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Primer, primer pair, method and reagent kit for simultaneously detecting four viruses

A primer pair, virus technology

Inactive Publication Date: 2014-08-13
TAIWAN SUGAR CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, most of the techniques for detecting Phalaenopsis viruses on the market are for detecting CyMV and ORSV, and there is no molecular detection method that can simultaneously detect CyMV, ORSV, CMV, and CaCV.

Method used

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  • Primer, primer pair, method and reagent kit for simultaneously detecting four viruses
  • Primer, primer pair, method and reagent kit for simultaneously detecting four viruses
  • Primer, primer pair, method and reagent kit for simultaneously detecting four viruses

Examples

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Effect test

example 1

[0060] Example 1 Primer Design

[0061] Degenerate primers were designed for the genes of Cucunber mosaic virus sheath protein (CMV-CP for short) and Capsicum chlorosis tospovirus nucleosheath protein (CaCV-NP for short), so that the primers can simultaneously detect target viruses with different gene mutations.

[0062] The published gene sequences of CMV-CP and CaCV-NP were searched by GenBank. 以CMV-CP基因序列(GenBank序列编号AJ131618、AJ131619、AJ871492、DQ004557、D28780、AJ810266、AY611027、FJ403473、EU128723、AF198622、DQ285569、AK172329、AY172328、EF620777、FJ896156、FJ896157、FJ896160、FJ896159)设计对CMV- CP gene-specific primer pair CM343-F (SEQ ID NO: 1) and CM343-R (SEQ ID NO: 2). Primers Ca157-F (SEQ ID NO: 3) and Ca157-R (SEQ ID NO: 4) were designed based on the CaCV-NP gene sequence (GenBank sequence numbers EF100595, EF100598, EF100597, EF137177, EF100600, EF602029, EU216023, EU095940).

[0063] The principle of primer design is that the GC content is more than 40%, and avoid the formation ...

example 2

[0066] Example 2RNA preparation

[0067] After lyophilization, the sample was ground with glass beads, and the ground powder was stored in a -80°C refrigerator. Take 0.1g ground sample and use Trizol (invitrogen; cat#15596018), Quick Gene RNA Tissue Kit II (FujiFilm; cat#RT-S2) and Dr. RNA Extraction Kit (Crystal Biotechnology Company; cat#8c2018) -01) RNA extraction, the extraction steps refer to the operation manual of each company.

example 3

[0068] Example 3 standard plasmid preparation

[0069] Using the nucleic acid of leaf specimens infected with CMV and CaCV viruses as templates, CM343-F / CM343-R and Ca157-F / Ca157-R degenerate primers were used for RT-PCR amplification, and Promega SV Gel and PCR Clean-Up System (Promega; cat#A9281) was used for recovery and purification of DNA fragments. The recovered amplified product was bonded to the TA vector, selected and colonized in the yT&A plasmid (Yisheng Biotech; cat#YC103), and sequenced after transformation into the corresponding Escherichia coli (Yisheng Biotech; cat#YE607). The CaCV sequencing sequence is SEQ ID NO: 14, which was confirmed as GenBank sequence number EF095725 after alignment; the CMV sequencing sequence was SEQ ID NO: 13, which was confirmed to be GenBank sequence number EU329011 after alignment. Take 3mL of the overnight cultured bacterial solution containing the plasmid, remove the supernatant after centrifugation, and then use the PureYield ...

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Abstract

The present invention relates to a primer, a primer pair, a method and a reagent kit for simultaneously detecting four viruses, namely cymbidium mosaic virus (CyMV), Odontglossum ringspot virus (ORSV), Cucumber mosaic virus (CMV) and Capsicum chlorosis tospovirus (CaCV).

Description

technical field [0001] The present invention relates to virus detection technology. Specifically, the present invention relates to a detection technique for simultaneously detecting several orchid viruses by utilizing orchid virus-specific primers and primer pairs. Background technique [0002] Orchid is an important flower product with great economic value, and Phalaenopsis is the main flower among them. For example, Taiwan's export volume has shown positive growth over the years. In 2009, the export value reached NT$2 billion. Like all plants, orchids may face the threat of pests and diseases during the cultivation process, resulting in loss of growth and quality. For most diseases that infect orchids, as long as traditional control methods such as application of pesticides are used, good control effects can be obtained, but viral diseases infected by viral viruses still lack effective treatment strategies so far. Therefore, the disease caused by the virus can be said t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C40B40/06C12R1/94
Inventor 黄怡仁陈郁璇
Owner TAIWAN SUGAR CORP
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