Apple stem groove virus visual detection system based on CRISPR-Cas12a technology and detection method thereof

A technology of apple stem groove virus and detection method, which is applied in the field of molecular biology, can solve the problems of time-consuming and high cost, and achieve the effect of improving specificity, low cost and high consistency rate

Active Publication Date: 2019-12-13
HENAN AGRICULTURAL UNIVERSITY
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Problems solved by technology

However, the CRISPR-based detection method consumes a lot of time when using fluorescence detection and requires large-scale equipment such as microplate readers. Although the visual detection based on test strips developed later is simple, it needs to add a large number of expensive fluorescent probes to observe, and the cost is relatively high. high

Method used

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  • Apple stem groove virus visual detection system based on CRISPR-Cas12a technology and detection method thereof
  • Apple stem groove virus visual detection system based on CRISPR-Cas12a technology and detection method thereof
  • Apple stem groove virus visual detection system based on CRISPR-Cas12a technology and detection method thereof

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Embodiment

[0045] Detection method of apple stem groove virus visual detection system based on CRISPR-Cas12a technology:

[0046] 1. AuNP-DNA complex preparation

[0047] ① Probe DNA and linker DNA sequence, DNA1 sequence is shown in SEQ ID NO.1, DNA2 sequence is shown in SEQ ID NO.2, Linker DNA sequence is shown in SEQ ID NO.3; provided by Sangon Biotechnology (Shanghai, China )synthesis:

[0048] ② Add 2 μL of 1 wt % Tween20 and 10 μL of 4 μM mPEG-SH (MW~5kDa) to 200 μL of 0.1 mg / ml 13 nm AuNP solution;

[0049] ③ Add 10 μL of 100 μM thiolated DNA1 or DNA2 to the mixture, and add 50 μL of 4M NaCl; age at room temperature for 60 min;

[0050] ④ Centrifuge in a centrifuge at 14000 rpm for 10 min at 4 °C to remove the supernatant;

[0051] ⑤ Resuspend the pellet with PBST solution, centrifuge and resuspend three times, then add 200 μL PBST solution to obtain AuNP-DNA1 or AuNP-DNA2;

[0052] ⑥ Mix 5 μL each of AuNP-DNA1 and AuNP-DNA2, and add 2.8 μL of 4 M NaCl solution to the mixture ...

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Abstract

The invention belongs to the field of molecular biology and relates to an apple stem groove virus visual detection system based on the CRISPR-Cas12a technology and a detection method thereof. CrRNA isdesigned according to an ASGV conserved sequence, AuNP-DNA for colorimetric detection is manufactured, the influence of a Cas12a protein in-vitro cleavage reaction time and linker DNA concentration on a detection effect is explored, and the optimal ASGV visual detection system and the detection method thereof are established. The method is carried out under a constant-temperature reaction condition, a virus is recognized through a CRISPR-Cas12a-crRNA complex to improve the specificity, the linker DNA has universality, expensive double-labeled probes are not needed, a plurality of samples canbe detected within 40 minutes by means of low-speed centrifugation, the lower detection limit is 5.62*102 copies / reaction, and the sensitivity is 100 times higher than that of RT-PCR.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a detection method for apple stem groove virus, in particular to a visual detection system and detection method for apple stem groove virus based on CRISPR-Cas12a technology. Background technique [0002] apple( Malus domesica ) is one of the fruits with the largest cultivation area and yield in the world. It is mainly planted in temperate regions of the world and has high commercial value. From 2018 to 2019, the world's total apple production is expected to be 68.6 million tons, and China's total apple production is expected to reach 31 million tons. At the same time, the apple industry is facing a serious problem of apple virus diseases. At present, 17 kinds of apple virus diseases are known. Among them, latent viruses such as ACLSV, ASPV, and ASGV are more common and more harmful in my country. Latent virus usually does not show obvious symptoms, but after the tree is infected,...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78C12Q1/70C12Q1/686C12Q1/6809C12Q1/6811
CPCC12Q1/6809C12Q1/6811C12Q1/686C12Q1/701G01N21/78C12Q2521/107C12Q2563/107C12Q2521/327C12Q2521/301
Inventor 郑先波焦健冯建灿黄松孔康康韩金蒙夏炎白团辉宋春晖王苗苗宋尚伟陈海燕张英丽
Owner HENAN AGRICULTURAL UNIVERSITY
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