Method for preventing nucleic acid contamination and indicating reaction result in nucleic acid isothermal amplification reaction

A constant-temperature amplification and nucleic acid technology, which is applied to the molecular biology detection of nucleic acid, prevents nucleic acid contamination and indicates reaction results. A wide range of applications, the effect of preventing false positives

Active Publication Date: 2013-02-20
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem that the amplified products are easy to be polluted and false positives are easy to be generated in the existing nucleic acid constant temperature amplification reaction, which leads to inaccurate in

Method used

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  • Method for preventing nucleic acid contamination and indicating reaction result in nucleic acid isothermal amplification reaction
  • Method for preventing nucleic acid contamination and indicating reaction result in nucleic acid isothermal amplification reaction
  • Method for preventing nucleic acid contamination and indicating reaction result in nucleic acid isothermal amplification reaction

Examples

Experimental program
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Effect test

experiment example 1

[0056] Experimental example 1. Detect whether the paraffin block and indicator (calcein FD) inhibit the LAMP reaction

[0057] To detect whether the paraffin block inhibits the LAMP reaction, the specific experimental method includes the following steps:

[0058] 1) Melt semi-refined solid paraffin (or fully refined solid paraffin) with a melting point of 42°C (40-60°C is acceptable), introduce it into a mold, then cool down to make it solidify, and make the paraffin suitable for the size of the reaction tube (diameter and columnar solid paraffin block with the same diameter as the reaction tube;

[0059] 2) The experiment is divided into six groups: negative control group (paraffin and calcein are not added, negative means that the template of LAMP reaction is double distilled water, which is for quality control, to ensure that the operation process and primers are not contaminated), plus FD positive group ( Add calcein, positive means that the template of LAMP reaction is t...

experiment example 2

[0063] Experimental Example 2. Using two different sets of LAMP primers to verify the blocking effect of paraffin blocks

[0064] Use two different sets of LAMP primers to verify the blocking effect of the paraffin block. The specific experimental method includes the following steps:

[0065] 1) Melt semi-refined solid paraffin (or fully refined solid paraffin) with a melting point of 42°C (40-60°C is acceptable), introduce it into a mold, cool down to make it solidify, and make the paraffin suitable for the size of the reaction tube (diameter and columnar solid paraffin block with the same diameter as the reaction tube;

[0066] 2) Experiments were divided into four groups: primer 1 plus paraffin group (primer 1 and paraffin added), primer 1 without paraffin group (primer 1 and paraffin added), primer 2 plus paraffin group (primer 2 and paraffin added), primer 2 No paraffin group (add primer 2 and paraffin);

[0067] 3) Add the reaction solution into the reaction tube. The ...

experiment example 3

[0070] Experimental example 3, the influence of different melting point paraffins on LAMP reaction

[0071] Detect the impact of different melting point paraffins on the LAMP reaction, the specific experimental method comprises the following steps:

[0072] 1) Melt semi-refined solid paraffins with different melting points (36°C, 44°C, 52°C, 60°C, 64°C) and introduce them into the mold respectively, then lower the temperature to make them solidify, and make the paraffin wax suitable for the size of the reaction tube (diameter and columnar solid paraffin block with the same diameter as the reaction tube;

[0073] 2) The experiment was divided into five groups according to the different melting points of paraffin: 36°C paraffin group (adding paraffin with a melting point of 36°C), 44°C paraffin group (adding paraffin with a melting point of 44°C), 52°C paraffin group (adding a melting point of 52 ℃ paraffin), 60 ℃ paraffin group (adding paraffin with a melting point of 60 ℃), 6...

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Abstract

The present invention provides a method for preventing nucleic acid contamination and indicating a reaction result in a nucleic acid isothermal amplification reaction. With application of a LAMP reaction as a sample, fully refined paraffin having a melting point of 40-60 DEG C or semi-refined paraffin is adopted, the principle that the paraffin is at a liquid state at a LAMP reaction temperature (60-65 DEG C) and recovers to a solid state after completing the reaction and cooling to a room temperature is adopted to prevent pollution, and turbidity can be adopted to determine a result, or an indicator can be adopted to indicate an isothermal amplification reaction result. According to the present invention, the easily-prepared wax block having a special melting point is adopted, price is low, a solid state sealing effect is good, and permanent sealing can be achieved, such that contamination on amplification products can be avoided, the false positive result of the nucleic acid isothermal amplification reaction can be effectively prevented, and the method is applicable for all nucleic acid isothermal amplification reactions.

Description

technical field [0001] The invention belongs to the molecular biological detection method of nucleic acid in the field of biotechnology, in particular to a method for preventing nucleic acid pollution and indicating reaction results in nucleic acid constant temperature amplification reaction. Background technique [0002] Nucleic acid constant temperature amplification methods currently mainly include the following: loop-mediated isothermal amplification (LAMP), nickase nucleic acid constant temperature amplification (nicking enzyme mediated amplification, NEMA), strand displacement amplification (strand displacement) amplification (SDA), rolling circle amplification (RCA), helicase-dependent isothermal DNA amplification (HAD), transcription-based amplification system (transcription-based amplification system, TAS), nucleic acid sequence-dependent amplification (NASBA), also known as autonomous sequence replication system (self-sustained sequence replication, 3SR), QB replic...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 袁静刘威黄留玉
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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