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Real-time fluorescence LAMP detection method for specific expression human CD46 transgenic pigs and kit

A transgenic pig, real-time fluorescence technology, applied in the field of molecular biology, can solve the problems of amplification, inability to long-chain DNA, difficult to amplify, etc., and achieve the effects of shortened reaction time, high specificity and high sensitivity

Active Publication Date: 2016-12-07
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since LAMP amplification is strand displacement synthesis, the length of the target sequence is preferably within 300bp, and it is difficult to amplify if it is greater than 500bp, so long-chain DNA cannot be amplified

Method used

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  • Real-time fluorescence LAMP detection method for specific expression human CD46 transgenic pigs and kit
  • Real-time fluorescence LAMP detection method for specific expression human CD46 transgenic pigs and kit
  • Real-time fluorescence LAMP detection method for specific expression human CD46 transgenic pigs and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Optimization of LAMP reaction system conditions

[0062] LAMP is a 25μL reaction system, and the general components and concentrations are initially determined: 2.5μL 10×Isothermal buffer (including: 20mM Tris-HCl, 50mM KCl, 10mM (NH 4 ) 2 SO 4 , 2mM MgSO4, 0.1% Tween-20), 0.8M betaine, 6mM MgSO4, 1.4mM per dNTPs, 8U Bst DNA polymerase, 0.5μL SYBR Green I (25×), two internal primers each 1.6μM, two Each outer primer is 0.2 μM, two loop primers are 0.8 μM, and 1 μL DNA template. The betaine, magnesium ion concentration, dNTPs concentration, primer concentration and ratio were optimized.

[0063] (1) Optimization of magnesium ion concentration: Mg in the LAMP reaction system 2+ (MgSO 4 ) final concentrations are: 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, that is, add 25mM MgSO to the reaction system 4 In order: 0μL, 1μL, 2μL, 3μL, 4μL, 5μL, 6μL. Repeat the experiment to determine the optimum concentration ( figure 1 ).

[0064] The results show that with the i...

Embodiment 2

[0071] Embodiment 2 Sensitivity experiment

[0072] The CD46 positive plasmid was diluted 10 times, and the dilution was 10 -1 to 10 -7 Seven dilutions, according to the LAMP reaction system optimized in Example 1, were used for LAMP amplification with ESEQuant TS tube scanner to determine the lowest dilution of the real-time fluorescent LAMP detection established in this study (see Figure 5 ); while using F3 and B3 as primers, research the sensitivity of ordinary PCR detection (see Image 6 ),Compare.

[0073] Sensitivity analysis results showed that the positive plasmid for CD46 was diluted to 10 -5 Dilution, when the concentration is 6pg / μL, the real-time fluorescence LAMP method can still detect the amplification curve, that is, the lowest detection concentration is 6pg / μL; ordinary PCR can only detect 10 -3 Dilution, the minimum detection concentration is 600pg / μL, thus confirming that the detection sensitivity of real-time fluorescent LAMP is 100 times that of conve...

Embodiment 3

[0074] Embodiment 3 specificity experiment

[0075] Using positive plasmids of CD46, CD39 (insulin-specific expression gene), LEA29Y (a novel fusion protein gene that efficiently inhibits T cell activity), and water as templates, the LAMP reaction system optimized in Example 1 was carried out with ESEQuant TS tube scanner LAMP amplification to determine the specificity of the detection method ( Figure 7 ).

[0076] The results showed that the real-time fluorescent LAMP detection method established in this study only had a specific amplification curve for the CD 46 gene, but no amplification curves for CD 39, LEA29Y, and water, showing a negative reaction, indicating that the method has good specificity.

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Abstract

The invention provides a real-time fluorescence LAMP detection method for specific expression human CD46 transgenic pigs and a kit. Real-time fluorescence LAMP detection is carried out on specific expression human CD46 transgenic pigs by screening six specific primers, and a detection system and reaction conditions are optimized. The method is applicable to on-site rapid screening, closed tube detection is carried out fluorescence amplification signals, cover opening operation after the reaction is avoided, the aerosol pollution risk is lowered, and false positive occurrence possibility is greatly reduced; meanwhile, reaction time is shortened, and a judgment result can be obtained within 45 min.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a real-time fluorescent LAMP detection method and a kit for transgenic pigs specifically expressing human CD46. Background technique [0002] According to the latest data released by the International Diabetes Federation (IDF) in 2015, a total of 415 million adults worldwide suffer from diabetes. Diabetes killed 5 million people in 2015, more than malaria, tuberculosis and HIV combined. The cost of treating diabetes and related complications is enormous, estimated at more than $670 billion per year, more than the entire military expenditure of the United States. Currently, treatments for diabetes include insulin injections and islet transplantation. The multi-gene modified pigs for diabetes treatment will provide human insulin for diabetes treatment, and will also provide a more ideal source of donors for clinical xenogeneic islet transplantation. [0003] Since there are Gal ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/6848C12Q1/6888C12Q2531/119C12Q2563/107C12Q2561/113
Inventor 李刚刘巍潘登科梁琳
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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