CN102363819A

CN102363819A - Primer for detecting sugarcane streak mosaic virus (SCSMV) and detection method thereof

Info

Publication number: CN102363819A
Application number: CN2011103496166A
Authority: CN
Inventors: 李文凤; 黄应昆; 王晓燕; 尹兴祥; 罗志明; 尹炯
Original assignee: Sugarcane Research Institute of Yunnan Academy of Agricultural Sciences
Current assignee: Sugarcane Research Institute of Yunnan Academy of Agricultural Sciences
Priority date: 2011-11-08
Filing date: 2011-11-08
Publication date: 2012-02-29

Abstract

The invention provides a primer for detecting sugarcane streak mosaic virus (SCSMV) and a detection method thereof. A nucleotide sequence of the primer is shown as a sequence list SCSMV-F&SCSMV-R. The invention further provides a method for detecting the sugarcane streak mosaic virus. The method comprises the following steps of: performing reverse transcription polymerase chain reaction (RT-PCR) by taking a sample as a total ribonucleic acid (RNA) as a template and utilizing the primer, and after reaction is finished, determining a result according to the position of a specific amplification fragment. The primer has high specificity; and the detection method is quick and simple and can be used for detecting a large number of sugarcane samples, so the primer provides a guarantee for introduced quarantine inspection of sugarcanes and the propagation dynamic monitoring of the sugarcane streak mosaic virus in sugarcane areas of China.

Description

Sugarcane striped mosaic virus detects with primer and detection method thereof

Technical field:

The present invention relates to Measurement for Biotechnique, relate to sugarcane striped mosaic virus specifically and detect with primer and detection method thereof.

Background technology:

Sugarcane striped mosaic virus (Sugarcane streak mosaic virus; SCSMV) be a kind of novel cause of disease that identifies from the mosaic of sugarcane plant; Belonging to one of marmor upsilon section (Potyviridae) newly belongs to---and Gramineae Tobamovirus (Poacevirus) is a kind of important infective pathogen that causes mosaic of sugarcane.Only there are India, Pakistan, the U.S., Thailand, Sri Lanka, Vietnam and Indonesia that report was arranged at present in the world.China is domestic not to be a lot of to this virus research, only has in sugarcane base, Guangdong from drawing the report of finding this virus from the sugarcane germ plasm resource of India.But in recent years; Because the development and the demand of sucrose industry science and technology and production; Each sugarcane district of China is in succession from frequently having introduced large quantities of sugar cane breeds and germplasm materials with domestic other sugarcane district overseas; When sugarcane is carried out breed improvement and germplasm innovation, also increased the risk of the dangerous disease diffusion of picture sugarcane striped mosaic virus class.If the sugarcane germ plasm resource to introducing a fine variety is not quarantined or quarantines not in place; Very likely cause this virus at China's sugarcane district internal diffusion; Cause potential threat for the Sustainable development of China's sucrose industry, therefore setting up this viral method for quick is the important measures of stopping this dangerous virus diffusion.

Summary of the invention:

The object of the present invention is to provide a kind of sugarcane striped mosaic virus to detect with primer and detection method thereof, the primer sequence and the detection method thereof that are used for sugarcane striped mosaic virus RT-PCR detection can be satisfied the demand.

The present invention is through analyzing the CP gene conserved regions sequence of the sugarcane striped mosaic virus of having reported, the design primer.Described primer is to being made up of forward primer and reverse primer, and its nucleotide sequence is shown in sequence table SCSMV-F&SCSMV-R.The nucleotides sequence of forward primer is classified as: SCSMV-F:5 '-ACAAGGAACGCAGCCACCT-3 ', the nucleotides sequence of reverse primer is classified as: SCSMV-R:5 '-ACTAAGCGGTCAGGCAAC-3 ', the purpose stripe size is 938bp.

The present invention has also further provided the RT-PCR detection method of using above-mentioned primer, and this method is a template with the total RNA of sample, carries out the RT-PCR amplification, transcribes synthetic cDNA, and reaction finishes the location determination result of back according to the 938bp dna fragmentation of specific amplification.

The RT-PCR amplification procedure is following:

The reaction system of RT is 10ul; The sterilization distilled water, 2.0ul 5 * ThermoScript II damping fluid, 1.0ul DNTP, reverse primer SCSMV-R 0.5ul (20um/ul), 0.5ul RNA enzyme inhibitors, 0.5ul ThermoScript II, the total RNA of 3ul that promptly in the PCR of 0.2ml reaction tubes, add 2.5ul, mixing.Response procedures is 25 ℃ of 10min, 42 ℃ of 60min, 98 ℃ of 5min, finishes the back and obtains reverse transcription product cDNA.

PCR reaction system 25ul promptly adds the sterilization distilled water of 9.5ul, 2 * PCR Taq mix of 2.0ul cDNA, forward primer SCSMV-F and each 0.5ul of reverse primer SCSMV-R (20um/ul), 12.5ul in the PCR of 0.2ml reaction tubes; Response procedures is 94 ℃ of 5min; 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 1min, 35 circulations; 72 ℃ are extended 10min.

Further, can also the invention described above primer and related reagent be assembled into test kit, use with convenient.

The present invention is according to the CP gene conserved regions sequences Design primer of sugarcane striped mosaic virus; The RT-PCR that is used for sugarcane striped mosaic virus detects; Through optimization to reaction system and reaction annealing temperature; Set up the RT-PCR detection method of sugarcane striped mosaic virus, reaction can be according to whether containing sugarcane striped mosaic virus in the segmental location determination sample of specific amplification after finishing.Detection method of the present invention, primer specificity is good, and the detection that detection method is fast simple, can be used for a large amount of sugarcane samples is for sugarcane is introduced a fine variety quarantine and the propagation dynamic monitoring of sugarcane striped mosaic virus in China sugarcane district provides assurance.

Description of drawings

Fig. 1 is the RT-PCR detected result of sugarcane striped mosaic disease, and wherein M is D2000plus DNA ladder, and 1 is the SCSMV positive control, and 2 is that SCMV positive control, 3 is the SrMV positive control, and 4 is healthy plant.

Fig. 2 is the test-results that the present invention detects a large amount of samples, and wherein M is D2000plus DNA ladder, and 1-8 is the field collected specimens, and 9 is the SCSMV positive control, and 10 is healthy plant.

Embodiment

Following examples of implementation are used for further specifying of the present invention, but are not used for limiting scope of the present invention.

Embodiment 1 primer design is with synthetic

According to the conserved sequence (accession number Y17738) of sugarcane striped mosaic virus genome sequence of having reported in the GenBank DB and coat protein gene, adopt Premier 5.0 design primers, primer sequence is:

SCSMV-F (forward): 5 '-ACAAGGAACGCAGCCACCT-3 '

SCSMV-R (oppositely): 5 '-ACTAAGCGGTCAGGCAAC-3 '

The extraction of embodiment 2 total RNA

1) gets fresh blade 0.1g+ liquid nitrogen grinding powdering, join in the 2.0ml centrifuge tube;

2) add 1000ul TRIZOL reagent, mixing gently, room temperature is placed 5min;

3) add the 200ul chloroform, concuss 15 seconds, room temperature is placed 5min, 4 ℃ of centrifugal 15min of 12000rpm;

4) get supernatant in the 1.5ml centrifuge tube, add the 600ul Virahol, behind the mixing (light mixing), room temperature is placed 10min, 4 ℃ of centrifugal 10min of 12000rpm, precipitated rna;

5) abandon supernatant, add 1000ul 75% washing with alcohol RNA deposition 1 time, the RNA that fully suspends during washing deposition (with letting the deposition levitating at the bottom of the light finger bomb tube), the centrifugal 5min of 8000rpm;

6) abandon supernatant after, be deposited in seasoning 6min in the air, use 30ul ddH ₂O dissolving RNA deposition, in 55 ℃ of dissolvings (in the water-bath) 10min ,-20 ℃ of refrigerators are preserved subsequent use.

The foundation of embodiment 3RT-PCR amplification method

With total RNA is template, carries out the RT-PCR reaction.The 10ul reaction system is carried out reverse transcription reaction; The sterilization distilled water, 2.0ul 5 * ThermoScript II damping fluid, 1.0ul DNTP, 0.5ul reverse primer SCSMV-R (20um/ul), 0.5ul RNA enzyme inhibitors, 0.5ul ThermoScript II, the total RNA of 3ul that promptly in the PCR of 0.2ml reaction tubes, add 2.5ul; Mixing; Put into the PCR appearance, response procedures is 25 ℃ of 10min, 42 ℃ of 60min, 98 ℃ of 5min, finishes the back and obtains reverse transcription product cDNA.

PCR reaction system 25ul promptly adds the sterilization distilled water of 9.5ul, 2 * PCR Taq mix of 2.0ul cDNA, 0.5ul forward primer SCSMV-F (20um/ul), 0.5ul reverse primer SCSMV-R (20um/ul), 12.5ul in the PCR of 0.2ml reaction tubes; Response procedures is 94 ℃ of 5min; 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 1min, 35 circulations; 72 ℃ are extended 10min.

The specificity of embodiment 4SCSMV RT-PCR is confirmed

The total RNA of sugarcane blade with SCSMV positive control plant is a template; With healthy leaves and other two kinds of cause of disease corn mosaic viruses (the Sugarcane mosaic virus that causes mosaic of sugarcane; SCMV) and sorghum mosaic virus (Sorghum mosaic virus; SrMV) the total RNA template of positive plant blade increases through RT-PCR as contrast, and reaction finishes the 938bp dna fragmentation result of determination of back according to specific amplification.

Test-results:

The total RNA of sugarcane blade with the SCSMV positive control; Detect the amplified fragments that can be observed 938bp through RT-PCR; Healthy leaves, SCMV and SrMV then all do not have the appearance of specific amplification band; Explain that the RT-PCR that sets up has good specificity to SCSMV, can SCSMV be come with other two kinds of cause of disease SCMV that cause mosaic of sugarcane and SrMV difference that the result sees Fig. 1.

The detection of embodiment 5SCSMV land for growing field crops sample

Totally 10 duplicate samples, comprising 8 parts from the field mosaic disease band disease sample and 1 normal healthy controls, 1 SCSMV positive control sample of random acquisition.Use the RT-PCR amplification method of being set up to detect to above-mentioned sample, detected result such as Fig. 2,8 land for growing field crops samples all detect band SCSMV.

Detection method of the present invention, primer specificity is good, and the detection that detection method is fast simple, can be used for a large amount of sugarcane samples is for sugarcane is introduced a fine variety quarantine and the propagation dynamic monitoring of sugarcane striped mosaic virus in China sugarcane district provides assurance.

The nucleotides sequence tabulation

< 110>Sugarcane Inst., Yunnan Prov. Agriculture Academy

< 120>sugarcane striped mosaic virus detects with primer and detection method thereof.

<160>2

<210>1

<211>19

<212>DNA

< 213>artificial sequence (forward primer)

<400>1

ACAAGGAACGCAGCCACCT 19

<210>2

<211>18

<212>DNA

< 213>artificial sequence (reverse primer)

<400>2

ACTAAGCGGTCAGGCAAC 18

Claims

1. a sugarcane striped mosaic virus detects and uses primer; It is characterized in that forming by forward primer and reverse primer; The nucleotides sequence of forward primer is classified as: SCSMV-F:5 '-ACAAGGAACGCAGCCACCT-3 '; The nucleotides sequence of reverse primer is classified as: SCSMV-R:5 '-ACTAAGCGGTCAGGCAAC-3 ', the purpose stripe size is 938bp.

2. one kind is adopted the said sugarcane striped of claim 1 mosaic virus to detect the detection method with primer, it is characterized in that comprising the steps:

(1) be template with the total RNA of sample, utilize the described SCSMV-R primer of claim 1 to carry out the RT-PCR amplification, cDNA is synthesized in reverse transcription;

(2) be template with synthetic cDNA, utilize above-mentioned SCSMV-F and SCSMV-R primer to carrying out pcr amplification, reaction finishes the location determination result of back according to the specific amplification dna fragmentation.

3. detection method as claimed in claim 2 is characterized in that the annealing temperature of pcr amplification in the RT-PCR reaction is 50 ℃, and cycle number is 35 times.

One kind according to claim 1 sugarcane striped mosaic virus detect test kit with primer.