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Multi-enzyme cleavage site mediated nucleic acid isothermal amplification detecting method

A constant temperature amplification detection and nicking enzyme technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of long time, several hours, non-specific amplification, and low amplification efficiency. Achieve rapid response, short detection time and simple detection process

Active Publication Date: 2014-02-12
QINGDAO NAVID BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] Nucleic acid detection has been widely used in many aspects such as clinical diagnosis, environmental monitoring, and prevention and control of infectious diseases. The high sensitivity of polymerase chain reaction (PCR) makes it the most widely used DNA amplification method at present. However, the existing conventional PCR technology has the following defects: first, repeated thermal denaturation is required to untangle the DNA double strands, and the application depends on high-quality thermal cyclers; second, multiple factors affect the amplification effect; third, it often causes Non-specific amplification; Fourth, the amplification reaction time is long, it takes several hours, it is difficult to popularize and apply at the grassroots level, and many other defects
In 2009, Kemin Wang used molecular beacons for nucleic acid detection, which uses target nucleic acids to open molecular beacons, and primers are extended and amplified on the opened molecular beacons. This method is linear amplification, the amplification efficiency is not high, and the sensitivity is not high. Gao; in 2009, Ashley R Connolly also used molecular beacons for nucleic acid detection. He added a nickase site to the molecular beacons to trigger an exponential reaction, but primers were needed in the reaction system, and the presence of primers could easily lead to Inevitable non-specific amplification, high background, low sensitivity

Method used

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  • Multi-enzyme cleavage site mediated nucleic acid isothermal amplification detecting method
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  • Multi-enzyme cleavage site mediated nucleic acid isothermal amplification detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: Verify the feasibility of the method and the correctness of its principle.

[0025] In this example, artificially synthesized microRNA Let-7a was used as the target nucleic acid, and molecular beacons were used as the template nucleic acid to verify the feasibility of the method and the correctness of the principle through fluorescence signals and electrophoresis results. The molecular beacon constant temperature amplification system composed of 200nM molecular beacon (sequence: 5'-FAM-AGGTAGTAGCCATCCTCAGCACTCCGAATCCTCAGCAAACTATACAACCTA

[0026] CTACCT-DABCYL-3' (SEQ ID NO.1), 1×NEB CutSmart TMBuffer (50mM Potassium Acetate20mM Tris-acetate10mM Magnesium Acetate100μg / ml BSA pH7.925℃), 0.1U / μL polymerase Klenow fragment (3′→5′exo-), 0.2U / μL nickase Nb.BbvCI, dNTPs100μM, Add 1×10 to the system -7 M, 1×10 -9 M target let-7a (sequence is 5'-UGAGGUAGUAGGUUGUAUAGUU-3', which is SEQ ID NO.2), using Bio-Rad CFX96 TM The real-time fluorescence quantitative PCR ...

Embodiment 2

[0027] Example 2: Detection of artificially synthesized oligonucleotide fragments by using double nickase site-mediated molecular beacon constant temperature amplification detection method.

[0028] This example detects different concentrations of artificially synthesized microRNA Let-7a to verify the sensitivity of the nucleic acid detection method. The molecular beacon constant temperature amplification system mediated by double nickase sites is composed of a 200nM molecular beacon (SEQ ID NO.1 ), 1 x NEB CutSmart TM Buffer, 0.1U / μL polymerase Klenow fragment (3′→5′exo-), 0.2U / μL nickase Nb.BbvCI, dNTPs 100μM, add 5×10 -9 M, 1×10 -9 M, 5×10 -10 M, 1×10 -10 M, 5×10 -11 M, 1×10 -11 M, 1×10 -13 M, 1×10 -15 M, 1×10 -17 Targets of M and 0M (SEQ ID NO.2), using Bio-Rad CFX96 TM The real-time fluorescent quantitative PCR instrument detects the fluorescent signal once per minute, and reacts for 85 minutes; the results are as follows: Figure 6 As shown, the concentration g...

Embodiment 3

[0029] Example 3: The specific detection of the target is realized by using the molecular beacon constant temperature amplification detection method mediated by double nickase sites.

[0030] This example detects four artificially synthesized let-7 family sequences (let-7b: 5'-UGAGGUAGUAGGUUGUGUGGUU-3', which have one or more base differences at different positions with the target nucleic acid sequence, namely SEQ ID NO.3; let -7c: 5'-UGAGGUAGUAGGUUGUAUGGUU-3', namely SEQ ID NO.4; let-7e: 5'-UGAGGUAGGAGGUUGUAUAGUU-3', namely SEQ ID NO.5; let-7i: 5'-UGAGGUAGUAGUUUGUGCUGUU-3', namely SEQ ID NO. ID NO.6;), each let-7 family sequence and the target nucleic acid sequence in Example 1 have one or more base differences at different positions, which are used to investigate the ability of the method of the present invention to distinguish base differences when detecting nucleic acids ability.

[0031] The molecular beacon constant temperature amplification system mediated by double ni...

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Abstract

The invention belongs to the technical field of molecular biology nucleic acid detection, and relates to a multi-enzyme cleavage site mediated nucleic acid isothermal amplification detecting method. Firstly, specific hybridization is conducted on a target nucleic acid and template nucleic acid molecules through base complementary pairing effect; polymerase and cutting enzyme are cooperatively and circularly amplified to continuously generate products 1 and products 2; the products 1 and template nucleic acid are combined for circular amplifying to continuously generate the products 2; finally, the generated products 2 are hybridized with the template nucleic acid to generate signals, and the generated signals are detected under the condition of constant temperature to achieve the detection of nucleic acid isothermal amplification. The detecting method has the advantages of simple detecting technique, quick reaction, short detecting time, high sensitivity and wide application range.

Description

Technical field: [0001] The invention belongs to the technical field of nucleic acid detection in molecular biology, and relates to a nucleic acid constant temperature amplification detection method mediated by multiple nickase sites. Background technique: [0002] Nucleic acid detection has been widely used in many aspects such as clinical diagnosis, environmental monitoring, and prevention and control of infectious diseases. The high sensitivity of polymerase chain reaction (PCR) makes it the most widely used DNA amplification method at present. However, the existing conventional PCR technology has the following defects: first, repeated thermal denaturation is required to untangle the DNA double strands, and the application depends on high-quality thermal cyclers; second, multiple factors affect the amplification effect; third, it often causes Non-specific amplification; Fourth, the amplification reaction time is long, requiring several hours, and it is difficult to popula...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2531/119C12Q2521/301
Inventor 石超马翠萍韩典昂王文硕
Owner QINGDAO NAVID BIOTECH CO LTD
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