RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit capable of detecting CymMV (Cymbidium Mosaic Virus) and ORSV (Odontoglossum Ringspot Virus) simultaneously and method thereof

Abstract

The invention provides a RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit capable of detecting a CymMV (Cymbidium Mosaic Virus) and an ORSV (Odontoglossum Ringspot Virus) simultaneously. The RT-PCR detection kit mainly comprises specific degenerate primers and a PCR reaction reagent, wherein the nucleotide sequences of the degenerate primers are shown as follows: an upstream primer: 5'-TYAAMAMTKKCGAGAAAG-3'; and a downstream primer: 5'-TYGKGWMCRGTGTTAAT-3'. By designing degenerate primers specific to two types of viruses, a one-step RT-PCR detection kit capable of detecting two types of cymbidium viruses simultaneously and a method thereof are established. As proved by experience, the method is efficient, rapid and accurate, and is a good virus detection method.

Description

(1) Technical field [0001] The invention relates to a RT-PCR detection kit capable of simultaneously detecting Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) and a detection method thereof. (2) Background technology [0002] Cymbidium mosaic virus (CymMV) is one of the two most common viruses infecting orchids. CymMV belongs to Flexiviridae and Potexvirus. CymMV virus particles are linear positive-sense single-stranded RNA (+ssRNA) with a size of about 475nm×13nm or 490nm×13nm. The virus shell has no envelope , the line is curved, the helix is ​​symmetrically extended, the thread is obvious, the pitch is 2.8nm, there is a methylation structure at the 5' end, and a poly(A) tail at the 3' end. The complete sequence of the viral RNA genome is about 6-7kb, and the entire genome consists of 5 An open reading frame (ORF), coded as RDRP (160KDa), TGB (26KDa / 13KDa / 10KDa) and CP (24KDa), where the CP gene is a complete ORF5 frame, about 700nt long, encoding a...

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Problems solved by technology

Orchid plants are severely damaged by CymMV and ORSV virus diseases and cannot be cured. The combined infection rate is high. Once infected with these two viruses, the leaves of orchids will form chlorotic stripes, depressed gray spots or necrotic circles. Spots and other symptoms lead to poor plant growth, small and less flowering, shortened flowering period, greatly reduced the ornamental value of the plant, and caused huge economic losses. important bottleneck of

Conventional virus serological detection methods generally have the disadvantage of low sensitivity, while for general molecular detection of viruses, the quality of RNA extraction has a great impact on the experimental results, and RNA is particularly easy to degrade, requiring high extraction techniques; at the same time, it is necessary to To detect two orchid viruses, it is necessary to design different primer pairs for the two viruses, and the detection process must be carried out separately, which is time-consuming and laborious

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