A kind of oncidium virus detection primer and method

A technology for virus detection and cymbidium ring spot virus, applied in the field of biology, can solve the problems of low detection sensitivity, high price of antiserum, false positive reaction, etc., and achieve the effects of high detection sensitivity, high detection efficiency and low cost

Active Publication Date: 2016-08-24
CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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Problems solved by technology

[0004] At present, antiserum-based enzyme-linked immunosorbent assay (ELISA method) is mostly used in the detection of oncidium virus. A certain degree of false positive reaction, relatively low detection sensitivity and other issues
In recent years, in order to improve the efficiency of virus detection, virus detection technology based on PCR technology is developing rapidly. However, single PCR detection technology can only detect one virus in one reaction. The detection of viruses has the problems of time-consuming, labor-intensive, and high cost; in view of this, in recent years, researchers at home and abroad have gradually applied multiple RT-PCR technology to plant virus detection. , multiplex PCR detection technology has the advantages of simultaneously detecting multiple viruses in one mRT-PCR reaction, simpler operation, shortened detection time and reduced experimental cost, etc.
However, there is no relevant literature report on the simultaneous detection of CyMV, ORSV and BYMV in Oncidium leaves using multiplex PCR detection technology

Method used

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  • A kind of oncidium virus detection primer and method
  • A kind of oncidium virus detection primer and method
  • A kind of oncidium virus detection primer and method

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Embodiment 1

[0024] The present invention is a method for detecting oncidium virus. Based on the conserved nucleotide sequences of coat protein genes of three viruses, namely, the orchid mosaic virus, the jaundice ring spot virus and the bean yellow mosaic virus, primers for the Jianlan mosaic virus are designed and synthesized respectively. Oncidium ring spot virus primer pair and common bean yellow mosaic virus primer pair, and then establish mRT-PCR detection system, and use the Jian orchid mosaic virus primer pair to detect the oncidium mosaic virus, the tooth orchid ring spot virus in the leaves of Oncidium The primer pair was used to carry out mRT-PCR reaction for the jaundice ring spot virus and the bean yellow mosaic virus in the leaves of Oncidium orchid, and finally the reaction products were subjected to 1% agarose gel electrophoresis. Analysis and clone sequencing alignment.

[0025] Wherein, each primer pair is specifically: (1) build orchid mosaic virus primer pair (as shown ...

Embodiment 2

[0038] The PCR amplification products obtained by the multiple PCR reaction system in step 400 of Example 1 were recovered by gel tapping, and then respectively connected with pMD18-T simple vector in vitro, and then positive clones were picked, and finally the plasmids were extracted and sequenced to check CyMV and ORSV. and BYMV amplification results of three viruses.

[0039] And the sequencing result is: the amplification products of CyMV, ORSV and BYMV are sequences consisting of 571, 325 and 212 nucleotides respectively, wherein the sequence of the CyMV amplification product is shown in SEQ ID NO: 7, the ORSV The sequence of the amplification product is shown in SEQ ID NO: 8, and the sequence of the BYMV amplification product is shown in SEQ ID NO: 9. The homology analysis by NCBI showed that the obtained amplification product sequence SEQ ID NO: 7 had a homology rate of 99% with the reference sequence, that is, the coat protein gene sequence of CyMV, and the sequence SE...

Embodiment 3

[0042] Application of multiplex mRT-PCR for simultaneous detection of CyMV, ORSV and BYMV:

[0043] Step 1: The sampling location is the Germplasm Resource Garden of the Flower Research Center of the Fujian Academy of Agricultural Sciences. Randomly select 8 oncidium leaf samples with toxic symptoms in the field, and the 8 oncidium leaf samples are all 0.2 g; then RNA extraction is performed on each sample (the operation is the same as step 200 in Example 1), and the RNA is reverse transcribed into cDNA (the operation is the same as step 300 in the first embodiment).

[0044] Step 2: Using the cDNA obtained by processing as a template, and using the three pairs of primers described in the present invention as primer pairs, single-plex and multiplex mRT-PCR amplification reactions are performed simultaneously, and the specific steps are the same as step 400 in the first embodiment.

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Abstract

The invention designs and synthesizes three pairs of corresponding characteristic primers respectively according to nucleotide sequences of coat protein genes of cymbidium mosaic virus, odontoglossum ringspot virus and bean yellow mosaic virus and also discloses a method for detecting cymbidium mosaic virus, odontoglossum ringspot virus and bean yellow mosaic virus existing in the leaves of oncidium at the same time by virtue of the three pairs of primers. The invention establishes a method in which the three oncidium viruses (cymbidium mosaic virus, odontoglossum ringspot virus and bean yellow mosaic virus) can be detected at the same time through one reaction. The method has the characteristics of high detection efficiency and low detection cost; even through the content of RNAs of the three oncidium viruses in a sample is very low, the three oncidium viruses still can be detected by the method; therefore, the method can ensure high detection sensitivity.

Description

【Technical field】 [0001] The invention relates to a plant virus detection primer and method in the field of biology, in particular to an oncidium virus detection primer and method. 【Background technique】 [0002] Cymbidium mosaic virus (CyMV), Odontoglossum ringspot virus (ORSV) and Bean yellow mosaic virus (BYMV) are the three main viruses that endanger oncidium. [0003] Oncidium has a beautiful flower shape and a long flowering period. It is a flower of high economic value with great competitiveness and development potential. However, the harm of virus diseases seriously affects its ornamental and utilization value, causing the plants to show symptoms such as mosaic leaves, necrosis, deformity, discoloration of petals, short flower ears, and shortened flowering period, which seriously degrades the quality of oncidium and has become a constraint on the development of the oncidium industry. main limiting factor. At present, there is no effective medicine for the preventio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor 黄敏玲樊荣辉钟淮钦叶秀仙罗远华吴建设林兵
Owner CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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