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Detection method for isothermal amplification of single or multiple target gene fragments

A gene fragment and target technology, which is applied in the field of amplifying the target gene fragment and detecting the amplification result, can solve the problems such as single amplification or multiple amplification detection is difficult to overcome, rapid detection cannot be realized, and promotion and use are restricted. Achieve strong multiple detection capabilities, fast detection speed, and easy results to read

Active Publication Date: 2015-10-07
ICDC CHINA CDC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method is cumbersome to operate and requires specific kits to purify LAMP products. The sequencing process requires special personnel, as well as sequencers and sequencing reagents that cannot be afforded by ordinary laboratories.
These disadvantages limit the widespread use of this method
In addition, none of the existing multiple LAMP detection technologies can achieve rapid detection, and it takes more than 2.5 hours to complete multiple LAMP detection
Due to the high sensitivity of the LAMP reaction, the operation of uncapping the LAMP product will cause great pollution to the subsequent LAMP experiments.
[0006] In summary, the LAMP in the prior art has insurmountable technical problems no matter in the detection of single amplification or multiple amplification.

Method used

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  • Detection method for isothermal amplification of single or multiple target gene fragments
  • Detection method for isothermal amplification of single or multiple target gene fragments
  • Detection method for isothermal amplification of single or multiple target gene fragments

Examples

Experimental program
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Effect test

Embodiment 1

[0080] Example 1. Single amplification of MERT-LAMP:

[0081] (1) The MERT-LAMP single amplification (ie standard reaction) system is as follows: the concentration of primers EFIP and FIP are each 20 pmol, the concentration of primer BIP is 40 pmol, the concentration of primers F3 and B3 is 5 pmol, and the concentration of primers LF and BF is 20pmol, 10mM Betain, 6mM MgSO 4 , 1mM dNTP, 2.5μl of 10×Bst DNA polymerase buffer, 8U strand displacement DNA polymerase, 15U DNA restriction endonuclease, 1μl template, and add deionized water to 25μl. The whole reaction was carried out in a fluorescence detector (Rotor-Gene Q Real Time System, Qiagen), and the reaction was terminated at 63~65℃ for 1h by isothermal amplification and 5min at 80℃.

[0082] (2) Feasibility verification:

[0083] Under standard system conditions, the MERT-LAMP primer and corresponding DNA template designed according to lmo0733 and smcL specific genes are added, and the lmo0733-MERT-LAMP reaction is used to amplif...

Embodiment 2

[0093] Example 2. MERT-LAMP multiple amplification

[0094] (1) The MERT-LAMP multiple reaction system is as follows: the concentration of primers EFIP and FIP are each 20pmol, the concentration of primer BIP is 40pmol, the concentration of primers F3 and B3 are 10pmol, the concentration of primers LF and BF is 10pmol, 10mM Betain 6mM MgSO 4 , 1mM dNTP, 2.5μl of 10×Bst DNA polymerase buffer, 8U strand displacement DNA polymerase, 15U DNA restriction endonuclease, 1μl template, and add deionized water to 25μl. The whole reaction was carried out in a fluorescence detector (Rotor-Gene Q Real Time System, Qiagen), and the isothermal amplification was carried out at 64°C for 1 h and 80°C for 5 min to terminate the reaction.

[0095] (2) Verify the multiple detection capability and sensitivity of MERT-LAMP

[0096] In order to realize the multiple amplification detection of MERT-LAMP technology, the present invention optimizes the standard system to adapt to multiple detection, and establ...

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Abstract

The invention discloses a detection method for isothermal amplification of single or multiple target gene fragments. According to the method, FIB and / or BIP primer 5' end is added with a restriction enzyme cutting site sequence during LAMP amplification and is marked with fluorophore, and other parts of the primer are marked with quenching groups; after amplification, amplification results of the corresponding target genes are displayed according to a fluorescence detection, thereby accomplishing detection of single or multiple target gene fragments by one-time amplification. Compared with traditional LAMP, the method provided by the invention has the advantages of single or multiple detection, high efficiency amplification, rapid reaction, specific detection and simple operation.

Description

Technical field [0001] The invention relates to a method for amplifying target gene fragments and detecting the amplification results, and belongs to the field of molecular biology. Background technique [0002] Loop-mediated isothermal amplification (LAMP) is a new nucleic acid specific amplification technology established by Notomi et al. It has the advantages of strong specificity, high sensitivity, simple operation and easy product detection. This technology has been widely used in the field of molecular diagnostics. LAMP designs 4 core primers for 6 specific sites of the target sequence, and uses Bst DNA polymerase with strand displacement activity to catalyze the synthesis of new strands under constant temperature conditions, thereby enabling efficient amplification of the target sequence. Two of the four core primers are inner primers, namely FIP (ForWard inner primer, FIP) and BIP (Backward inner primer, BIP). FIP contains Flc and F2 (complementary sequence of F2c regio...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 叶长芸王毅王艳
Owner ICDC CHINA CDC
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