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Visual detection system, reagent or kit and detection method for detecting target nucleic acid molecules

A target nucleic acid and kit technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of harsh reaction conditions, lack of visual detection methods, lack of visual detection process, etc., to achieve low detection cost, applicable Wide range of effects and high sensitivity

Active Publication Date: 2022-02-25
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technology mainly relies on guide ssDNAs, gene editing enzyme Pyrococcus furiosus Argonaute (PfAgo) and fluorescent reporter nucleic acid detection method. and other expensive instruments, and lack the most intuitive visual detection process
[0005] At present, the PCR method is still a common method for detecting TTV at present. The reaction conditions are relatively harsh, and the laboratory needs expensive instruments such as PCR instrument and Q-PCR instrument, and lacks the most intuitive visual detection method.

Method used

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  • Visual detection system, reagent or kit and detection method for detecting target nucleic acid molecules
  • Visual detection system, reagent or kit and detection method for detecting target nucleic acid molecules
  • Visual detection system, reagent or kit and detection method for detecting target nucleic acid molecules

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Experimental program
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Effect test

Embodiment 1

[0150] Preparation of detection reagent and detection method

[0151] In this embodiment, a kit for the nucleic acid detection method based on the dead nuclease Clostridium perfringensArgonaute (dAGO) of the present invention and its usage method are provided.

[0152] 1.1 Detection reagents and kits

[0153] In this embodiment, taking the detection of the wild type TTV gene as an example, the corresponding specific target nucleic acid sequence 5'-TCCTGGGGCGTGTCTACGAGGTCTATATAAGCAACAGCGGTGACGAAT-3', SEQ ID No.:1.

[0154] Based on the method of the present invention, the corresponding detection reagents include the following:

[0155] (1), specific guide ssDNA (gDNA), the specific sequence is as follows:

[0156] 5'P-ATTCGTCACCGCTGTTGCTTAT-3' (SEQ ID No.2)

[0157] (2), present deoxynucleic acid (DNAer), the specific sequence is as follows:

[0158] 5'-ATAGACCTCGTAGACACGCCCCAGGA-3'B (SEQ ID No.3)

[0159] (3), color reaction reagents: such as streptavidin-horseradish pero...

Embodiment 2

[0173] Detection of different concentrations of nucleic acids to be detected

[0174] Dilute the specific target nucleic acid (SEQ ID NO.: 1), and dilute it into standard mother solutions of 0 μM, 2 μM, 4 μM, 6 μM and 8 μM respectively. Nucleic acid standard mother solutions of different concentrations were added to the reaction system described in Example 1, and the sample addition reaction was carried out step by step, and the optical density signal value at 450 nm was detected by a microplate reader.

[0175] The result is as Figure 6 As shown, the sample control is the non-target nucleic acid which is the total DNA extracted from normal human serum. The results showed that even adding irrelevant non-target nucleic acids to the system did not produce positive results.

[0176] The result is as Figures 7A-7B shown. in, Figure 7A The concentrations of the target nucleic acids in the samples were 0 μM, 2 μM, 4 μM, 6 μM, 8 μM, respectively, Figure 7B Standard curve fo...

Embodiment 3

[0179] Specificity test and typing detection

[0180] Different types of target nucleic acid solutions with a concentration of 200pM were prepared, which were two types of TTV virus, TS14 and TS15, respectively.

[0181] Configure the typing mixed detection system: add 2 pairs of specific amplification primers to the detection amplification system, and add the target detection nucleic acid to the detection amplification system in the following 6 groups: (1) Target TS14+gDNA TS14; ( 2) TargetTS15+gDNA TS14; (3) blank control; (4) Target TS14+gDNA TS15. (5) Target TS15+gDNA TS15; (6) Blank control. (The specificity of the guide ssDNA and the presented deoxynucleic acid are respectively verified in Figure 8, and typing detection can be performed in one reaction system)

[0182] The result is as Figures 8A-8B shown. The results showed that: when the detection target nucleic acid was a blank control, the optical density signal value was very low, and the visualized color was v...

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Abstract

The invention relates to a visual detection system, a reagent or a kit and a detection method for detecting a target nucleic acid molecule. The visual detection system for detecting the target nucleic acid molecule comprises guide ssDNA (gDNA), a death gene editing enzyme Clostridium perfringens Argonaute (dAGO) and a presentation deoxynucleic acid (DNAer). Compared with the prior art, the nucleic acid detection method disclosed by the invention is high in sensitivity, good in specificity and low in price, and can be widely applied to various fields such as molecular medical diagnosis, food safety detection and environment monitoring.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a visual detection method for viruses based on prokaryotic dead AGO nuclease (dAGO) and an application thereof. Background technique [0002] Viruses have always been a threat to humans. The nucleic acids of viruses include double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), double-stranded RNA (dsRNA), positive single-stranded RNA (+ssRNA), and negative single-stranded RNA (-ssRNA). different types. For example, TT virus (TTV) is a ssDNA virus first discovered by Japanese scholars in the serum of a patient with elevated transaminases due to blood transfusion in 1997. There are about 10% of TTV infected people in the world, so it is very necessary to detect and study this virus. . DNA is currently the main method for diagnosing TTV infection. Due to the differences in the conservation of base sequences in different gene regions, the application of PCR is still a c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2521/327C12Q2563/103Y02A50/30
Inventor 冯雁李蓉
Owner SHANGHAI JIAO TONG UNIV