CN103757140A - Detection kit for sugarcane streak mosaic virus (SCSMV) and detection method of SCSMV - Google Patents

Abstract

The invention relates to a detection kit for sugarcane streak mosaic virus (SCSMV) and a detection method of SCSMV, and is dedicated to detection of SCSMV. The detection kit comprises a forward primer, a reverse primer, RTBuffer, an RNA enzyme inhibitor, reverse transcription enzyme, dNTPs, 2*PCR Taq mix, a positive control, a negative control and RNase-freeddH2O. According to the invention, a specific primer is designed based on CP (coat protein) gene sequence of the SCSMV; the SCSMV is detected through an RT-PCR (reverse transcription polymerase chain reaction) technology; the detection is quick, accurate, sensitive, and high in operability; the preparation method for the detection kit is simple and convenient; the detection kit is not only suitable for fast test and detection of a sugarcane introducing quarantine sample, but also used for detection, diagnosis, epidemic situation monitoring, prewarning and forecast of SCSMV in sugarcane production, and broad in application prospect.

Description

Sugarcane stripe mosaic virus detection kit and detection method thereof

Technical field:

The present invention relates to a kind of sugarcane stripe mosaic virus detection kit and detection method thereof, belong to technical field of biological, not only be applicable to sugarcane to introduce a fine variety the rapid detection of quarantine sample, also can be applicable to detection diagnosis, epidemic situation monitoring and the early-warning and predicting of stripe mosaic virus in sugarcane production.

Background technology:

Sugarcane stripe mosaic virus (Sugarcane streak mosaic virus, SCSMV) be the novel cause of disease of one identifying from mosaic of sugarcane plant, belong to new genus of marmor upsilon section (Potyviridae)---Gramineae Tobamovirus (Poacevirus), it is the important infective pathogen of one that causes mosaic of sugarcane, main by sugarcane asexual propagation material (germplasm) propagation, research shows that this virus has abundant genetic diversity.There were report in Jin You India, Pakistan, the U.S., Thailand, Sri Lanka, Vietnam and Indonesia in the world at present.China is domestic few to this virus research, only has the report that detects this virus of discovery on Guangdong and Yunnan Cong Yinzi India, Japan, Indonesian sugarcane germplasm.But in recent years, along with sugar industry development and international co-operation are strengthened, cane seedling and breeding material worldwide extensively exchange, introduce a fine variety frequently in domestic sugarcane interval, when promoting sugar cane breed improvement and germplasm innovation, also increased the risk of the dangerous disease diffusion of picture sugarcane stripe mosaic virus type.If it is not in place that the sugarcane germplasm of introducing a fine variety is not quarantined or quarantined, very likely cause this virus at China's sugarcane district internal diffusion, cause potential threat to the Sustainable development of China's sugar industry.Therefore, strengthen the research of sugarcane stripe mosaic virus detection techniques, set up generation and the expanding of quick, accurate and sensitive detection method for this virus of prevention, the safety in production of protection sugarcane, is extremely important.So far, few to the detection method research of SCSMV, there is not yet and be specifically designed to the molecular detection kit that detects SCSMV.

Summary of the invention:

The object of the present invention is to provide a kind of sugarcane stripe mosaic virus detection kit and detection method thereof, can carry out diagnostic detection fast and accurately to stripe mosaic virus on domestic and international Introduced Sugarcane Varieties material and field sugarcane.

Technical solution of the present invention is as follows:

(1) test kit detecting for sugarcane stripe mosaic virus, is characterized in that, described test kit comprises: 1) upstream primer: concentration is 20 μ mol/L, and the base sequence of described upstream primer is as shown in SEQ ID NO:1 in sequence table;

2) downstream primer: concentration is 20 μ mol/L, and the base sequence of described downstream primer is as shown in SEQ ID NO:2 in sequence table;

3)RT buffer：5×；

4) RNA enzyme inhibitors: 40U/ μ L;

5) ThermoScript II: 200U/ μ L;

6）dNTPs：2.5μmol/L；

7）Taq PCR mix：2×；

8）RNase-free ddH ₂O

(2) the above-mentioned detection method for sugarcane stripe mosaic virus detection kit.It is characterized in that, comprise the following steps:

1) reverse transcription reaction: by add in proper order RNase-free ddH in PCR pipe ₂o3.5 μ L, 5 × RT buffer2 μ L, concentration are dNTPs1 μ L, RNA enzyme inhibitors 0.5 μ L, ThermoScript II 0.5 μ L, the total RNA2 μ of the testing sample L of downstream primer 0.5 μ L, the 2.5 μ mol/L of 20 μ mol/L, then put PCR instrument into, 25 ℃ of 10min, 42 ℃ of 60min, 98 ℃ of 5min, be cooled to 16 ℃, synthetic cDNA;

2) PCR reaction: by add in proper order RNase-free ddH in PCR pipe ₂o9.5 μ L, 2 × Taq PCR mix12.5 μ L, concentration are the each 0.5 μ L of upstream and downstream primer, the synthetic cDNA2 μ L of step 1 of 20 μ mol/L, and making to react cumulative volume is 25 μ L; Mixed solution is put PCR instrument into, 94 ℃ of denaturation 5min, then 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ extend 60s, 10min is extended in latter 72 ℃ of 35 circulations again, reaction finishes;

3) after pcr amplification product electrophoresis detection: PCR reaction finishes, getting PCR product 10 μ L 1.5 agarose gel electrophoresis detects, after ethidium bromide staining, at gel imaging system, observe and record experimental result, the sample electrophoresis detected result that contains sugarcane stripe mosaic virus is at 938bp place, to occur bright DNA band.

The present invention is according to the coat protein CP gene order design Auele Specific Primer of sugarcane stripe mosaic virus, through a large amount of screening experiments, optimal upstream primer and downstream primer have been determined, by optimization, optimum response system and response procedures have been determined again, detection sugarcane stripe mosaic virus that can be quick, accurate, sensitive.Sugarcane stripe mosaic virus detection kit provided by the present invention and detection method beneficial effect thereof are: 1) high specificity, highly sensitive, the virus-specific that the sugarcane stripe mosaic virus that infects sugarcane and other can be caused to mosaic of sugarcane makes a distinction; 2) simple, the Applied economy of detection kit making method, can be used for the rapid detection of a large amount of sugarcane samples, has good actual application value.

In sequence table shown in SEQ ID NO:1 is the base sequence of upstream primer.

In sequence table shown in SEQ ID NO:2 is the base sequence of downstream primer.

Accompanying drawing explanation:

Fig. 1 is the sugarcane stripe mosaic virus detected result of embodiment 2.Wherein 1: positive control; 2: negative control; 3 and 4: the sample that carries sugarcane stripe mosaic virus.

Fig. 2 is the specificity the result of embodiment 3.Wherein 1: sugarcane stripe mosaic viral sample; 2: corn mosaic virus sample; 3: sorghum mosaic virus sample; 4: health cane Plant samples

Fig. 3 is the detected result of the land for growing field crops sample of embodiment 4.Wherein 1-6: the mosaic disease sample of Yuanjiang County of Yunnan sugarcane field random acquisition; 7: positive control; 8: negative control; 9: field gather without disease sample.

Embodiment

Examples of implementation are used for further illustrating of the present invention below, but are not used for limiting the scope of the invention.

Embodiment 1: the preparation (20 detection limits) of sugarcane stripe mosaic virus detection kit

1) upstream primer: concentration is 20 μ mol/L, 1 pipe (30 μ L), upstream primer base sequence as shown in SEQ ID NO:1 in sequence table;

2) downstream primer: concentration is 20 μ mol/L, 1 pipe (30 μ L), base sequence as shown in SEQ ID NO:2 in sequence table;

3) RT buffer:5 ×, 1 pipe (50 μ L);

4) RNA enzyme inhibitors: 40U/ μ L, 1 pipe (15 μ L);

5) ThermoScript II: 200U/ μ L, 1 pipe (15 μ L);

6) dNTPs:2.5 μ mol/L, 1 pipe (25 μ L);

7) Taq PCR mix:2 ×, 1 pipe (300 μ L);

8) positive control sample of sugarcane stripe mosaic virus: 1 pipe (30 μ L);

9) not containing the negative control sample of sugarcane stripe mosaic virus: 1 pipe (30 μ L);

10) RNase-free ddH ₂o:1 manages (1mL);

Embodiment 2: the detection method of sugarcane stripe mosaic virus detection kit

The detection method of above-mentioned sugarcane stripe mosaic virus detection kit, comprises the following steps:

1) reverse transcription reaction: by add in proper order RNase-free ddH in PCR pipe ₂o3.5 μ L, 5 × RT buffer2 μ L, concentration are dNTPs1 μ L, RNA enzyme inhibitors 0.5 μ L, ThermoScript II 0.5 μ L, the total RNA2 μ of the testing sample L of downstream primer 0.5 μ L, the 2.5 μ mol/L of 20 μ mol/L, after mixing, put PCR instrument into, 25 ℃ of 10min, 42 ℃ of 60min, 98 ℃ of 5min, be cooled to 16 ℃, synthetic cDNA;

2) PCR reaction: by add in proper order RNase-free ddH in PCR pipe ₂o9.5 μ L, 2 × Taq PCR mix12.5 μ L, concentration are the each 0.5 μ L of upstream primer, downstream primer, the synthetic cDNA2 μ L of step 1 of 20 μ mol/L, and making to react cumulative volume is 25 μ L; After mixing, put PCR instrument into, 94 ℃ of denaturation 5min, then 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ extend 60s, 10min is extended in latter 72 ℃ of 35 circulations again, reaction finishes;

3) pcr amplification product detects: after PCR reaction finishes, getting PCR product 10 μ L 1.5 agarose gel electrophoresis detects, after ethidium bromide staining, at gel imaging system, observe and record experimental result, the sample electrophoresis detected result that contains sugarcane stripe mosaic virus is at 938bp place, to occur bright DNA band, otherwise without (Fig. 1).

Embodiment 3: the specificity checking of sugarcane stripe mosaic virus detection kit

1) extraction of the total RNA of sugarcane sample: respectively to carry sugarcane stripe mosaic virus (SCSMV), corn mosaic virus (Sugarcane mosaic virus, SCMV), sorghum mosaic virus (Sorghum mosaic virus, SrMV) and the sugarcane sample of healthy plant be material, each material is got respectively fresh blade 0.1g+ liquid nitrogen grinding powdering, joins in 2.0mL centrifuge tube; Add 1000 μ L TRIZOL reagent, mix gently, room temperature is placed 5min; Enter 200 μ L chloroforms, concuss 15 seconds, room temperature is placed 5min, 4 ℃ of centrifugal 15min of 12000rpm; Get supernatant liquor in 1.5mL centrifuge tube, add 600 μ L Virahols, after mixing (gently mixed), room temperature is placed 10min, 4 ℃ of centrifugal 10min of 12000rpm, precipitated rna; Abandon supernatant, add 1000 μ L75% washing with alcohol RNA precipitation 1 time, the RNA that fully suspends during washing precipitation (with finger flick the pipe end allow precipitate levitating), the centrifugal 5min of 8000rpm; Abandon after supernatant, be deposited in seasoning 6min in air, with 30 μ L ddH ₂o dissolves RNA precipitation, and in 55 ℃ of dissolvings (in water-bath) 10min ,-20 ℃ of Refrigerator stores are standby.

2) responsive transcription: by add in proper order RNase-free ddH in PCR pipe ₂o3.5 μ L, 5 × RT buffer2 μ L, concentration are dNTPs1 μ L, RNA enzyme inhibitors 0.5 μ L, ThermoScript II 0.5 μ L, the total RNA2 μ of the testing sample L of downstream primer 0.5 μ L, the 2.5 μ mol/L of 20 μ mol/L, after mixing, put PCR instrument into, 25 ℃ of 10min, 42 ℃ of 60min, 98 ℃ of 5min, be cooled to 16 ℃, synthetic cDNA;

3) PCR reaction: by add in proper order RNase-free ddH in PCR pipe ₂o9.5 μ L, 2 × Taq PCR mix12.5 μ L, concentration are the each 0.5 μ L of upstream and downstream primer, the synthetic cDNA2 μ L of step 1 of 20 μ mol/L, and making to react cumulative volume is 25 μ L; After mixing, put PCR instrument into, 94 ℃ of denaturation 5min, then 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ extend 60s, 10min is extended in latter 72 ℃ of 35 circulations again, reaction finishes;

4) pcr amplification product detects: after PCR reaction finishes, getting PCR product 10 μ L 1.5 agarose gel electrophoresis detects, after ethidium bromide staining, at gel imaging system, observe and record experimental result (Fig. 2), as seen from Figure 2, there is bright DNA band in sugarcane stripe mosaic viral sample only at 938bp place, other viral sample and the equal nothing of health cane sample, illustrate that test kit of the present invention has good specificity.

The detection of embodiment 4 Yuanjiang County of Yunnan land for growing field crops sample SCSMV

From 6 parts of mosaic of sugarcane band disease samples of Yuanjiang County of Yunnan random acquisition and 1 part, without disease sample, detect after adopting the method for embodiment 3 to extract total RNA, 6 parts of band disease samples all detect SCSMV as a result, without disease sample, do not detect SCSMV.

Sequence table

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

<120> sugarcane stripe mosaic virus detection kit and detection method thereof

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<170> PatentIn version 3.3

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<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> upstream primer

<400> 1

acaaggaacg cagccacct 19

<210> 2

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> downstream primer

<400> 2

actaagcggt caggcaac 18

Claims (2)

1. a sugarcane stripe mosaic virus detection kit, it is characterized in that, described test kit comprises: 1) upstream primer: 20 μ mol/L, the base sequence of described upstream primer is as shown in SEQ ID NO:1 in sequence table, 2) downstream primer: 20 μ mol/L, the base sequence of described downstream primer as shown in SEQ ID NO:2 in sequence table, 3) RT Buffer:5 ×; 4) RNA enzyme inhibitors: 40U/ μ L; 5) ThermoScript II: 200U/ μ L; 6) dNTPs:2.5 μ mol/L; 7) Taq PCR mix:2 ×; 8) RNase-free ddH ₂o.

2. the detection method of sugarcane stripe mosaic virus detection kit claimed in claim 1, is characterized in that, comprises the following steps:

1) reverse transcription reaction: by add in proper order RNase-free ddH in PCR pipe ₂o3.5 μ L, 5 × RT buffer2 μ L, concentration are dNTPs1 μ L, RNA enzyme inhibitors 0.5 μ L, ThermoScript II 0.5 μ L, the total RNA2 μ of the testing sample L of downstream primer 0.5 μ L, the 2.5 μ mol/L of 20 μ mol/L, after mixing, put PCR instrument into, 25 ℃ of 10min, 42 ℃ of 60min, 98 ℃ of 5min, be cooled to 16 ℃, synthetic cDNA;

2) PCR reaction: by add in proper order RNase-free ddH in PCR pipe ₂o9.5 μ L, 2 × Taq PCR mix12.5 μ L, concentration are the each 0.5 μ L of upstream primer, downstream primer, the synthetic cDNA2 μ L of step 1 of 20 μ mol/L, and making to react cumulative volume is 25 μ L; After mixing, put PCR instrument into, 94 ℃ of denaturation 5min, then 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ extend 60s, 10min is extended in latter 72 ℃ of 35 circulations again, reaction finishes;

3) after pcr amplification product electrophoresis detection: PCR reaction finishes, getting PCR product 10 μ L 1.5 agarose gel electrophoresis detects, after ethidium bromide staining, at gel imaging system, observe and record experimental result, the sample electrophoresis detected result that contains sugarcane stripe mosaic virus is at 938bp place, to occur bright DNA band.

Images