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Locked nucleic acid modified LAMP primer composition and application thereof

A primer composition and nucleic acid-locking technology, applied in the field of nucleic acid amplification, can solve the problems of non-specific amplification limiting practical application value, exacerbating non-specific amplification of primer sources, and difficulty in realizing high-throughput detection, etc., achieving good results , Improve specificity and sensitivity, and increase the effect of amplification speed

Inactive Publication Date: 2017-11-10
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in recent years, the actual application of LAMP technology in the laboratory is relatively limited, and the progress is relatively slow. According to the previous research, the inventors found that there are two important reasons that limit its wide application, specifically: (1) the current LAMP system is difficult to The realization of high-throughput detection in the true sense needs to be further improved; (2) Although the LAMP system does not require heating and cooling during the detection process, the reaction speed can be greatly accelerated, and efficient and rapid detection can be realized. However, the stability of the LAMP system is relatively low. Poor, and due to the large number of primers in LAMP amplification, the problem of non-specific amplification from primers is exacerbated, including non-specific amplification inside the primers (false positives appear in the blank control system) and non-specific binding amplification between primers and templates , although there are relatively few literature reports in this area, the problem of non-specific amplification severely limits the practical application value of this technology

Method used

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  • Locked nucleic acid modified LAMP primer composition and application thereof
  • Locked nucleic acid modified LAMP primer composition and application thereof
  • Locked nucleic acid modified LAMP primer composition and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] This example is the application of the locked nucleic acid modified LAMP primer composition in the constant temperature amplification detection of Datura genotype.

[0040] 1) Preparation of datura nucleic acid sample: extract and prepare according to the following steps:

[0041] a. Get a single seed to grind and pulverize;

[0042] b. Grind finely in liquid nitrogen and transfer to a centrifuge tube, add 500-800μL TES [100mM Tris (pH8.0), 10mM EDTA, 2wt% SDS]; add 50-100μg protein kinase K and mix well, place at 55-60℃ Water bath for 60-90min, and gently mix several times during the period;

[0043] c. To adjust the salt concentration, add 1 / 10 volume of 10% cetyltriethylammonium bromide (CTAB), and place in a water bath at 65°C for 10-20min;

[0044]d. Add an equal volume of SEVGA (chloroform: isoamyl alcohol = 24:1) to mix, and incubate at 0°C for 30 minutes;

[0045] e. Centrifuge, take the supernatant, add 3-6 μL RNase A, and bathe in water at 37°C for 30 minut...

Embodiment 2

[0058] This example is the establishment of a CALR gene type 2 mutant LAMP system, which contains locked nucleic acid modified primers, formamide, and a hot-start enzyme.

[0059] In this embodiment, the target sequence of CALR gene type 2 mutation and the sequences of its amplification primers are shown in the following table:

[0060] Table 1 Base sequence list of primers and target sequences for detecting CALR gene type 2 mutation

[0061]

[0062] Wherein, the nucleic acid containing the CALR gene type 2 mutation target sequence is extracted according to conventional techniques, and the primers for amplifying the target sequence are also designed according to conventional methods, and the locked nucleic acid is modified at the 3' end of the FIP primer and the BIP primer.

[0063] The configuration of the LAMP amplification reaction system is as follows: 25ul

[0064] Set different reaction time (10, 20, 30, 40, 60, 80min) and temperature (57°C, 60°C, 63°C, 65°C, 68°C) ...

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Abstract

The invention discloses a locked nucleic acid modified LAMP primer composition. In the LAMP primer composition, the locked nucleic acid modification position is at least one of three basic groups on the outmost side of the end 3' of an FIP primer and a BIP primer. The invention further relates to an LAMP reaction system comprising the LAMP primer composition. The system comprises formamide and hot start Taq. The invention further relates to an amplification method adopting the LAMP primer composition, and the amplification temperature is 58-72 DEG C. For solving the LAMP non-specific amplification problem, by adopting the technical scheme, while the amplification efficiency is improved slightly, non-specific amplification is greatly lowered, the stability and specificity of the LAMP reaction system are greatly improved, and the great significance in further improvement and clinic application of the LAMP technology is achieved.

Description

technical field [0001] The invention relates to the technical field of nucleic acid amplification, in particular to a nucleic acid-locked modified LAMP primer composition and its application. Background technique [0002] With the development of laboratory medicine, some molecular biology techniques, such as PCR and strand displacement amplification, have played an important role in medical examination and diagnosis. However, due to the high requirements for instruments and equipment and complicated operating procedures for the detection of nucleic acids by molecular biology techniques, it limits its development and promotion as a rapid diagnostic method. It solves the problems of expensive equipment and complicated procedures in nucleic acid diagnosis. [0003] The LAMP reaction only needs a DNA polymerase with strand displacement activity (such as Bst, Gsp, etc.) and 4-6 primers designed for 6-8 regions of the target gene (required for F3 / B3 and FIP / BIP, LF / LB optional) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2531/119C12Q2525/307C12Q2527/125C12Q2527/101C12Q2521/101
Inventor 曹国君关明方雪恩周连群张威康志华邓萱赵缜邢志芳孔继烈
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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