rate, and the antigens that cause allergic reactions are called allergens, also known as sensitizers. Natural allergens present in food ingredients can cause allergic reactions in 6% to 8% of children and 2% of adults, which can be severe and life-threatening. Since prepackaged foods are mostly deep-processed products with complex ingredients, if they contain allergen ingredients and are not clearly announced, it may ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 曹际娟徐君怡曹冬梅孙铭英
Owner 曹际娟