Quick, simple and convenient detection method for transgenic cows with human lactoferrin gene

A technology of transhuman lactoferrin and human lactoferrin, which is applied in the field of rapid and simple detection, can solve the problems of high detection cost and high technical requirements for detection personnel, and achieve high detection rate, high sensitivity, and increased specific effect

Inactive Publication Date: 2013-02-13
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the detection of nucleic acid levels of exogenous genes, PCR amplification and electrophoresis detection are usually required. However, ordinary PCR, nested PCR and fluorescent quantitative PCR with higher sensitivity must rely on precision instruments such as PCR machines. The cost is high, and there are high technical requirements for the testing personnel, so it cannot be carried out in the basic laboratories with poor conditions.

Method used

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  • Quick, simple and convenient detection method for transgenic cows with human lactoferrin gene
  • Quick, simple and convenient detection method for transgenic cows with human lactoferrin gene
  • Quick, simple and convenient detection method for transgenic cows with human lactoferrin gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Amplification of the target fragment of human lactoferrin gene

[0039] 1. DNA extraction and purification: human blood samples and bovine blood samples were collected according to conventional methods, using the reported phenol-chloroform extraction method (refer to Sam Brook, translated by Huang Peitang. Molecular cloning experiment guide [M]. Science Press. Beijing: 2005 Version method) separately extract human and bovine genomic DNA to obtain human or bovine genomic DNA.

[0040] 2. Routine PCR amplification

[0041] Using conventional PCR amplification method (Sam Brook, translated by Huang Peitang. Molecular cloning experiment guide [M]. Science Press. Beijing: 2005), with two outer primers numbered as F3 (SEQ ID NO: 3), The primers of B3 (SEQ ID NO: 4 in the sequence listing) amplify the target fragment of the lactoferrin gene in human genomic DNA (for the nucleotide sequence see SEQ ID NO: 5 in the sequence listing or figure 1 As shown, the full length of t...

Embodiment 2

[0059] Example 2: Test of detection limit of human lactoferrin LAMP detection method

[0060] Using the plasmid pMD18T-LTF containing the human lactoferrin gene as a template, the detection limit of the LAMP method for detecting human lactoferrin was verified and compared with the detection limit of the PCR method. Specific steps are as follows:

[0061] 1. Construction of plasmid pMD18T-LTF

[0062] 1) Recovery and purification of PCR products of the target fragment of human lactoferrin gene

[0063] The PCR method was used to amplify the target fragment of the lactoferrin gene in human genomic DNA (its nucleotide sequence) with two external primers, namely primer F3 (SEQ ID NO: 3 in the sequence listing) and primer B3 (SEQ ID NO: 4 in the sequence listing). See SEQ ID NO: 5 or Figure 8 As shown, the full length of the sequence is 246bp). Reaction system: upstream primer F30.2μM, downstream primer B30.2μM, 1μl PCR buffer, 1.5mM Mgcl 2 , 75μM dNTPs, 0.5U DNA polymerase, 1μL of huma...

Embodiment 3

[0086] Example 3: LAMP detection of transgenic cattle with human lactoferrin

[0087] LAMP detection was performed on 14 transgenic cattle with human lactoferrin, and PCR method was used as control at the same time. The genomic DNA of transgenic cattle and human genomic DNA samples used in the experiment were both diluted to 20ng / μl.

[0088] 1) LAMP amplification process: FIP (SEQ ID NO: 1), BIP (SEQ ID NO: 2) as inner primers, F3 (SEQ ID NO: 3), B3 (SEQ ID NO) :4) The external primers are used to amplify human lactoferrin DNA at a constant temperature, the reaction system is: 1M betaine, 400μM dNTPs, 2.5μl 10×BstBuffer, Mg 2+ 2mM, 8U BstNDA polymerase large fragment, 1μl template, 0.2μM for outer primer, 1.6μM for inner primer, supplemented with ddH 2 O to 25μl, mix all the above reagents except enzymes and put them in a 200μl EP tube, react at 95℃ for 5min, immediately ice bath for 1-2min, add 8U Bst NDA polymerase large fragment, react at 59℃ for 60min, react at 80℃ Stop the r...

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Abstract

The invention belongs to the technical field of transgenic component detection and relates to a quick detection method for a transgenic component of transgenic cows with a human lactoferrin gene. The invention is associated with a loop-mediated isothermal amplification reaction. The method comprises the following steps: (1) designing a primer, namely designing a specific primer according to the sequence of the human lactoferrin gene, wherein the sequence of the primer is shown by sequences SEQID NO.1, SEQID NO.2, SEQID NO.3 and SEQID NO.4; (2) establishing a loop-mediated isothermal amplification reaction method, namely, performing isothermal amplification of the human lactoferrin gene by using FIP and BIP as internal primers and F3 and B3 as external primers to obtain a specifically amplified fragment; and (3) analyzing the product of the loop-mediated isothermal amplification, namely analyzing the amplification product by agarose gel electrophoresis and fluorescent dye dying, determining if there is a trapezoidal band according to the observation of a gel image or according to the result of the observation of the product of the loop-mediated isothermal amplification in sunlight by naked eye and determining if the product contains a transgenic component. The method is simple, convenient, quick, high in specificity, low in cost and suitable for basic use.

Description

Technical field [0001] The invention belongs to the technical field of detection of genetically modified components, and specifically relates to a fast and simple method for detecting genetically modified components of transgenic cattle with human lactoferrin. Background technique [0002] The safety evaluation of genetically modified organisms is the prerequisite for the marketization and commercialization of genetically modified organisms and their products. The detection of genetically modified ingredients is an important content in the safety evaluation. The establishment of fast, simple and accurate detection methods for genetically modified organisms lays the foundation for safety evaluation and management. basis. [0003] Human lactoferrin is a natural protein in human breast milk. It has important functions such as iron supplementation, antibacterial, anti-cancer, and immunity enhancement. It is an indispensable functional component for the growth and development of infants...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): C12Q1/68
Inventor刘榜翟珊莉刘楚新
OwnerHUAZHONG AGRI UNIV