Primer, plasmid and method for detection of mulberry mosaic type atrophy-associated viruses

A technology for virus detection and wasting disease, which is applied in the application field of molecular detection of viruses related to mulberry leaf wasting disease, achieves good practical promotion and application prospects, convenient use, and reliable detection results

Inactive Publication Date: 2018-05-15
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the use of PCR technology to detect geminiviruses has been reported (Xie Yan et al., 2002, Ye Qingjing et al., 2009), but the virus associated with mulberry leaf wilt disease is a new species of geminiviruses (Ma Yuxin, 2015), and its PCR detection The technology needs further research, and the research of using PCR technology to detect mulberry virus has not been carried out so far.

Method used

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  • Primer, plasmid and method for detection of mulberry mosaic type atrophy-associated viruses
  • Primer, plasmid and method for detection of mulberry mosaic type atrophy-associated viruses
  • Primer, plasmid and method for detection of mulberry mosaic type atrophy-associated viruses

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1 detection primer design and establishment of PCR amplification method

[0044] 1. Primer design

[0045] On the basis of obtaining the whole genome of the mulberry leaf dwarf disease-associated virus, several pairs of primers are designed, and the preferred two pairs of primers in the present invention are determined. And through a large number of specificity and sensitivity tests, it has been proved that the 2 pairs of primers have excellent specificity and sensitivity. The primer sequences are as follows:

[0046] (1) MCP746f / MCP1148r primer set:

[0047] Upstream primer MCP746f (SEQ ID NO.1):

[0048] 5'-CGAGTTTGGCAAGAAGGAAGAG-3'

[0049] Downstream primer MCP1148r (SEQ ID NO.2):

[0050] 5'-TTGGCTCCCACTAAATGAAAGG-3'.

[0051] (2) 13MCP156f / 13MCP1464r primer set:

[0052] Upstream primer 13MCP156f (SEQ ID NO.4): 5'-TAAAGCAGATTCGCCGACAG-3'

[0053] Downstream primer 13MCP1464r (SEQ ID NO.5): 5'-GGGACTCCCATCCAGAGGTA-3'

[0054] 2. Establishment of ...

Embodiment 2

[0069] Example 2 Construction of positive control plasmid 13MCP-pEASY-Bblunt

[0070] The coat protein gene sequence of the mulberry leaf dwarf disease-associated virus DNA is shown in SEQ ID NO.6. The present inventor analyzed and summarized the nucleic acid sequence of the virus associated with mulberry mosaic leaf atrophy, and designed primers for amplifying and selecting the 13MCP fragment comprising the coat protein gene sequence (shown in SEQ ID NO.7), including upstream primer 13MCP156f and downstream primer 13MCP1464r , the amplification conditions are as the system and conditions of the 13MCP156f / 13MCP1464r primer set in Example 1. 13MCP was used to construct the 13MCP-pEASY-Blunt positive plasmid according to the pEASY-Blunt Cloning Kit kit of Quanshijin Biotech Co., Ltd., which is used as a positive control during detection, and its nucleotide sequence is shown in SEQ ID NO.5.

Embodiment 3

[0071] The specific detection of embodiment 3 primer MCP746f / MCP1148r

[0072] 1. Separate fungi and bacteria from mulberry leaves, and powdery mildew pathogenic bacteria (Phyllactinia moricola) and mulberry sclerotinia pathogenic bacteria (Ciboria carunculoides) that are the same as the pathogenic fungi of mulberry trees. Beauveria bassiana, Beauveria bassiana, Ralstonia solanacearum, the pathogenic bacterium of mulberry wilt, was used as a control group. PCR amplification was carried out by the method of Example 1 using primers MCP746f / MCP1148r, and the results were detected by agarose gel electrophoresis after the amplification was completed.

[0073] 2. The amplification results of the primers are as attached figure 1 shown. The results showed that only the DNA of the mulberry leaf dwarf disease-associated virus had a band at the target position (403bp). It indicated that the primer set could specifically detect the virus associated with mulberry leaf-shrinking disease. ...

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Abstract

The invention discloses a primer, a plasmid and a method for the detection of mulberry mosaic type atrophy-associated viruses. The primer comprises an upstream primer MCP746f and a downstream primer MCP1148r, and nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.1 and SEQ ID NO.2; meanwhile, the positive plasmid for detection is constructed,the fragment construction plasmid 13MCP-pEASY-Bblunt with a size of 1309 bp is obtained by the amplification of an upstream primer 13MCP156f and a downstream primer 13MCP1464r, and nucleotide sequences of the plasmid and the primers are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5. The primer disclosed by the invention can specifically detect the mulberry mosaic type atrophy-associated viruses, is reliable in detection results, easy to operate, strong in specificity and high in sensitivity, particularly has an important practical application significance in the detection of diseased leaves at the early stage of infection (during the latent incubation period), meanwhile can detect pathogenic bacteria existing on leaves, petioles, buds and branches of diseased plants and has a practical guiding significance in the prevention and the control of the mulberry mosaic atrophy-associated viruses.

Description

technical field [0001] The invention relates to the technical field of pathogen molecule detection. More specifically, it relates to an application in the molecular detection of viruses associated with mulberry leaf wasting disease. Background technique [0002] Mulberry is an important economic crop in my country. With the comprehensive utilization of mulberry resources, in addition to being the main food for silkworms, mulberry has more food and medicinal value and ecological restoration functions. With the growth of the mulberry industry, mulberry diseases will gradually become the focus of our attention. [0003] As one of the most important mulberry virus diseases, mulberry mosaic leaf atrophy is common and serious in silkworm regions in my country. The disease has a great impact on the yield of mulberry leaves. The promotion of hybrid mulberry varieties has led to a decrease in the planting area of ​​disease-resistant varieties, which eventually led to a large increa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12N15/63C12R1/94
CPCC12Q1/686C12Q1/701C12Q2600/166C12Q2565/125
Inventor 刘吉平孙勋勋杨宏宇
Owner SOUTH CHINA AGRI UNIV
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