PCR primer combination for Passiflora edulia Sims cucumber mosaic virus and detecting method

A combination technology of cucumber mosaic virus and primers, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problems of difficult identification of diseased plant materials, low detection cost, etc., and achieve high sensitivity High, avoid false negatives, strong stability

Active Publication Date: 2019-08-13
三明市农业科学研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although passion fruit CMV-infected plants can show obvious characteristics, the onset of CMV requires certain climatic conditions, and it is difficult to identify diseased plant materials
The use of PCR technology for CMV vir

Method used

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  • PCR primer combination for Passiflora edulia Sims cucumber mosaic virus and detecting method
  • PCR primer combination for Passiflora edulia Sims cucumber mosaic virus and detecting method
  • PCR primer combination for Passiflora edulia Sims cucumber mosaic virus and detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] plant material

[0035] The leaves of CMV-positive passion fruit in Sha County, Sanming were used as materials.

[0036] Total RNA extraction and cDNA synthesis from leaves to be tested

[0037] Cut 0.5 g of CMV-positive passion fruit leaves, extract total RNA with 1 mL Trizol reagent (Invitrogen), detect RNA quality with 1% agarose, and measure RNA concentration with a UV spectrophotometer. Take 0.5 μg of total RNA, and use RevertAidFisrst Strand cDNA Synthesis Kit (Fermentas) for cDNA first-strand synthesis (10 μL system).

[0038] Primer synthesis

[0039] The conserved nucleotide sequence was designed according to the CMV coat protein sequence, and specific primers were designed using Primer 5.0. Primers were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. The primer sequences and expected target fragment sizes are listed in Table 1.

[0040] Table 1 Primer sequences and expected target fragment sizes

[0041]

[0042] PCR amplification and det...

Embodiment 2

[0052] plant material

[0053] The test materials come from the passion fruit leaves of virus-free seedlings in the passion fruit seedling base of Huizhou Xinwo Agricultural Technology Co., Ltd., Guangdong Province ( Figure 3-1 ); Sanming Academy of Agricultural Sciences, Fujian Province suspected CMV 'golden fruit' passion fruit leaves ( Figure 3-2 ), ‘Tainong No. 1’ passion fruit leaves ( Figure 3-3 ), ‘Uranus’ passion fruit leaves ( Figure 3-4 ). Passion fruit CMV positive control leaves in Sha County, Sanming City.

[0054] Total RNA extraction and cDNA synthesis from leaves to be tested

[0055] 0.5 g of passion fruit leaves to be tested and CMV positive control leaves were cut respectively, total RNA was extracted with 1 mL Trizol reagent (Invitrogen), RNA quality was detected with 1% agarose, and RNA concentration was determined with a UV spectrophotometer. Take 0.5 μg of total RNA and use RevertAid Fisrst Strand cDNA Synthesis Kit (Fermentas) for cDNA first-st...

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Abstract

The invention provides a PCR primer combination for Passiflora edulia Sims cucumber mosaic virus and a detecting method. Particularly, the PCR primer combination comprises 3 Passiflora edulia Sims cucumber mosaic virus coat protein specific primers and 2 Passiflora edulia Sims general reference gene EF1a specific primers. Through the design of the 3 Passiflora edulia Sims cucumber mosaic virus coat protein specific primers, 3 specific sites of the virus can be detected at the same time, and occurrence of false positive is avoided; by using the two Passiflora edulia Sims EF1a specific primers,the Passiflora edulia Sims cDNA synthesis quality can be detected, and occurrence of false negative is avoided. The whole detection process can be completed within 4 hours, PCR result detection can beconducted by using 1% agarose, an expensive fluorescent quantatitive PCR instrument is not needed, and the PCR primer combination has the advantages of being fast, economical, high in sensitivity andhigh in stability.

Description

technical field [0001] The invention relates to the field of plant virus detection, in particular to a combination of PCR primers and a detection method for passion fruit cucumber mosaic virus. Background technique [0002] passion fruit( Passifloraspp .) Also known as passion fruit, egg fruit, as a fruit that can be eaten fresh and processed, it is widely planted in tropical and subtropical regions all over the world. [0003] Passion fruit is susceptible to a variety of viral diseases, among which cucumber mosaic virus ( Cucumber mosaic virus, CMV) are the most common. CMV is a very serious RNA-like virus disease. The virus can reach any part except the growth point. Passion fruit has been propagated through cuttings or grafting for a long time, which can easily cause the spread of large-scale diseased plants. In terms of chemical planting, usually the whole plant can only be directly destroyed if the virus disease is found in passion fruit. Therefore, the key to the p...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/94
CPCC12Q1/701C12Q1/686C12Q2600/166C12Q2545/101C12Q2565/125Y02A50/30
Inventor 江金兰叶炜杨学匡云波颜沛沛王培育周建金廖承树罗晓锋乔锋赖瑞联
Owner 三明市农业科学研究院
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