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Kit and method for visual detection of citrus huanglongbing based on RPA-CRISPR-Cas12a system

A technology of citrus huanglongbing and kits, applied in the direction of microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of unsatisfactory application effect and low detection sensitivity, and achieve low cost and high sensitivity , the effect of convenient operation

Active Publication Date: 2022-02-15
ZHONGKAI UNIV OF AGRI & ENG
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  • Description
  • Claims
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Problems solved by technology

Chinese patent CN112280879A discloses a kind of RPA primer, kit and its detection method and application for rapid detection of citrus Huanglongbing Asian species. Although the detection time can be shortened, the detection sensitivity is low. Due to the low concentration of pathogenic bacteria in the initial stage of citrus Huanglongbing infection, This method does not work well in practice

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  • Kit and method for visual detection of citrus huanglongbing based on RPA-CRISPR-Cas12a system
  • Kit and method for visual detection of citrus huanglongbing based on RPA-CRISPR-Cas12a system
  • Kit and method for visual detection of citrus huanglongbing based on RPA-CRISPR-Cas12a system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 RPA primer design screening and amplification system determination

[0037] 针对柑橘黄龙病菌基因组中的16s rRNA序列保守区域(5'-ACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGCGTATGCGAATACGAGCGGCAGACGGGTGAGTAACGCGTAGGAATCTACCTTTTTCTACGGGATAACGCATGGAAACGTGTGCTAATACCGTATACGCCCTATTGGGGGAAAGATTTTATTGGAGAGGGATGAGCCTGCGTTGGATTAGCTAGTTGGTAGGGTAAGAGCCTACCAAGGCTACGATCTATAGCTGGTCTGAGAGGACGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGAGTGAAGAAGGCCTTAGGGTTGTAAAGCTCTTTCGCCGGAGAAGATAATGACGGTAT-3'),根据所述靶标序列设计得到了一系列满足RPA引物设计原则的引物对,由华大生物合成(PAGE纯化) ; After a series of preliminary detection and screening. The primer screening results are as follows:

[0038] RPA-CLA-F: 5'-GTGAGTAACGCGTAGGAATCTACCTTTTTCTA-3';

[0039] RPA-CLA-R: 5'-ATCCCTCTCCAATAAAATCTTTCCCCCAATA-3';

[0040] The size of the amplified fragment is 105bp;

[0041] After determining the optimal primer set, the RPA amplification system was established, and conditions such as primer concentr...

Embodiment 2

[0043] Example 2 crRNA screening and CRISPR / cas12a system optimization

[0044] Among the amplified 105bp fragments, a 27bp fragment of TTTV- (PAM site) that meets the detection requirements of cas12a was selected and determined as the target of the crRNA guide sequence. The specific target nucleotide sequence is 5'-TTTCTACGGGATAACGCATGGAAACGT- 3', while designing and synthesizing the corresponding crRNA according to the specific target nucleotide.

[0045] The crRNA sequence is as follows:

[0046] Unmodified IVTcrRNA:

[0047] 5'-CUUAAUUUCUACUAAGUGUAGAUUACGGGAUAACGCAUGGAAACGU-3';

[0048] And CM crRNA modified with three thio and methoxy groups at both ends:

[0049] 5'-mC*U*UAAUUUUCUACUAAGUGUAGAUUACGGGAUAACGCAUGGAAAC*G*mU-3', * is sulfo modification, m is methoxy modification; it is consistent with the above self-synthesized target, but the synthesis method and modification are different. The chemical synthesis is by GenScript Biotechnology company synthesized and RNase...

Embodiment 3

[0060] Example 3 Establishment of RPA-CRISPR / cas12a method

[0061] 1. Method

[0062] First, dilute the standard to the specified concentration: use RNase free H 2 O Dilute the plasmid containing the 16s rRNA gene fragment of Huanglongbing bacteria to a concentration of 10 to the 0 to 10 power copy number / μL. Then use the CRISPR / cas12a system described in Example 2 to prepare the Cas12a / crRNA complex (RNP), and finally add 2.5 μL of 10 μM ssDNA fluorescent probe and the above-mentioned diluted standard product amplified by RPA in Example 1 or 7.5 μL of standard not amplified by RPA. React at 37°C for 2 hours. Use a microplate reader or qPCR instrument to track and measure the fluorescence value under the blue-green excitation light, or take a photo to judge directly with the naked eye. The ssDNA fluorescent probes are 3 kinds of modified oligonucleotides:

[0063] ssDNA-reporter1: 5'-FAM-TTTTTT-BHQ1-3';

[0064] ssDNA-reporter2: 5'-FAM-ggTTggTgTgg-BHQ1-3';

[0065] ssD...

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Abstract

The invention discloses a kit and a method for visual detection of citrus huanglongbing on the basis of an RPA-CRISPR-Cas12a system. The kit comprises an RPA primer and a crRNA guide sequence. The method comprises the following steps: carrying out RPA amplification on a to-be-detected sample, guiding a CRISPR-Cas12a system to carry out recognition combination on an RPA amplification product under the mediation of a crRNA sequence, cutting target double-stranded DNA to activate a non-specific nuclease function, randomly cutting an ssDNA fluorescent probe in the system to obtain a split product, and finally carrying out judgment through color development detection of the split product. The citrus huanglongbing can be duplexly and specifically detected through RPA amplification and crRNA recognition, high specificity is realized, sensitivity can be as low as 2 copies / [mu]L, plants infected with the citrus huanglongbing can be found earlier, and the citrus huanglongbing can be prevented and treated.

Description

technical field [0001] The invention relates to the technical field of plant pathological molecular diagnosis, and more specifically, to a kit and method for visually detecting citrus Huanglongbing based on the RPA-CRISPR-Cas12a system. Background technique [0002] Citrus Huanglongbing (Citrus Huanglongbing) is one of the most serious diseases in citrus production at present, causing huge economic losses to the citrus industry in the world. The disease is currently thought to be caused by a Gram-negative bacterium confined to the phloem sieve tube cells, which belongs to the prokaryotes, Phyllobacteria, candidate Phloembacteria genus of the class Proteobacteriacea ( Candidatus Liberibacter sp.), three species were found on citrus: Asian species ( Candidatus Liberibacterasiaticus), African species ( Candidatus Liberibacterafricanus) and American species ( Candidatus Liberibacter americaus), of which the Asian species " Candidatus "Liberibacter asiaticus" is currently the...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6844C12Q2521/507C12Q2522/101C12Q2521/327
Inventor 林进添梁芳吴仲真舒本水宾淑英杨清
Owner ZHONGKAI UNIV OF AGRI & ENG
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