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A detection method for loop-mediated isothermal amplification in transgenic sfat-1 animals
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A ring-mediated isothermal and sfat-1 technology, applied in the field of warm amplification detection, can solve the problems of high technical requirements for detection personnel and high detection costs, and achieve the effects of low cost, high accuracy and increased specificity
Active Publication Date: 2019-03-19
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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[0007] Although the PCR reaction has the characteristics of high sensitivity, high specificity, and high efficiency, it is the main detection method in the initial screening stage of genetically modified products. It requires two steps of PCR amplification and electrophoresis detection, but it must rely on precision instruments such as PCR machines. High, there are high technical requirements for the testing personnel, and it cannot be carried out in the grassroots laboratories with poor conditions
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Embodiment 1
[0060] In order to verify the detection sensitivity of the LAMP method, the FAT genome was used as the detection object, and the LAMP and PCR reactions were performed on templates with different concentrations, and the results were detected by the chromogenic method (adding calcein) and the turbidity method, respectively.
[0061] 1. The specific steps of LAMP detection sensitivity are as follows:
[0062] 1. Positive template DNA samples are preserved by the Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences;
[0063] After quantifying the positive template DNA samples, perform 10-fold dilutions so that the final nucleic acid concentrations are 126ng / μl, 12.6ng / μl, 1.26ng / μl, 126pg / μl, 12.6pg / μl, 1.26pg / μl, 0.126 pg / μl.
[0064] 2. LAMP amplification
[0065] 1) The volume of the reaction system is 25 μl, and the components are: 1 μl of templates with different concentrations, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2...
Embodiment 2
[0091] Test samples to be tested
[0092] 1. Take the tissues of the pigs to be tested with labels 1, 2, 4, 5, 9, 10, 11, 12, 13, 14, 19, and 27, and extract the genome with a kit or a conventional extraction method to obtain 1, 2 , 4, 5, 9, 10, 11, 12, 13, 14, 19, 27 and other 12 template genomes to be detected, the concentration of the sample genomes are all above 1.26ng / μl;
[0093] 2. LAMP amplification
[0094] 1) The volume of the reaction system is 25μl, and the components are: 1μl of templates with different labels, 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 0.1% Triton X-100, 0.8M Betaine, 8mM MgSO 4 , 1.4mMdNTPs, 8U Bst DNA polymerase, 40pmol FIP and 40pmol BIP, 5pmol 18F3 and 5pmol 18B3, 20pmol LB and 20pmol LF, and finally distilled water to volume.
[0095] 2) Reaction procedure: place the mixture at a constant temperature of 65°C for 60 minutes.
[0096] During the reaction process, real-time turbidity meter LA-320c was used to detect its turbi...
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Abstract
The invention relates to the field of molecular detection of transgenic ingredients in a transgenic animal, in particular to an sFat-1 transgenic animal LAMP (Loop Mediated Isothermal Amplification) detecting method. According to the method, primers 18F3, 18B3, FIP, BIP, 18LFand 18LB are used for performing LAMP on a template to be detected; and products can be detected. The sFat-1 transgenic animal LAMP detecting method provided by the invention is used for detecting sFat-1 genes in an sFat-1 gene animal; by aiming at the sFat-1 genes, three pairs of primers are designed, the specific region of a target gene sequence of the template to be detected can be specifically recognized; the LAMP can be performed under the certain temperature condition; the products can be directly distinguished through the turbidity or the color; and precise instruments such as a PCR (Polymerase Chain Reaction) instrument. The method has the advantages that the operation is simple and convenient; the sensitivity is high; the specificity is high; the consumed time is short; and the cost is low.
Description
technical field [0001] The invention relates to the field of molecular detection of transgenic components in transgenic animals, in particular to a detection method for ring-mediated isothermal amplification of transgenic animals with sFat-1 gene. Background technique [0002] The safety evaluation of GMOs is the prerequisite for the marketization and commercialization of GMOs and their products. The detection of GMO ingredients is an important content in the safety evaluation. The establishment of fast, simple and accurate detection methods for GMOs lays a solid foundation for safety evaluation and management. Base. [0003] Omega-3 polyunsaturated fatty acids (PUFAs) have been proven to play an important role in the prevention and treatment of cardiovascular diseases, arthritis, cancer and other diseases, participate in a variety of physiological and pathological processes, reduce the occurrence of cardiovascular diseases, and have Has an inhibitory effect. The ω-3 fatty...
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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2531/119C12Q2565/113C12Q2563/173
Inventor 李奎周荣陶晨雨郑新民唐中林任红艳李训碧
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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