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DHAV-1 and DHAV-3 detection system based on RT-RPA and CRISPR/Cas and application

A DHAV-3, DHAV-1 technology, applied in the field of pathogen detection, can solve the problem of no application of CRISPR/Cas detection technology, and achieve the effect of high sensitivity and cost reduction

Active Publication Date: 2021-03-23
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the application of CRISPR / Cas detection technology to DHAV detection

Method used

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  • DHAV-1 and DHAV-3 detection system based on RT-RPA and CRISPR/Cas and application
  • DHAV-1 and DHAV-3 detection system based on RT-RPA and CRISPR/Cas and application
  • DHAV-1 and DHAV-3 detection system based on RT-RPA and CRISPR/Cas and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Design of crRNA targeting virus-specific sites

[0027] The core of the CRISPR / Cas detection method is to identify the crRNA at the specific site of the virus. The sequence of the crRNA includes: the anchoring scaffold sequence combined with the Cas protein and the crRNA guide sequence, wherein the guide sequence matches the fragment in the target sequence. In order to screen crRNAs with better effects, we designed some crRNAs for the VP0 gene of DHAV-1 and the VP3 gene of DHAV-3, and found that the crRNAs shown in Table 1 have good detection effects.

[0028] The crRNA targeting the DHAV-1 sequence recognized by Cas12a: its sequence is shown in SEQ ID NO.1;

[0029] The crRNA targeting the DHAV-3 sequence recognized by Cas13a: its sequence is shown in SEQ ID NO.2;

[0030] Table 1 crRNA that recognizes specific sites of DHAV-1 and DHAV-3

[0031]

[0032] (Note: The uppercase letters in the table are the anchoring scaffold sequences bound by the Cas pro...

Embodiment 2

[0034] Embodiment 2: The establishment of DHAV-1 and DHAV-3 detection system and its detection method based on RT-RPA and CRISPR / Cas

[0035] 1. Establishment of DHAV-1 and DHAV-3 detection system based on RT-RPA and CRISPR / Cas

[0036]DHAV-1 and DHAV-3 detection systems based on RT-RPA and CRISPR / Cas (abbreviated as "DHAV-1 and DHAV-3 detection systems") include RT-RPA isothermal amplification systems and CRISPR / Cas detection systems, where RT -RPA isothermal amplification system (25 μl) is shown in Table 2:

[0037] Table 2 RT-RPA isothermal amplification system

[0038]

[0039] Among them, RNase Inhibitor, RNase free water, Rehydration Buffer and magnesium acetate (MgAc) come from Basic RT RPA kit (TwistDx, product number: TABASRT01KIT);

[0040] RT-RPA amplification primers for DHAV-1:

[0041] RPA-F1: 5'-GTTCCAAATGATGATTATTATGCCTGCCTAC-3' (SEQ ID NO.3)

[0042] RPA-R1: 5'-TTAGACTCCTTTCTCCAACATTTGCATGGAT-3' (SEQ ID NO.4)

[0043] RT-RPA amplification primer sequ...

Embodiment 3

[0070] Embodiment 3: DHAV-1 and DHAV-3 detection system based on RT-RPA and CRISPR / Cas detect the specificity analysis of DHAV-1 and DHAV-3

[0071] Using AxyPrep from Axygen TM Body Fluid Viral DNA / RNA Miniprep Kit Extraction of Duck Tambusu Virus (DTMUV), Avian Influenza Virus H9 Subtype (AIV-H9), New Duck Reovirus (NDRV), Duck Plague Virus (DPV) and DHAV-1 and the RNA of DHAV-3, according to the DHAV-1 and DHAV-3 detection system and program based on RT-RPA and CRISPR / Cas system of the embodiment of the present invention 2, carry out RT-RPA amplification and CRISPR / Cas detection sequentially to the above samples DHAV-1 and DHAV-3.

[0072] The result is as figure 1 As shown, the fluorescence signal values ​​of common duck susceptible virus samples such as DTMUV, AIV-H9, NDRV, and DPV before and after the reaction were basically unchanged and similar to the negative control, while the fluorescence signal values ​​of DHAV-1 and DHAV-3 samples after the reaction Much highe...

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Abstract

The invention discloses a DHAV-1 and DHAV-3 detection system based on RT-RPA and CRISPR / Cas and application. The DHAV-1 and DHAV-3 detection system based on RT-RPA and CRISPR / Cas comprises a RT-RPA system and a CRISPR / Cas system. The RT-RPA system comprises a RT-RPA amplification primer RPA-F1 / RPA-R1 of DHAV-1 and a RT-RPA amplification primer RPA-F3 / RPA-R3 of DHAV-3. The CRISPR / Cas system comprises a CRISPR / / Cas12a protein and a CRISPR / / Cas13a protein, Cas12a is LbCas12a, and Cas13a is LwCas13a. DHAV-1 and DHAV-3 can be rapidly and accurately detected based on the DHAV-1 and DHAV-3 detectionsystem of RT-RPA and CRISPR / Cas, samples containing single copies of DHAV-1 and DHAV-3 can be detected, the sensitivity is high, DHAV-1 and DHAV-3 can be comprehensively detected at a time, whether serum type 1 or serum type 3 infection or co-infection exists is distinguished, the detection system can react at a constant temperature of 37 DEG C, the requirement on the equipment is low, and the DHAV-1 and DHAV-3 detection system is made into a kit which is suitable for popularization and application.

Description

technical field [0001] The invention belongs to the technical field of pathogen detection, and in particular relates to a DHAV-1 and DHAV-3 detection system and application based on RT-RPA and CRISPR / Cas. Background technique [0002] Duck viral hepatitis caused by duck hepatitis A virus (Duck hepatitis A virus, DHAV) mainly affects ducklings aged 1 to 3 weeks. After being infected with the virus, the onset process is rapid, and the ducklings will become ill within about 24 hours of infection, and the virus is highly contagious and has a high lethality rate, and the mortality rate of ducklings after infection can be as high as 90%. had a serious impact. There are currently three serotypes of DHAV, among which serotype 1 (DHAV-1) and serotype 3 (DHAV-3) are prevalent in my country. At present, the DHAV-1 vaccine has been widely used, and there have been research reports on the successful acquisition of DHAV-3 attenuated vaccine candidate strains. Therefore, real-time monit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/706C12Q1/6844C12Q2531/119C12Q2521/507C12Q2521/327C12Q2563/107C12Q2521/107Y02A50/30
Inventor 吴凤瑶张小飞卢凤英刘青涛潘群兴孙华伟赵莎徐彬沈丽雅
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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