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Two single-labeled primer detection technology and kit

A technology of technology detection and product technology, applied in the field of nucleic acid amplification PCR detection, can solve problems such as low yield, affecting technology popularization, and increasing clinical diagnosis and treatment costs

Active Publication Date: 2017-03-08
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Taqman probe method has good specificity, but the dual-labeled probe requires high oligonucleotide synthesis technology and low yield, resulting in the cost of Taqman probes being dozens of times higher than that of ordinary primers, and the cost of a single component often accounts for the entire detection. More than 60% of the cost, increasing the cost of clinical diagnosis and treatment, affecting the further popularization of this technology

Method used

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  • Two single-labeled primer detection technology and kit
  • Two single-labeled primer detection technology and kit
  • Two single-labeled primer detection technology and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: the design of primer

[0020] Design primers according to the sequence of hepatitis B virus, the forward primer is SEQ ID NO.1 in the sequence listing, and the fluorescent reporter group FAM is marked on the 16th base dG; the reverse primer is SEQ ID NO.2 in the sequence listing , the 16th base dA is labeled with the fluorescent quenching group TAMRA. The two primers were synthesized by a commercial sequence synthesis company and purified by HPLC.

Embodiment 2

[0021] Embodiment 2: the composition of amplification system

[0022] The PCR amplification system consists of Tris-HCl (pH8.3) 10mM, KCl 50mM, dATP, dGTP, dCTP, dTTP each 0.2mM, MgCl 2 3mM, 0.15μM each primer, and contains 1U Taq DNA polymerase and 10 4 Copy the HBV DNA template in a total reaction volume of 30 μl.

Embodiment 3

[0023] Embodiment 3: Reaction program setting and result judgment

[0024] Put the prepared reaction system tube on the real-time fluorescent PCR instrument, pre-denature at 94°C for 2min, then run according to the program of 94°C for 15sec and 60°C for 30sec for 40 times, and detect the fluorescence signal of the FAM channel at 60°C. After the run is finished, it can be judged that the reaction system contains the target nucleic acid sequence according to the weakening of the fluorescence signal of the FAM channel.

[0025]

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Abstract

Belonging to the field of biotechnologies, the invention relates to a double single-label primer detection technology and a kit. The method involves two primers, which are respectively labeled with a reporter group and a quenching group and used for hepatitis B virus detection. With the technology and the kit provided in the invention, the cost of fluorescent PCR method can be significantly reduced, and at the same time, the sensitive and specific characteristics of fluorescence PCR can be maintained.

Description

technical field [0001] The invention relates to a method and a kit for performing nucleic acid amplification PCR detection by using two primers respectively labeled with a reporter group and a quencher group. Background technique [0002] In recent years, nucleic acid amplification technology, especially polymerase chain reaction (PCR), has developed rapidly in experimental detection and clinical diagnosis, especially real-time fluorescent PCR technology, which can be amplified and judged by automated instruments, which improves the sensitivity of detection. [0003] Currently, there are two main methods for real-time fluorescent PCR detection technology. One is to add fluorescent dyes, such as sybgreen, to the PCR reaction system. As the PCR progresses, the fluorescent dyes penetrate into the double-stranded amplification products and emit light. However, there are defects in the specificity of this method. The background fluorescence of the dye itself and many non-specific...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 夏懿
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD