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Real-time fluorescent lamp detection method and kit for transgenic pigs specifically expressing human cd46

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

Active Publication Date: 2019-12-20
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • 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 fluorescent lamp detection method and kit for transgenic pigs specifically expressing human cd46
  • Real-time fluorescent lamp detection method and kit for transgenic pigs specifically expressing human cd46
  • Real-time fluorescent lamp detection method and kit for transgenic pigs specifically expressing human cd46

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. The general components and concentration are preliminarily 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 inner primers each 1.6μM, two The outer primers are 0.2μM each, the two loop primers are 0.8μM, and the DNA template is 1μL. Optimize betaine, magnesium ion concentration, dNTPs concentration, primer concentration and ratio.

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

[0064] The results show that as the concentrat...

Embodiment 2

[0071] Example 2 Sensitivity experiment

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

[0073] The sensitivity analysis result showed that the CD46 positive plasmid was diluted to 10 -5 When the dilution is 6pg / μL, the real-time fluorescent LAMP method can still detect the amplification curve, that is, the minimum detection concentration is 6pg / μL; ordinary PCR only detects 10 -3 Dilution, the lowest detection concentration is 600pg / μL, which confirms that the detection sensitivity of real-time fluorescent LAMP is 100 times that of conventional PCR.

Embodiment 3

[0074] Example 3 Specific Experiment

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

[0076] The results show that the real-time fluorescent LAMP detection method established in this study only has a specific amplification curve for CD 46 gene, and CD 39, LEA29Y and water have no amplification curve, which is 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 kit for specifically expressing human CD46 transgenic pigs. Background technique [0002] According to the latest data released by the International Diabetes Federation (IDF) in 2015, 415 million adults worldwide have diabetes. In 2015, 5 million people died of diabetes, surpassing the deaths from malaria, tuberculosis and HIV combined. The cost of treatment of diabetes and related complications is huge, estimated to exceed 670 billion US dollars per year, which is higher than the total military expenditure of the United States. Currently, treatments for diabetes include insulin injections and islet transplantation. Multi-gene modified pigs used for diabetes treatment will provide human insulin for the treatment of diabetes, and will also provide a more ideal source of donors for clinical xenotransplantation treatment. [0003] Since ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844C12N15/11
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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