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Real-time fluorescent quantitative LAMP primer group for detecting purple leaf curl disease phytoplasma of sisal hemp and application thereof

A phytoplasma and primer set technology, applied in the biological field, can solve problems such as a large number of non-specific bands, and achieve high sensitivity and specificity, high sensitivity, and strong specificity

Active Publication Date: 2021-01-12
ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the technical defects of a large number of non-specific bands and a large number of false positive sequences in the existing phytoplasma universal primers when detecting phytoplasma related to sisal purple leafroll disease, in order to detect more quickly, accurately and practically Sisal purple leaf curl phytoplasma

Method used

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  • Real-time fluorescent quantitative LAMP primer group for detecting purple leaf curl disease phytoplasma of sisal hemp and application thereof
  • Real-time fluorescent quantitative LAMP primer group for detecting purple leaf curl disease phytoplasma of sisal hemp and application thereof
  • Real-time fluorescent quantitative LAMP primer group for detecting purple leaf curl disease phytoplasma of sisal hemp and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, detection of sisal purple leafroll disease phytoplasma (Phytoplasma) real-time fluorescence quantitative LAMP group primer preparation

[0036] 1. Primer design:

[0037] According to this experiment, the Phytoplasma (Phytoplasma) 16Sr DNA sequence (sequence 5 in the sequence listing) obtained from the purple leafroll diseased plant was cloned and compared with the false positive sequence in the sisal plant for multiple comparisons, and the polymorphic-rich region was selected for primers design. Primers were designed around the specific region and its flanking sequences using the online software Primer-Primer Explore (Fujitsu Ltd., Tokyo, Japan) (http: / / primer explorer.jp / e / ).

[0038] Design primer set of the present invention, be:

[0039] F3 upstream primer sequence: CCACGCCGTAAACGATGAG (from the 657-669th nucleotide at the 5' end of sequence 5 in the sequence listing, i.e. sequence 1 in the sequence listing);

[0040] The sequence of the downstream...

Embodiment 2

[0049] Embodiment 2, real-time fluorescence quantitative LAMP detection sisal phytoplasma (Phytoplasma) system optimization

[0050] Using the primer set prepared in Example 1, four factors and four levels of orthogonal experiments were carried out using the sisal purple leafroll disease phytoplasma plasmid DNA as a template (Table 1, Table 2).

[0051]

[0052]

[0053]

[0054] The above 25 μL LAMP reaction system was quickly placed at 63° C. for real-time fluorescent quantitative LAMP reaction. The system is as follows:

[0055]

[0056]

[0057] The QuantStudio 6Flex real-time fluorescence quantitative instrument is used for detection, and the LAMP reaction result can be judged by observing the amplification curve: if the detection result has no Ct value and no typical amplification curve, it means that the sample does not contain phytoplasma of sisal purple leafroll; If a Ct value ≤ 35.0 is observed and a typical amplification curve appears, it indicates t...

Embodiment 3

[0060] Embodiment 3: detect the specificity of the real-time fluorescent quantitative LAMP primer set of sisal purple leafroll disease phytoplasma (Phytoplasma)

[0061]Utilize embodiment 1 to prepare the gained primer set, with sisal purple leafroll disease strain, sisal zebra stripe fungus (Phytophthora nicotianae), sisal stalk rot fungus (Aspergilus niger), coffee dew wetting fungus (Paramyrothecium roridum) , coffee palm Pestalotiopsis trachicarpicola, coffee Fusarium solani, coffee anthracnose (Coletotrichum gloeosporioides), sugarcane red rot (Coletotrichum falcatum), sugarcane smut (Ustilago scitaminea), rice Magnaporthe oryzae, Xanthomonas oryzaepv. Oryzicola, and Bacilus subtilis DNA were subjected to LAMP reaction, and the specificity of the primer set provided by the present invention was analyzed.

[0062] DNeasy Plant Mini Kit (QIAGEN, Germany) was used to extract the DNA of the above-mentioned sisal purple leafroll diseased strain samples. Fungal DNA extraction ...

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Abstract

The invention discloses a real-time fluorescent quantitative LAMP primer group for detecting purple leaf curl disease phytoplasma of sisal hemp and application thereof. The LAMP primer group for the purple leaf curl disease phytoplasma consists of nucleotide shown as a sequence 1 in a sequence table, nucleotide shown as a sequence 2 in the sequence table, nucleotide shown as a sequence 3 in the sequence table and nucleotide shown as a sequence 4 in the sequence table. An LAMP molecular detection method has the advantages of strong specificity, high sensitivity, easiness in operation and reliable result.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to a real-time fluorescent quantitative LAMP primer pair, a kit and a detection method for detecting sisal purple leafroll disease phytoplasma. Background technique [0002] Sisal purple leaf curl is a devastating disease that can cause huge losses to the sisal industry. The disease was first discovered in Qingkan Farm, Changjiang, Hainan in November 2001, and it spread rapidly to the whole farm and surrounding farms in less than half a year. Sporadic diseased sisal plants occurred in the company and state-owned Torch, Wuyi and other farms and surrounding rural areas. Since then, the disease has gradually spread to the entire Guangdong Province. According to incomplete statistics, as of 2018, only 100,000 mu of sisal purple leaf roll disease had been reported in Zhanjiang, Guangdong, and 30,000 mu of sisal had been eliminated due to severe disease, and the incide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/107C12Q2561/113
Inventor 吴伟怀易克贤鹿鹏鹏王桂花贺春萍梁艳琼习金根黄兴郑金龙谭施北
Owner ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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