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Quick and simple detection method suitable for converting humlysozyme gene cow and pig

A technology of human lysozyme and a detection method, which is applied in the field of quick and easy detection, can solve the problems of high detection cost and high technical requirements of detection personnel, and achieves the effects of high detection rate, high sensitivity and simple detection.

Active Publication Date: 2013-11-27
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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 and simple detection method suitable for converting humlysozyme gene cow and pig
  • Quick and simple detection method suitable for converting humlysozyme gene cow and pig
  • Quick and simple detection method suitable for converting humlysozyme gene cow and pig

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Amplify the target fragment of human lysozyme gene

[0038] 1. DNA extraction and purification

[0039] Human blood samples and bovine blood samples were collected according to conventional methods, and the reported phenol-chloroform extraction method (refer to Sam Brook, translated by Huang Peitang. Molecular cloning experiment guide [M]. Science Press. Beijing: 2005 edition method) was used to extract human and cattle respectively. Genomic DNA, to obtain human or bovine genomic DNA.

[0040] 2. Routine PCR amplification

[0041] Using conventional PCR amplification methods (Sam Brook, translated by Huang Peitang. Molecular cloning experiment guide [M]. Science Press. Beijing: 2005) Amplify human genomic DNA with two external primers, namely primers numbered F3 and B3 The target fragment of lysozyme gene (see SEQ ID NO:1 and the nucleotide sequence figure 1 As shown, the full length of the sequence is 190 bp). At the same time, bovine genomic DNA and pig genomic D...

Embodiment 2

[0059] Example 2: Sensitivity test of human lysozyme LAMP detection method

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

[0061] 1. Construction of plasmid pMD18T-LYZ

[0062] 1) Recovery and purification of PCR products of human lysozyme gene target fragments

[0063] The PCR method was used to amplify the target fragment of the lysozyme gene in human genomic DNA with two external primers, namely primer F3 (see SEQ ID NO: 4 for its sequence) and primer B3 (see SEQ ID NO: 5 for its sequence) (see for its nucleotide sequence) Sequence Listing SEQ IDNO: 1 or figure 1 As shown, the full length of the sequence is 190 bp). Reaction system: upstream primer F30.2μM, downstream primer B30.2μM, 1μl PCR buffer, 1.5mM Mgcl 2 , 75μM dNTPs, 0.5U DNA polymerase, 1μL human genomic DNA,...

Embodiment 3

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

[0087] LAMP detection was performed on 5 transgenic cattle with human lysozyme and 2 transgenic pigs with human lysozyme. The genomic DNA and human genomic DNA samples of the transgenic cattle used in the experiment were diluted to 20ng / μl.

[0088] 1) LAMP amplification process: FIP (sequence list SEQ ID NO: 2), primer BIP (sequence list SEQ ID NO: 3) as inner primers, F3 (sequence list SEQ ID NO: 4), B3 (sequence list SEQ ID NO) :5) The external primers are used to amplify human lysozyme DNA at a constant temperature. The reaction system is: 1M Betaine, 400μM dNTPs, 2.5μl 10×Bst Buffer, Mg 2+ 2mM, 8U Bst DNA polymerase large fragment, 1μl template, 0.2μM for outer primers, 1.6μM for inner primers, supplemented with ddH2O to 25μl, mix all the above reagents except enzymes and put them in a 200μl EP tube, react at 95℃ 5min, immediately ice bath for 1-2min, then add 8UBstDNA polymerase large fragment, react ...

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Abstract

The invention belongs to the technical field of the molecular detection of a transgenic ingredient in a transgene animal, which particularly relates to a quick and simple detection method suitable for converting human lysozyme gene cow and pig. According to the quick and simple detection method disclosed by the invention, the human lysozyme gene in the transgene cow and pig can be quickly and accurately detected by a loop-mediated isothermal amplification principle. According to the quick and simple detection method disclosed by the invention, no special equipment is required, and a quick and simple method is provided for basic layer detection personnel. According to the quick and simple detection method, a practical molecular detection method is simultaneously provided for the safety evaluation and the management of a transgene creature and the product of the transgene creature.

Description

Technical field [0001] The invention belongs to the technical field of detection of genetically modified components, and specifically relates to a quick and simple method for detecting genetically modified components of transgenic lysozyme cattle and pigs. 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. Human lysozyme is a type of lysozyme, which mainly exists in human body fluids and tissues. The activity of human lysozyme is more than 3000 times higher than that of livestock such as pigs and cattle. In addition to natural antibacterial activity, human lysozyme is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 刘榜翟珊莉刘楚新张庆德
Owner HUAZHONG AGRI UNIV