Unlock instant, AI-driven research and patent intelligence for your innovation.

Method for detecting L-trpyptophan based on rolling circle amplification technology

A rolling circle amplification technology, rolling circle amplification technology, applied in the field of detection of L-tryptophan based on rolling circle amplification technology, can solve the problems of cumbersome operation, human error, high requirements for equipment maintenance and standardization, and achieve operational Simple, easy-to-step effects

Active Publication Date: 2015-03-25
中国医科大学
View PDF2 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these detection and analysis methods all require large-scale professional analytical instruments and equipment, which not only require professionals to operate, but also have relatively high requirements for equipment maintenance and standardization
Cannot prepare convenient miniaturized kits
At present, the only ELISA method that can be used to detect tryptophan with a kit, this method uses the combination of tryptophan antibody and tryptophan to detect tryptophan, although the requirements for large-scale professional equipment are reduced, but this detection method needs to include Covering, plating, sample addition, antibody addition, color development, and multiple washing steps between each step are cumbersome and prone to human error

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for detecting L-trpyptophan based on rolling circle amplification technology
  • Method for detecting L-trpyptophan based on rolling circle amplification technology
  • Method for detecting L-trpyptophan based on rolling circle amplification technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The present invention quantitatively measures the relationship between L-Trp concentration and RCA inhibitory rate ( image 3 ). Specific steps are as follows:

[0027] (1) Preparation of the ring template:

[0028] Prepare 10 μl of ligation reaction system buffer (50mM Tris pH 8.0, 10mM MgCl 2 , 5mM DTT and 0.1mM ATP), add 10pmol linear single-stranded template DNA "ON-CT", 10pmol linear single-stranded primer DNA "ON-P" and 175U T4 DNA ligase, and incubate at 16°C for 1 hour. See Table 1 for the DNA sequence.

[0029] (2) Preparation of samples:

[0030] Prepare L-tryptophan aqueous solutions with molar concentrations of 0, 5, 10, 20, 40, 60 and 80 μM.

[0031] (3) Rolling circle amplification detection:

[0032] Prepare 100 μl of polymerization reaction system buffer (50mM Tris pH 7.5, 10mM MgCl 2 , 10mM (NH 4 ) 2 SO 4 , 4mM DTT, SYB Green II (1:10000), 3U phi29DNA polymerase, 0.5pmol ligation reaction product and trpR2.5μM), add different amounts of L-Trp ...

Embodiment 2

[0037] The present invention specifically identifies L-Trp and other 19 kinds of amino acids ( Figure 4 ) The specific steps are as follows:

[0038] (1) Preparation of the ring template:

[0039] Prepare 10 μl of ligation reaction system buffer (50mM Tris pH 8.0, 10mM MgCl 2 , 5mM DTT and 0.1mM ATP), add 10pmol linear single-stranded template DNA "ON-CT", 10pmol linear single-stranded primer DNA "ON-P" and 175U T4 DNA ligase, and incubate at 16°C for 1 hour. See Table 1 for the DNA sequence.

[0040] (2) Preparation of samples:

[0041] Preparation molar concentration is the aqueous solution of the L-tryptophan of 80 μ M, and the preparation molar concentration is glycine (Gly), L-alanine (L-Ala), L-isoleucine (L-Ile), L-leucine ( L-Leu), L-methionine (L-Met), L-valine (L-Val), L-arginine (L-Arg), L-histidine (L-His), L-lysine (L-Lys), L-aspartic acid (L-Asp), L-glutamic acid (L-Glu), L-asparagine (L-Asn), L-glutamine (L-Gln), L-serine ( L-Ser), L-threonine (L-Thr), L-...

Embodiment 3

[0048] The present invention qualitatively identifies L-Trp and six kinds of L-Trp analogs ( Figure 5 ). Specific steps are as follows:

[0049] (1) Preparation of the ring template:

[0050] Prepare 10 μl ligation reaction system buffer (50mM Tris pH 8.0, 10mM MgCl 2 , 5mM DTT and 0.1mM ATP), add 10pmol linear single-stranded template DNA "ON-CT", 10pmol linear single-stranded primer DNA "ON-P" and 175U T4 DNA ligase, and incubate at 16°C for 1 hour. See Table 1 for the DNA sequence.

[0051] (2) Preparation of samples:

[0052] Preparation molar concentration is the aqueous solution of L-tryptophan of 80 μ M, preparation molar concentration is L-kynurenine (L-Kynurenine), indole acetic acid (IAA), melatonin (Melatonin), 5-hydroxytryptamine (5- HT) in aqueous solution, and an aqueous solution of trytamine (Trytamine) and D-tryptophan (D-Trp) with a molar concentration of 400 μM.

[0053] (3) Rolling circle amplification detection:

[0054] Prepare 100 μl of polymeriza...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a method for detecting L-trpyptophan based on a rolling circle amplification technology. According to the method, L-trpyptophan is combined with trp repressor (trpR) to lead to conformational change of trpR and can be specifically combined on a recognition sequence of DNA double chains, so that RCA is inhibited. By detecting the amplification efficiency of RCA, the concentration of L-Trp is calculated. The method provides a fast, simple and quantitative measuring technology for detecting L-Trp and has an important scientific value and a broad application prospect.

Description

technical field [0001] The invention relates to detection methods for biochemistry, molecular biology, and amino acid composition confirmation, in particular to a method for detecting L-tryptophan based on rolling circle amplification technology. Background technique [0002] L-tryptophan (L-Trp) is one of the 20 natural amino acids that make up proteins and one of the eight essential amino acids for the human body. As an important nutrient, tryptophan is contained in many foods (raw materials, semi-finished products, products, etc.), beverages and condiments. How to conveniently, quickly and accurately quantitatively analyze tryptophan is of great significance in food testing. [0003] In addition to protein synthesis, tryptophan has many metabolites that have important biological functions and significance. Tryptophan in the human body is mainly metabolized through two pathways: the serotonin pathway and the kynurenine pathway. In the serotonin metabolic pathway, namely...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2531/125C12Q2525/307
Inventor 赵国杰关一夫
Owner 中国医科大学