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An application of a Cas protein, and a method and kit for detecting a target nucleic acid molecule

A detection method and target nucleic acid technology, applied in biochemical equipment and methods, microbial measurement/inspection, genetic engineering, etc., can solve problems such as increasing operational difficulty and achieve high sensitivity

Active Publication Date: 2017-12-19
SHANGHAI TOLO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above method involves the detection of RNA. If only the detection of DNA is required, the operation difficulty may be increased due to the instability of RNA.

Method used

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  • An application of a Cas protein, and a method and kit for detecting a target nucleic acid molecule
  • An application of a Cas protein, and a method and kit for detecting a target nucleic acid molecule
  • An application of a Cas protein, and a method and kit for detecting a target nucleic acid molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Cas12a protein detects the target of single-stranded DNA (ssDNA) (probe FAM label)

[0077] Single-stranded DNA (target-T1-R) was selected as the target sequence to test the response value of different Cas12a proteins to its detection.

[0078]1. Preparation of crRNA: First, the transcription template was prepared by annealing T7-crRNA-F and synthetic oligonucleotide T7-T1-24-R, as shown in Table 5. Specifically, paired oligonucleotides (4 μM) were annealed in 1× PCR buffer (Transgen Biotech) in a total volume of 50 μL, followed by an annealing procedure: initial denaturation at 95 °C for 5 min, followed by cooling from 95 °C to 20 °C, using a thermal cycler to decrease 1 °C per minute. crRNA was synthesized using the T7 High Yield Transcription Kit, and the reaction was performed overnight (approximately 16 h) at 37°C. RNA was then purified using the RNA Purification and Concentration Kit, quantified with NanoDrop 2000C (Thermo Fisher Scientific), diluted t...

Embodiment 2

[0081] Example 2 Cas12a protein detects the target of single-stranded DNA (ssDNA) (the probe has HEX, BHQ1 double labeling)

[0082] Single-stranded DNA (target-T1-R) was selected as the target sequence to test the response value of different Cas12a proteins to its detection.

[0083] 1. Preparation of crRNA: First, the transcription template was prepared by annealing T7-crRNA-F with the synthetic oligonucleotide T7-T1-24-R (Table 5). Specifically, paired oligonucleotides (4 μM) were annealed in 1×PCR buffer (TransgenBiotech) in a total volume of 50 μL, followed by an annealing procedure: initial denaturation at 95°C for 5 min, followed by cooling from 95°C to 20°C °C, decreasing 1 °C per minute using a thermal cycler. crRNA was synthesized using the T7 High Yield Transcription Kit, and the reaction was performed overnight (about 16h) at 37°C. The RNA was then purified using the RNA Purification and Concentration Kit, quantified with NanoDrop2000C, diluted to a concentration...

Embodiment 3

[0086] Example 3 Cas12a protein detects the target of double-stranded DNA (dsDNA)

[0087] Double-stranded DNA (target-T1) was selected as the target sequence to test the response value of different Cas12a proteins to its detection.

[0088] 1. Preparation of crRNA: First, the transcription template was prepared by annealing T7-crRNA-F with the synthetic oligonucleotide T7-T1-24-R (Table 5). Specifically, paired oligonucleotides (4 μM) were annealed in 1×PCR buffer (TransgenBiotech) in a total volume of 50 μL, followed by an annealing procedure: initial denaturation at 95°C for 5 min, followed by cooling from 95°C to 20°C °C, decreasing 1 °C per minute using a thermal cycler. crRNA was synthesized using the T7 High Yield Transcription Kit, and the reaction was performed overnight (about 16h) at 37°C. The RNA was then purified using the RNA Purification and Concentration Kit, quantified with NanoDrop2000C, diluted to a concentration of 10 μM and stored in a -80°C refrigerator...

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Abstract

An application of a Cas protein, and a method and kit for detecting a target nucleic acid molecule are provided. The method includes adding a guide RNA, Cas12a and a nucleic acid probe into a reaction system containing a target nucleic acid molecule to be detected, and performing detection after a reaction is finished. Through the method, whether a sample contains a target nucleic acid sequence or not can be detected rapidly. Through combination with a nucleic acid amplification technique, the sensitivity of the method is greatly increased. According to characteristics of the method, the method is named as HOLMES (one HOur Low-cost Multiplex Efficient Simple assay), namely an assay method that is low-cost, multiplex, efficient and simple and can be achieved in about one hour. The method can be used for rapidly detecting pathogenic microorganisms, gene mutations, specific target DNAs, and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a method for detecting target nucleic acid molecules. Background technique [0002] Specific detection of nucleic acid molecules (Nucleic acid detection) method has important application value, such as detection of pathogens, genetic disease detection and so on. In terms of pathogen detection, since each pathogenic microorganism has its unique characteristic nucleic acid molecular sequence, nucleic acid molecular detection for specific species can be developed, also known as nucleic acid diagnostics (NADs, nucleic acid diagnostics), in food safety, environment Microbial contamination detection, human pathogen infection and other fields are of great significance (O'Connor and Glynn 2010, Scheler, Glynn et al. 2014). Another aspect is the detection of single nucleotide polymorphisms (SNPs, single nucleotide polymorphisms) in humans or other species. Understandi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6816C12Q2521/327C12Q2525/161C12Q2563/107C12Q1/6823C12N2310/20C12Q1/683Y02A50/30C12Q2521/301C12Q2522/101C12N9/22C12N15/113C12Q1/6886
Inventor 成秋香
Owner SHANGHAI TOLO BIOTECH CO LTD
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