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Multiplex RT-PCR method for simultaneously detecting four corn viruses

A technique of RT-PCR, corn, applied in the directions of microorganism-based methods, biochemical equipment and methods, determination/inspection of microorganisms, etc.

Active Publication Date: 2020-03-24
YUNNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But at present, the symptoms of maize plants infected by MATV or MaYMV alone have not been reported.

Method used

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  • Multiplex RT-PCR method for simultaneously detecting four corn viruses
  • Multiplex RT-PCR method for simultaneously detecting four corn viruses
  • Multiplex RT-PCR method for simultaneously detecting four corn viruses

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Embodiment 1

[0038] Sources of plant material and viruses: Four viruses (MCMV, SCMV, MaYMV, MATV) co-infected maize samples were collected from Yunnan Province in 2017, and the leaves were collected into clean bags and stored in a -80°C refrigerator ; 138 samples were collected from Daejeon, including 52 in Gansu and 86 in Yunnan, and the leaves were stored in a -80°C refrigerator; the total RNA of maize used the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa BIO INC, Japan) kit For extraction, the extracted RNA was reverse-transcribed into cDNA with M-MLV Reverse Transcriptase (TaKaRa BIO INC, Japan) reverse transcriptase, and the obtained cDNA could be directly subjected to PCR reaction or stored at -80°C for future use.

[0039] 1. RNA extraction and cDNA synthesis

[0040] Corn RNA extraction steps (refer to TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa BIO INC, Japan) kit instructions):

[0041] (1) Put an appropriate amount of corn leaves in a mortar, add liquid nitroge...

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Abstract

The invention discloses a multiplex RT-PCR method for simultaneously detecting four corn viruses, and belongs to the technical field of plant virus detection. According to the method disclosed by theinvention, corn elongation factor 1 alpha (Elongation factor 1 alpha, EF 1 alpha) gene is used as a reference gene; on the basis of optimizing the annealing temperature and the primer concentration ofa multiplex PCR detection system, a multiplex RT-PCR detection method for simultaneously detecting four kinds of maize viruses MCMV, SCMV, MaYMV and MATV is established. The multiplex PCR detection system disclosed by the invention is a detection method which is simple, rapid, economical and low in time consumption, and can be used for simultaneously detecting four viruses (MCMV, SCMV, MaYMV andMATV) of corn; the method can be widely applied to agriculture to diagnose the four viruses on the corn in advance, and has very important significance in controlling the occurrence of diseases and increasing economic benefits.

Description

technical field [0001] The invention relates to a multiple RT-PCR method for simultaneously detecting four corn viruses, belonging to the technical field of plant virus detection. Background technique [0002] Maize is an important economic crop and is widely planted all over the world, mainly in North America, Asia, Africa and Latin America. China is the world's second largest corn consumer and producer, with production expected to increase to 25 million tons in 2020. However, more than 50 viruses have been reported to infect maize and cause significant yield loss during maize growth. These viruses often form compound infections in maize, including compound infections of two or more viruses. For example, maize lethal necrosis (MLND) is caused by a co-infection between maize chlorotic mottle virus (MCMV) and a virus from the family Potatoviridae, in which MCMV forms a complex with sugarcane mosaic virus (SCMV) Infestation is most common. [0003] Lethal necrosis of maize...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12R1/94
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2600/166C12Q2537/143C12Q2521/107
Inventor 王建光李小琴李妤李英娟胡文丽陈穗云
Owner YUNNAN UNIV
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