Method for detecting PCR amplification product

A detection method and a technique for amplifying products, which are applied in biochemical equipment and methods, and the determination/inspection of microorganisms, etc., can solve problems such as the inability to quickly identify the presence or absence of PCR reaction products, achieve low cost, reduce experimental pollution, and simplify experiments program effect

Pending Publication Date: 2020-06-09
JINLING INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Purpose of the invention: In order to solve the problem that the presence or absence of PCR reaction products cannot be quickly identified, the present invention provides a detection method for PCR amplification products

Method used

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  • Method for detecting PCR amplification product
  • Method for detecting PCR amplification product
  • Method for detecting PCR amplification product

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1 Adding different concentrations of Trion X-100 to the impact of PCR result identification

[0026] Take anticoagulant blood from male pigeons, and use Dzup (blood) Genomic DNA Rapid Extraction Kit (Shanghai Shenggong) to extract DNA according to the instructions. With pigeon DNA as a template, with pigeon CHD gene primers F (5'-AGAACGTGGCAACAGAGTAC-3', the nucleotide sequence is shown in SEQ ID NO: 1) and R (5'-TGAAATGATCCAGTGCTTGT-3', the nucleotide sequence is as shown in Shown in SEQ ID NO:2) carry out PCR amplification. Composition of PCR reaction system (50 μL): 2×Master Mix 25 μL, 10 μM CHD primers F and R 1 μL each, fluorescent dye 4S Red Plus 10000 × stock solution diluted 800 times and then take 2 μL, for comparison with and without Trion X-100 For the impact on the identification of PCR results, samples 1, 3, and 5 were added with 1 μL of pigeon blood DNA template, and samples 2, 4, and 6 were not added as a control, and the volume concentration was...

Embodiment 2

[0027] Embodiment 2 adds Trion X-100 to directly identify the presence or absence of PCR reaction

[0028]The anticoagulant blood of male and female pigeons was collected respectively, and the DNA was extracted with the Dzup (blood) Genomic DNA Rapid Extraction Kit (Shanghai Sangong) according to the instructions. Using pigeon DNA as a template, with female pigeon W chromosome specific primer EE0.6P260 F (5'-TCCTATGCCTACCACCTTCC-3', the nucleotide sequence is shown in SEQ ID NO: 3) and R (5'-ATGATTCTAAAACATTTGTTGTTTG-3', The nucleotide sequence is shown in SEQ ID NO: 4) for PCR amplification. Since male pigeons have no W chromosome, only female pigeons can amplify a 260bp band. Composition of PCR reaction system (50 μL): 2×MasterMix 25 μL, pigeon blood DNA template 1 μL, 10 μM primers EE0.6P260 F and R 1 μL each, fluorescent dye SYBR GREEN I10000 × stock solution diluted 800 times and take 2 μL, the volume concentration is 5% Add 2, 3, 4, and 5 μL of Trion X-100 respectively,...

Embodiment 3

[0029] Embodiment 3 adds NP-40 to directly identify the presence or absence of PCR reaction

[0030] Anticoagulant blood was taken from male and female pigeons respectively, and DNA was extracted with the Dzup (blood) Genomic DNA Rapid Extraction Kit (Shanghai Sangong) according to the instructions. Using pigeon DNA as a template, with female pigeon W chromosome specific primer EE0.6P260 F (5'-TCCTATGCCTACCACCTTCC-3', the nucleotide sequence is shown in SEQ ID NO: 3) and R (5'-ATGATTCTAAAACATTTGTTGTTTG-3', The nucleotide sequence is shown in SEQ ID NO: 4) for PCR amplification. Since male pigeons have no W chromosome, only female pigeons can amplify a 260bp band. PCR reaction system (50 μL) composition: 2×MasterMix 25 μL, pigeon blood DNA template 1 μL, 10 μM primers EE0.6P260 F and R 1 μL each, 4S Red Plus 10000× stock solution diluted 800 times to take 2 μL, 5% NP-40 3 μL, Sterilized deionized water 17 μL. PCR reaction conditions: ①94°C for 3min; ②94°C for 30s; 53°C for 30...

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Abstract

The invention discloses a method for detecting a PCR amplification product. The method is characterized by comprising the following steps: (1) adding a fluorescent dye and a nonionic detergent into aPCR reaction system, and then carrying out PCR amplification, wherein the concentration of the fluorescent dye in the PCR reaction system is 0.1-1X, and the concentration of the nonionic detergent inthe PCR reaction system is 0.1-0.5%; and (2) observing the system obtained in the step (1) by naked eyes in a dark field under irradiation of an ultraviolet lamp, or observing the system subjected tophotographing by a fluorescent imaging system, and judging whether the PCR amplification product exists or not through the color of the system. For PCR identification which is judged by whether reacting occurs or not, the method can be used for direct judging by observation in a PCR tube by using the fluorescent imaging system or naked eyes without gel electrophoresis, so that experimental procedures are reduced, experimental operation is reduced, time is saved, and efficiency is improved.

Description

technical field [0001] The invention relates to identification of amplified products, in particular to a method for detecting PCR amplified products. Background technique [0002] PCR (polymerase chain reaction) is a molecular biology technique used to amplify specific DNA fragments. It is widely used in life science related fields, and the analysis, detection and determination methods of its products directly affect PCR. detection efficiency. At present, the most common application of PCR technology in production is to identify specific genes, genotypes or genomes by determining the presence or absence of specific product fragments, such as identification of pathogenic microorganisms, identification of specific animal source components, gender identification, and identification of transgenic components. Wait. There are mainly the following methods for product identification, analysis and identification after PCR amplification: [0003] 1. Gel electrophoresis can be divid...

Claims

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Application Information

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IPC IPC(8): C12Q1/686
CPCC12Q1/686
Inventor 徐世永陈清刘京鸽茅慧华李斯旋崔凛徐嘉雯
Owner JINLING INST OF TECH
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