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Nucleic acid detection method based on normal-temperature prokaryotic Argonaute protein and application thereof
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A normal temperature, protein technology, applied in the biological field, can solve the problems of complex design principles, high cost, and long time consumption
Pending Publication Date: 2022-04-05
SHANGHAI JIAO TONG UNIV
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[0005] At present, for the extremely small amount of nucleic acid RNA to be detected, there is still an urgent need to develop a nucleic acid detection method with high sensitivity, good specificity, fast and efficient, regardless of the shortcomings of time-consuming, high cost, and complicated design principles.
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Embodiment 1
[0191] Preparation and detection method of nucleic acid detection reagent based on PbAgo
[0192] In this embodiment, the reagents used in the nucleic acid detection method based on the gene editing enzyme Paenibacillus borealisArgonaute (PbAgo) of the present invention and their usage methods are provided.
[0194] 在本实施例中,以检测SRAS-COV2 1b基因为例子,相应的特异性目标核酸序列为5’-GGGGAUAAAAGUGCAUUAACAUUGGCCGUGACAGCUUGACAAAUGU UAAAAACACUAUUAGCAUAAGCAGUUGUGGCAUCUCCUGAUGAGGUUC CACCUGGUUUAACAUAUAGUGAACCGCCACACAUGACCAUUUCACUCA AUACUUGAGCACACUCAUUAGCUAAUCUAUAGAAACGGUGUGACAAGC UACAACACGUUGU-3’,SEQ ID No.:1。
[0195] Based on the method of the present invention, the corresponding detection reagents include the following:
[0196] (1), reverse transcription primer R-primer, the specific sequence is as follows:
[0197] R-primer: 5'-ACAACGTGTTGTAGC-3' (SEQ ID No.2)
[0198] (2), specific guide ssDNAs pair, including sense strand guide ssDNA and nonsense strand guide ssDNA, ...
Embodiment 2
[0228] Preparation and detection method of nucleic acid detection reagent based on KmAgo
[0229] In this embodiment, the reagents used in the nucleic acid detection method based on the gene editing enzyme Kurthia massiliensis Argonaute (KmAgo) of the present invention and their usage methods are provided.
[0230] 2.1 Detection reagent
[0231] 在本实施例中,以检测SRAS-COV2 1b基因为例子,相应的特异性目标核酸序列为5’-GGGGAUAAAAGUGCAUUAACAUUGGCCGUGACAGCUUGACAAAUGU UAAAAACACUAUUAGCAUAAGCAGUUGUGGCAUCUCCUGAUGAGGUUC CACCUGGUUUAACAUAUAGUGAACCGCCACACAUGACCAUUUCACUCA AUACUUGAGCACACUCAUUAGCUAAUCUAUAGAAACGGUGUGACAAGC UACAACACGUUGU-3’,SEQ ID No.:1。
[0232] Based on the method of the present invention, the corresponding detection reagents include the following:
[0233] (1), reverse transcription primer R-primer, the specific sequence is as follows:
[0234] R-primer: 5'-ACAACGTGTTGTAGC-3' (SEQ ID No.2)
[0235] (2), specific guide ssDNAs pair, including sense strand guide ssDNA and nonsense strand guide ssDNA, t...
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Abstract
The invention provides a nucleic acid detection method based on normal-temperature prokaryotic Argonauteprotein and application of the nucleic acid detection method. Specifically, the invention provides a detection system for detecting a target nucleic acid molecule, and the system comprises (a) a guide ssDNA pair; (b) a normal-temperature Argonauteprotein (Ago enzyme), wherein the normal-temperature Argonauteprotein (Ago enzyme) is a normal-temperature Argonaute protein (Ago enzyme) which is used for guiding the shearing of a target DNA (Deoxyribonucleic Acid) by taking the DNA as a guide; (c) fluorescence report nucleic acid, wherein the fluorescence report nucleic acid carries a fluorophore and a quenching group; wherein the target nucleic acid molecule is a target RNA (Ribonucleic Acid). The method is simple, convenient and good in compatibility, and can be widely applied to the fields of detection of infectious diseases such as major infectious diseases and pathogen infectious diseases (viruses and pathogenic bacteria) and the like.
Description
technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nucleic acid detection method based on a normal-temperature prokaryotic Argonaute protein and an application thereof. Background technique [0002] Nucleic acid detection technology is a general term for a series of technologies that convert trace amounts of nucleic acid into intuitive and readable signals mainly through direct amplification of target nucleic acid or in combination with other signal amplification technologies. It is widely used in many fields such as molecular medical diagnosis, food safety detection and environmental monitoring. Especially to help precision medicine, including pathogen detection, genotyping, disease course monitoring, etc., it is urgent to develop sensitive and efficient nucleic acid detection technology. [0003] Integrating existing nucleic acid detection technologies, in addition to sequencing, nucleic acid imprinting and various n...
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