CN101215611A

CN101215611A - Primer for detecting southern cowpea mosaic virus

Info

Publication number: CN101215611A
Application number: CNA2008100561138A
Authority: CN
Inventors: 陈红运; 林石明; 赵文军; 陈青; 朱水芳; 陈洪俊
Original assignee: Prof China Xiamen Entry-Exit Inspection And Quarantine Bureau; Chinese Academy of Inspection and Quarantine CAIQ
Current assignee: Prof China Xiamen Entry-Exit Inspection And Quarantine Bureau; Chinese Academy of Inspection and Quarantine CAIQ
Priority date: 2008-01-11
Filing date: 2008-01-11
Publication date: 2008-07-09
Anticipated expiration: 2028-01-11
Also published as: CN101215611B

Abstract

The invention provides a primer for detecting southern cowpea mosaic virus, and the ribonucleotide sequence of the primer is showed by sequence table SEQ ID No.1&2. The invention provides a process for detecting southern cowpea mosaic virus, which using sample general RNA as mold, proceeding RT-PCR augmentation by utilizing specific primers, and judging results according to the position of specific augmentation DNA section after the reaction being finished. The invention has excellent primer specificity, fast and simple detection process and high sensibility, which provides guarantees for import and export safety.

Description

Primer for detecting southern cowpea mosaic virus

Technical field

The present invention relates to Measurement for Biotechnique, relate in particular to primer for detecting southern cowpea mosaic virus.

Background technology

(Southern cowpea mosaic virus SCPMV) is one of important virus of Sobemovirus (Sobemovirus) to southern cowpea mosaic virus, and main natural host is a leguminous plants.SCPMV is highly stable, and fatal temperature 90-95 ℃, dilution end point 10 ^-5-10 ^-6, longevity in vitro is the longest to reach 165 days, and should virus also can be by seed dispersal.In September, 2007, Xiamen Entry-Exit Inspection and Quarantine Bureau intercepts and captures this virus first from the cowpea seed of import, and this also is that China detects this virus first from the seed that enters the territory.Because domestic scholars is less to the research of this virus, related data and experience of prevention and treatment lack.

The SCPMV antigenicity is stronger, develops the specific corrosioning anteserum of high titre easily, generally can reach 1/2048 to 1/4096.(Southern beanmosaic virus SBMV) has very close serological relation, therefore is difficult to by the serology detection SCPMV and SBMV accurately be identified for SCPMV and generic bean mosaic virus 4.(polymerase chain reaction PCR) provides solution for realizing this goal to be widely used in the polymerase chain reaction technology in Molecular Detection field in recent years.

The present invention selects southern cowpea mosaic virus coat protein gene and 3 ' end non-translational region sequence, be designed for the Auele Specific Primer that RT-PCR detects, set up the molecular detecting method of southern cowpea mosaic virus by reverse transcription and pcr amplification, reaction finishes the location determination result of back according to the specific amplification dna fragmentation.

Summary of the invention

The object of the invention is to be provided for the primer sequence that southern cowpea mosaic virus RT-PCR detects.

The present invention is by analyzing SCPMV coat protein gene and 3 ' the end non-translational region sequence of having reported, design SCPMV Auele Specific Primer.Described primer is to being made of its nucleotide sequence such as sequence table SEQ ID No.1﹠amp forward and reverse primer; Shown in 2.

The present invention has also further provided the RT-PCR detection method of using above-mentioned primer, and it is a template with the total RNA of sample, carries out the RT-PCR amplification, and reaction finishes the 979bp dna fragmentation result of determination of back according to specific amplification.

Specifically the present invention is a template with the total RNA of sample, carries out the RT-PCR reaction.The RT-PCR reaction comprises reverse transcription and two processes of pcr amplification.

At first carry out reverse transcription reaction, promptly in the reaction tubes of 0.2mL, add 6.75 μ L DEPC treating water, the total RNA of 4 μ L in 20 μ L reaction systems, 1 μ L SCPMV-RP (10 μ mol/L), 1 μ L dNTP (10mmol/L) in 65 ℃ of maintenance 5min, transfers to quenching 5min on ice rapidly behind the mixing; In reaction tubes, add 2 μ L DTT (0.1mmol/L) then, 4 μ L, 5 * reverse transcription damping fluid, 1 μ L RNA enzyme inhibitors (40 U/ μ L), 0.25 μ L ThermoScript II (200U/ μ L), 42 ℃ of reaction 50min; Obtain reverse transcription product cDNA after 72 ℃ of 15min deactivation ThermoScript II.

The PCR reaction system is 50 μ L, promptly in the PCR of 0.2mL reaction tubes, add 30.5 μ L DEPC treating water, 2 μ L cDNA, 2 μ L SCPMV-FP (10 μ mol/L), 2 μ LSCPMV-RP (10 μ mol/L), 10 * PCR damping fluid, 5 μ L, 8 μ L dNTP (2.5mmol/L), 0.5 μ L archaeal dna polymerase (5U/ μ L).Reaction conditions is: 94 ℃, and 5min; 94 ℃ of 30s, 52 ℃ of 45s, 72 ℃ of 1min, totally 30 circulations; 72 ℃ are extended 5min.

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

The present invention is according to southern cowpea mosaic virus coat protein and 3 ' end decomplier region sequence design primer, and this primer specificity is good, and the RT-PCR that is used for SCPMV detects.Detection method of the present invention has high specific and sensitivity, its rapidly and accurately judgement sample whether SCPMV is arranged, for imports and exports safety provides assurance.

Description of drawings

Fig. 1 uses the result that RT-PCR detects southern cowpea mosaic virus, and wherein, 1 is 100bp ladder, and 2 is the SCPMV positive control, and 3,4 for SCPMV infects disease plant, and 5 is healthy plant, and 6 is the SBMV positive control, and 7 is the SBMV positive control; 1～6 is the amplification of SCPMV specificity RT-PCR product; 7 is the SBMV primer amplified.

Fig. 2 is the sensitivity test result of detection method of the present invention, and wherein, 1 is 100bpladder, and 2～7 are the morbidity sample, and total RNA is respectively 5 μ L, 4 μ L, 3 μ L, 2 μ L, 1 μ L, 0.5 μ L in the 50 μ L reaction systems.

Embodiment

Following embodiment is used for further specifying of the present invention, but is not used for limiting the scope of the invention.

Embodiment 1 design of primers and synthetic

According to the open sequence in SCPMV coat protein and 3 ' end decomplier district (GenBank No.M23021), adopt primer-design software Primer 5.0 design primers, primer sequence is:

SCPMV-FP (forward): 5 '-atg tcc ggt cta ttc cat c-3 '

SCPMV-RP (oppositely): 5 '-cea ttc gga tag cgc tc-3 '

The extraction of embodiment 2 total RNA

1) get the 0.2g incidence of leaf, be cut into segment, powdered with liquid nitrogen grinding, move in the 1.5ml centrifuge tube of sterilization, add the Trizol reagent of 1ml then, concuss shakes up;

2) 4 ℃, the centrifugal 10min of 12000g to be removing insoluble composition, and supernatant liquor is changed in the new 1.5ml centrifuge tube;

3) keep 5min under the room temperature, add the 0.2ml chloroform, thermal agitation 15s, at room temperature keep 2-15min then after, 4 ℃, the centrifugal 15min of 12000g;

4) with the upper water phase transition in new 1.5ml centrifuge tube, add the 0.5ml Virahol, put upside down mixing, keep 15min under the room temperature;

5) 4 ℃, the centrifugal 10min of 12000g, RNA will form precipitation in the sidewall and the bottom of pipe;

6) outwell supernatant liquor, add 75% washing with alcohol precipitation, 4 ℃ then, the centrifugal 5min of 7500g (suspend as precipitation, then use 12000g) discards ethanol;

7) be deposited under the room temperature after the thorough drying, be dissolved in 40 μ l dH ₂Among the O (DEPC processing) ,-20 ℃ of preservations are standby.

The foundation of embodiment 3 RT-PCR amplification methods

With total RNA is template, carries out the RT-PCR reaction.20 μ L reaction systems are carried out reverse transcription reaction, promptly add 6.75 μ L DEPC treating water, the total RNA of 4 μ L in the reaction tubes of 0.2mL, 1 μ L SCPMV-RP (10 μ mol/L), 1 μ L dNTP (10mmol/L) in 65 ℃ of maintenance 5min, transfers to quenching 5min on ice rapidly behind the mixing; In reaction tubes, add 2 μ L DTT (0.1mmol/L) then, 4 μ L, 5 * reverse transcription damping fluid, 1 μ LRNA enzyme inhibitors (40U/ μ L), 0.25 μ L ThermoScript II (200U/ μ L), 42 ℃ of reaction 50min; Obtain reverse transcription product cDNA after 72 ℃ of 15min deactivation ThermoScript II.

The specificity of embodiment 4 SCPMV RT-PCR methods is determined

The total RNA of cowpea blade with reveal any symptoms is a template, with healthy leaves and SBMV positive material in contrast, increases by RT-PCR.Reaction finishes the location determination result of back according to SCPMV specific amplification dna fragmentation (979bp).

According to the open sequence in SBMV coat protein and 3 ' end decomplier district (GenBank No.DQ836714), adopt primer-design software Primer 5.0 design primers, primer sequence is:

SBMV-FP (forward): ATG GCG AAG AGG TTG AC

SBMV-RP (oppositely): ACT TTT GGA TTA CGC TCC ATC

SBMV amplification condition and SCPMV are identical, amplified fragments 930bp.

Test-results:

The total RNA of cowpea blade with reveal any symptoms is a template, detects the specific amplification fragment that can be observed 979bp by RT-PCR, and normal healthy controls and SBMV positive material then do not have the appearance of specific amplification band.Illustrate that the RT-PCR that sets up has good specificity to SCPMV, can make a distinction SCPMV and its generic SBMV.The results are shown in Figure 1.

The sensitivity of embodiment 5 SCPMV RT-PCR methods is determined

Use the Trizol reagent of 1ml from the 0.2g incidence of leaf, to extract total RNA, dissolve total RNA, carry out relative sensitivity and detect with 40 μ LDEPC treating water.In 50 μ L reaction systems, total RNA is respectively 5 μ L, 4 μ L, 3 μ L, 2 μ L, 1 μ L, 0.5 μ L.Detected result as shown in Figure 2.The RT-PCR method that shows foundation can successfully detect SCPMV from the total RNA of 0.5 μ L.Has higher sensitivity, the requirement of coincidence detection.

Sequence table

＜110〉Xiamen Entry-Exit Inspection and Quarantine Bureau, China Inst. of Quarantine Inspection Sciences

＜120〉primer for detecting southern cowpea mosaic virus

<130>

<160>2

<170>PatentIn?version?3.3

<210>1

<211>19

<212>DNA

＜213〉artificial sequence

<400>1

atgtccggtc?tattccatc 19

<210>2

<211>17

<212>DNA

＜213〉artificial sequence

<400>2

ccattcggat?agcgctc 17

Claims

1. primer for detecting southern cowpea mosaic virus, its nucleotides sequence is classified as:

SCPMV-FP：5’-atg?tcc?ggt?cta?ttc?cat?c-3’

SCPMV-RP：5’-cca?ttc?gga?tag?cgc?tc-3’

2. method that detects southern cowpea mosaic virus, this method is a template with the total RNA of sample, utilizes the described primer of claim 1 to carry out the RT-PCR amplification, reaction finishes the location determination result of back according to the specific amplification dna fragmentation.

3. method as claimed in claim 2 is characterized in that, the temperature of reverse transcription reaction is 42 ℃ in the reaction process of RT-PCR, and the annealing temperature of pcr amplification is 52 ℃, and elongating temperature is 72 ℃.

4. the test kit that contains the described primer for detecting southern cowpea mosaic virus of claim 1.

5. the application of the described primer of claim 1 in southern cowpea mosaic virus detects.