Direct-spread RPA (recombinase polymerase amplification) on-site visibility detection method

A detection method and amplification reaction technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc.

Inactive Publication Date: 2018-08-03
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although the existing RPA detection methods are widely used, there are still some defects, among which, the most important performance is in the detection of products.

Method used

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  • Direct-spread RPA (recombinase polymerase amplification) on-site visibility detection method
  • Direct-spread RPA (recombinase polymerase amplification) on-site visibility detection method
  • Direct-spread RPA (recombinase polymerase amplification) on-site visibility detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1 is used to detect the acquisition of the RPA primer set of transgenic maize TC1507

[0100] Primers are designed according to the conserved sequence of transgenic maize TC1507 found in GMDD (GMO Detection method Database). Due to the immature development of RPA technology, there is no special software for designing primers like PCR or LAMP. Therefore, the primer design of RPA requires manual Manually designed, further verified in experiments, and screened out the most suitable primer pairs.

[0101] When designing RPA primers, the following principles need to be followed:

[0102] 1) The length of the primer is 30-35bp. The length of the primer affects the activity of the recombinase. Too short a primer will reduce the efficiency of the recombinase. Possibility of primer secondary structure formation, which does not necessarily increase efficiency;

[0103] 2) The GC content is 40-60%. Among them, the presence of cytosine in the 3-5 nucleotides at the 5' end...

Embodiment 2

[0124] Embodiment 2RPA primer screening

[0125] In this example, three pairs of primers for TC1507 will be designed according to Twist DX The instruction manual of Liquid BasicKit was used to carry out the experiment, and a pair of primer sets that amplified better target bands were screened out.

[0126] Preparation of RPA reaction system: ddH 2 O 0.2 μL, Primer F / R 2.4 μL at a concentration of 10 μM, 25 μL of 2×ReactionBuffer, 9 μL of dNTPs at a concentration of 10 mM, 5 μL of 10×Basic E-mix, 2.5 μL of 20×Core Reaction Mix and 2.5 μL of MgOAc at a concentration of 280 mM, extracted Genomic DNA 1 μL.

[0127] The RPA reaction program is: 37°C, 20min.

[0128] The RPA results were detected by agarose gel electrophoresis, and the target fragment lengths of T6, T9, and T10 were 158bp, 159bp, and 161bp, respectively. Test results such as figure 1 As shown, T6, T9 and T10 are samples of different primers of transgenic maize TC1507 respectively, and N6, N9 and N10 are negati...

Embodiment 3

[0130]1) Use a DNA extraction kit (Shanghai Boman Biotechnology Co., Ltd.) and refer to the kit instructions to extract genomic DNA from transgenic corn TC1507.

[0131] 2) Prepare RPA reaction system

[0132] Set up a negative control (ddH 2 O) system, with the genome containing the target sequence of transgenic maize TC1507 as positive, prepare the RPA reaction system, set up a negative control to prove that the reaction system is not contaminated by other genomes, achieve the purpose of distinguishing positive samples, and verify the correctness of the measurement results.

[0133] In order to prevent false positive results in the experiment, when preparing the RPA reaction system and the control system, partition operations were performed.

[0134] When preparing the reaction system, without adding template, first prepare the outer primer F, inner primer R, 2×Reaction Buffer, dNTPs, 10×Basic E-mix and mix well, then add 20×Core Reaction Mix on the cap of the tube, Mix we...

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Abstract

The invention discloses a direct-spread RPA (recombinase polymerase amplification) on-site visibility detection method which comprises the following steps: 1) roughly extracting genomic DNA (deoxyribonucleic acid) of a to-be-tested sample; 2) designing an RPA specific primer for a target gene; 3) adding the RPA specific primer into an RPA reaction system, and performing an PRA amplification reaction, wherein the concentrations of components in the RPA reaction system are as follows: the concentration of an outer primer F is 0.12-0.48 mu M, the concentration of an inner primer R is 0.12-0.48 muM, the concentration of dNTPs is 1.4-1.8mM, the concentration of MgOAc is 8.4-14mM, the concentration of template DNA is 1-2 mu L; the reaction procedure is carried out at 32-42 DEG C for 5-10 minutes; 4) identifying amplification results, namely putting an SYBR Green I dye into an amplification product, directly observing color change with day light or sunshine, and judging that samples of greenare positive and samples of orange are negative. The method can be applied to RPA detection on all genes from animals, plants and microorganisms, and rapid, simple, convenient and visible identification on RPA amplification products can be achieved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and specifically relates to a direct expansion RPA on-site visual detection method that can be used for all gene detection, and also relates to a direct expansion RPA on-site visual detection method for detecting transgenic corn TC1507 and Staphylococcus aureus. Background technique [0002] At present, when it comes to gene detection, traditional molecular diagnostic methods mainly include PCR, real-time fluorescent quantitative PCR, gene chip and loop-mediated isothermal amplification method (LAMP), and recombinase polymerase amplification technology (Recombinase polymerase amplification, RPA). RPA is a new type of isothermal amplification technology. Its main principle is to use recombinase and primers to form microfilaments to search for a sequence that is completely complementary to it on the template DNA, and to make the template DNA with the help of single-stranded DNA b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/6895C12Q1/689C12Q1/14C12N15/11
CPCC12Q1/6844C12Q1/689C12Q1/6895C12Q2531/119C12Q2563/107C12Q2527/137C12Q2527/143C12Q2527/146
Inventor 赵凯范小瑞赵笑杜亚楠张琪张晓霞吴文静张皖静赵庆史斌赵桓震赵斌安潘磊
Owner SHANGHAI ACAD OF AGRI SCI
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