Method and kit for amplifying and detecting nucleic acid

A target nucleic acid and polymerase technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of inability to realize target detection of highly mutated species, influence, and limit popularization and application

Pending Publication Date: 2021-08-06
青岛简码基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

LAMP technology is well known for its high sensitivity and specificity, but this technology is prone to sample contamination, and the design of primers is difficult, making it impossible to detect highly mutated species targets
However, the reaction system of HDA technology requires two enzymes, and the dual-enzyme system is likely to cause non-specific amplification, which affects the judgment of the experimental results.
These shortcomings limit the popularization and application of these technologies to a certain extent.

Method used

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  • Method and kit for amplifying and detecting nucleic acid
  • Method and kit for amplifying and detecting nucleic acid
  • Method and kit for amplifying and detecting nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0220] 5.2 Example 2: Optimization of polymerase concentration for fast strand displacement amplification (SEA) reaction system.

[0221] The following studies were performed to test the effect of polymerase concentration on the rate of fast SEA reactions.

[0222] The same primers (SEQ ID NO: 1 and 2) were designed against the same target sequence in the above L. monocytogenes genome (SEQ ID NO: 1) as described in Example 1 above. Prepare the primers and the L. monocytogenes genome as described above and mix with the other PCR reactions to form a 10 µL amplification mix as shown in Table 2 below. In order to achieve the optimal amplification rate with the polymerase concentration, four amplification mixtures containing different enzyme concentrations were prepared, each containing a final concentration of 3.0×10 -6 M with primers and final concentrations of 0.16 U / μL, 0.20 U / μL, 0.24 U / μL, and 0.28 U / μL (corresponding to 8 U / μL enzyme stock solution of 0.20 μL, 0.25 μL, 0.30...

Embodiment 6

[0255] 5.6 Example 6: Comparison of isothermal SEA reaction under constant temperature conditions and fast SEA reaction under rapid thermal cycle conditions

[0256] The following study compares the isothermal SEA reaction carried out under constant temperature conditions (such as the procedure described in CN 109136337A) and the fast SEA reaction under the current fast thermal cycle conditions.

[0257] Specifically, the same primers (SEQ ID NO: 1 and 2) were designed for the same L. monocytogenes genome (SEQ ID NO: 1) target sequence according to the method described above. The primers and L. monocytogenes genomic material were obtained according to the method mentioned above, and mixed with other PCR reactions to form a 10 μL amplification mixture, the components of which are shown in Table 5 below. In addition, this study set up a series with different initial concentrations (1.0×10 -11 M, 1.0×10 -12 M, 1.0×10 -13 M, 1.0×10 -14 M, 1.0×10 -15 M, 1.0×10 -16 M, 1.0×10 ...

Embodiment 7

[0266] 5.7 Example 7: Primer Design

[0267] Use NUPACK network tool (www.nupack.org), DNAMelt Web (http: / / unafold.rna.albany.edu / ?q=DINAMelt), NOVOPRO www.novopro.cn / tools / rev_comp.html) and NCBI website ( The BLAST algorithm at www.ncbi.nlm.nih.gov / tools / primer-blast) was used for primer design and evaluation.

[0268] DNA primers were synthesized by Personal Biotechnology Co., Ltd. (Shanghai, China). The SEA detection kit was purchased from Navid Biotechnology Co., Ltd. (Qingdao, China). DNA extraction kit was purchased from Tiangen Biotechnology Co., Ltd. (Beijing, China). Other reagents and buffers were of analytical grade.

[0269] traditional PCR reaction : Genomic DNA of Mycoplasma pneumoniae, Chlamydia trachomatis, pork, Bacillus cereus and Staphylococcus aureus were extracted using TIANamp DNA Extraction Kit (Beijing Tiangen Biotechnology Co., Ltd., Beijing) according to the instructions provided by the manufacturer. Using CFX Connect TM Real-time quantitative...

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Abstract

The invention relates to a method and a kit for amplifying and detecting nucleic acid. The invention provides a method for denaturation bubble mediated target nucleic acid amplification and a related kit and use thereof. According to the method, generation of denatured bubbles in double-stranded target nucleic acid molecules is promoted through rapid temperature-changing thermal circulation, so that the strand exchange amplification (SEA) reaction is accelerated. The kit comprises a specially designed primer and a polymerase for performing the method. The method and the kit disclosed herein can be used in various situations, such as diagnosis of infectious or genetic diseases, sample quality control, and single nucleotide polymorphism (SNP) analysis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an improved method for amplifying target nucleic acid mediated by denatured bubbles, a special kit and application thereof. [0002] 1. Background technology [0003] Nucleic acid can be divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and is the basic element of all life forms. DNA carries genetic information and is responsible for encoding amino acids, the building blocks of proteins. RNA plays an important role in the coding, decoding, regulation and expression of genes. Therefore, nucleic acids have been used as important biomarkers in biological research and medical diagnosis. Nucleic acid amplification technology provides an important theoretical basis for the detection of pathogenic microorganisms, the traceability and authenticity of biological materials (such as meat), and other genetic testing. Establishing a simple, easy-to-operate, sensitive and rapi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/689
CPCC12Q1/6844C12Q1/689C12Q2527/101C12Q2531/119C12Q2527/149
Inventor 石超刘蒙蒙李阳马翠萍
Owner 青岛简码基因科技有限公司
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