Multi-nucleic acid aptamers for detecting pathogenic microorganisms and combinations thereof

A technology of pathogenic microorganisms and nucleic acid aptamers, applied in measuring devices, recombinant DNA technology, instruments, etc., can solve problems such as evaluating the ability of nucleic acid aptamers to bind to other proteins, and achieve convenient modification and substitution, small molecular weight, The effect of good affinity

Active Publication Date: 2022-07-29
中国科学院基础医学与肿瘤研究所(筹)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the patent does not evaluate the ability of nucleic acid aptamers to bind to other proteins

Method used

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  • Multi-nucleic acid aptamers for detecting pathogenic microorganisms and combinations thereof
  • Multi-nucleic acid aptamers for detecting pathogenic microorganisms and combinations thereof
  • Multi-nucleic acid aptamers for detecting pathogenic microorganisms and combinations thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Preparation of nucleic acid aptamers

[0061] 1. Construction of screening library

[0062] The ssDNA library used in this study is 76 nt in full length, consisting of 20 nt fixed primer fragments at both ends and a 30 nt random fragment in the middle; the 5' FITC fluorophore of the forward primer is modified with the 5' of the original library. The fixed sequence at the ' end is the same; the 5' end of the reverse primer is labeled with Biotin, and the sequence is reverse complementary to the fixed sequence at the 3' end of the original library;

[0063]The sequence of the screening library targeting SARS-CoV-2 nucleocapsid protein is:

[0064] SEQ ID NO. 5:

[0065] CTTCTGCACGCCTCCTTCC (N 35 ) GGAGACGAGATCGGCGGACACT.

[0066] The primers used to construct the library are:

[0067] Forward primer: SEQ ID NO.6: CTTCTGCACGCCTCCTTCC;

[0068] Reverse primer: SEQ ID NO. 7: AGTGTCCGCCGATCTCGTCTCC.

[0069] 2. Conjugation of protein and magnetic beads

[0070] Take s...

Embodiment 2

[0087] Sandwich combination selection of different aptamers and antibodies

[0088] After testing and screening, the selected nucleic acid aptamer combinations are shown in Table 1.

[0089] Table 1 Nucleic acid aptamer combination scheme

[0090] group combination 1 N35 / N2 2 N35 / N2-62 3 N35 / N10 4 N2 / N2-62 5 N2 / N10 6 N2-62 / N10 7 N35 / N2 / N2-62 8 N35 / N2 / N10 9 N35 / N2-62 / N10 10 N2 / N2-62 / N10 11 N35 / N2 / N10 / N2-62

Embodiment 3

[0092] Preparation of Nucleic Acid Aptamers Labeled with Latex Microspheres

[0093] The streptavidin (SA) modified latex particles (mass fraction 1%, provided by Hangzhou Ustar) were diluted twice with ultrapure water to a mass fraction of 0.5%. Then, a 4-fold excess of biotin-modified nucleic acid aptamer (biotin-modified nucleic acid aptamer was synthesized by Shanghai Sangon Biotechnology Co., Ltd.) was added to the SA-modified latex particles with a mass fraction of 0.5% for cross-linking. The obtained cross-linked product was centrifuged to remove nucleic acid aptamers unbound to latex particles in the supernatant, and the centrifugation conditions were 4°C, 10000 rpm, 10 min. Then, the cross-linked product was washed twice with ultrapure water by centrifugation, and the final volume was made up with 50 μL of dispensing solution for subsequent use.

[0094] Among them, there are two marking methods, namely:

[0095] 1) Mixed labeling, that is, different nucleic acid ap...

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Abstract

The invention discloses a multi-nucleic acid aptamer for detecting pathogenic microorganisms and a combination of the multi-nucleic acid aptamer, and belongs to the technical field of biological detection. The nucleic acid aptamer and the combination thereof disclosed by the invention can be specifically combined with pathogenic microorganisms, so that the nucleic acid aptamer and the combination thereof can be used for identifying and identifying the pathogenic microorganisms. The aptamer is screened by using an index enrichment ligand systematic evolution technology, and when the screened aptamer is used for detecting pathogenic microorganisms, the specificity is high, and the sensitivity is high. In addition, the aptamer has the characteristics of small molecular weight, better stability, easiness in modification, no immunogenicity, short preparation period and the like, and is more suitable for detecting pathogenic microorganisms compared with an antibody.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a polynucleic acid aptamer for detecting pathogenic microorganisms and a combination thereof. Background technique [0002] The new coronavirus antigen test, like the viral nucleic acid test, determines whether there is a new coronavirus by detecting the components of the virus. In the new coronavirus particles, the content of specific structural proteins is thousands of times that of viral nucleic acid molecules. The new coronavirus antigen detection method directly detects the structural protein of the new coronavirus, and there is no amplification and amplification process, which saves the amplification step that takes a long time in the nucleic acid detection process, so it is fast, simple, convenient, economical, early diagnosis, reliable High degree of characteristics. At present, the detection of novel coronavirus antigens mainly uses antibodies that can specifically ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/569
CPCC12N15/115G01N33/56983C12N2310/16G01N2333/165Y02A50/30
Inventor 谭蔚泓杨秋霞付晓艺彭瑞资李恒轩宋明慧罗昭峰符婷
Owner 中国科学院基础医学与肿瘤研究所(筹)
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