Novel isothermal multiple self-pairing triggering amplification technology

A technology of isothermal nucleic acid amplification and RT-IMSA, which is applied in the field of warm nucleic acid amplification technology, can solve problems such as obstacles, and achieve the effects of visualization of results, high detection specificity, and low chance of contamination

Inactive Publication Date: 2015-03-04
中国疾病预防控制中心病毒病预防控制所
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although PCDR has rapid detection, it is hindered in POCT application like ordinary PCR

Method used

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  • Novel isothermal multiple self-pairing triggering amplification technology
  • Novel isothermal multiple self-pairing triggering amplification technology
  • Novel isothermal multiple self-pairing triggering amplification technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0022] Embodiment 1: The specificity of detecting CVA16, EV71 and human type A H7N9 influenza based on RT-IMSA

[0023] Nucleic acid extraction: According to the instructions of the Qiagen kit, different sources of HFMD pathogenic RNA or influenza pathogenic RNA were extracted. For the detection of different pathogens, the following IMSA amplification reaction system was established: 12.5 μL (2×) reaction solution (Guangzhou Diao Biotechnology Co., Ltd.), 1.0 μL each of 6 IMSA primers corresponding to specific genes [primer concentration is DsF and DsR (5pmol / μL), FIT and RIT (20pmol / μL), SteR and SteF (40pmol / μL)], 1μL Bst2.0DNA polymerase (8U / μL, NEB company), 0.5μLAMV reverse transcriptase (10U / μL , Promega) and 4 μL of RNA samples, nuclease-free water was added to make the total volume of the system 25 μL. Mix evenly, place in a constant temperature device at a temperature of 63°C such as a water bath or a turbidimeter from Nippon Sakae Co., Ltd. to react for 1.5 hours, a...

Embodiment approach 2

[0024] Embodiment 2: Sensitivity of detecting CVA16, EV71 and human type A H7N9 influenza based on RT-IMSA

[0025] 10-fold gradient dilution corresponding to different pathogenic RNA extracted from the sample, respectively to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 , and 10 -7 And dilution, the system that RT-IMSA detects is the same as embodiment 1. The sensitivity of CVA16VP1 gene detection can reach 10 -7 Dilution RNA, EV71VP1-based gene detection can reach 10 -5 Dilution RNA, the sensitivity of H7N9HA gene detection can reach 10 -6 Dilution RNA.

Embodiment approach 3

[0026] Embodiment 3: Visual RT-IMSA detection of specific pathogen results determination

[0027]Visual judgment method 1: After the RT-IMSA reaction is completed, take out the reaction tube directly, observe the white precipitate at the bottom of the tube with the naked eye, or use 20 μL to measure the absorption value at 650 nm; visual judgment method 2: add HNB before RT-IMSA amplification , it can be seen that the color of the positive reaction tube turns into sky blue, while the color of the negative reaction tube is purple; visual judgment method 3: adding HNB modified dye (GeneFinder TM When mixing dye with HNB), it can be seen that the color of the positive reaction tube becomes dark green, while the color of the negative reaction tube is gray.

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[0030]

[0031]

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Abstract

The invention relates to a novel isothermal nucleic acid amplification technology, namely an isothermal multiple self-pairing triggering amplification technology (IMSA). According to the technology, amplification of a pathogeny gene can be realized under the condition of isothermality and under the action of a single functional enzyme without a thermal cycle instrument. The result determination of the isothermal multiple self-pairing triggering amplification technology (IMSA) can be realized by simple measures such as observing white precipitate of by-product magnesium pyrophosphate, adding a chromogenic agent and observing the color change before or after amplification. The most unique characteristic of the technology is that multiple oligonucleotide structures which can perform self-pairing and then trigger circular amplification will be generated during the process of amplification so that the triggering probability of following circular amplification is markedly increased, the amplification efficiency and detection sensitivity are promoted. On the basis of the isothermal multiple self-pairing triggering amplification technology (IMSA) we establish detection method of hand-foot-and-mouth disease pathogen coxsackievirus type A16 (CVA16) and human enterovirus type EV71 (EV71) VP1 gene, and rapid detection method of human influenza a virus H7N9 HA gene. By the application of the invention, simple, rapid, highly sensitive and highly specific detection can be realized.

Description

Technical field: [0001] The invention relates to a novel isothermal nucleic acid amplification technology, which is mainly characterized in that multiple special nucleotide structures can be generated during the amplification process under isothermal conditions, which can self-match and then trigger cyclic amplification. The invention can be applied to the rapid, high-sensitivity and high-specificity detection of pathogenic genes, so as to realize rapid on-site detection of disease pathogens. Background technique: [0002] In recent decades, nucleic acid amplification technology has made revolutionary contributions to molecular biology research and detection of pathogenic microorganisms. So far, nucleic acid amplification technology is mainly based on two strategies: one is to complete the three processes of template nucleic acid unzipping, primer annealing to template, and subsequent primer extension by heating and cooling with a thermal cycler; the other is The aforementi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6844C12Q2527/101C12Q2537/155
Inventor 马学军丁雄聂凯许文波石磊
Owner 中国疾病预防控制中心病毒病预防控制所
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