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
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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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