CN102660653A

CN102660653A - PCR (polymerase chain reaction) primer and method for detecting southern rice black-streaked dwarf virus in bodies of plant hoppers

Info

Publication number: CN102660653A
Application number: CN2012101699955A
Authority: CN
Inventors: 周倩; 高必达; 朱俊子
Original assignee: Hunan Agricultural University
Current assignee: Hunan Agricultural University
Priority date: 2012-05-29
Filing date: 2012-05-29
Publication date: 2012-09-12
Anticipated expiration: 2032-05-29
Also published as: CN102660653B

Abstract

The invention discloses a primer and a method for detecting southern rice black-streaked dwarf virus in bodies of plant hoppers. By the aid of the primer and the method, the southern rice black-streaked dwarf virus in bodies of the plant hoppers can be detected accurately and quickly and can be distinguished from common rice black-streaked dwarf virus accurately, and particularly, whether the southern rice black-streaked dwarf virus is carried in bodies of Sogatella furcifera or not can be determined accurately and quickly. Accordingly, the primer and the method can be applied to disease diagnosis and forecast.

Description

Detect the PCR primer and the method thereof of southern rice black-streaked dwarf virus in the plant hopper body

Technical field

The invention belongs to the agricultural cience and farming techniques field, relate to a kind of PCR primer and method thereof that detects southern rice black-streaked dwarf virus in the plant hopper body.

Background technology

The southern rice black-streaked dwarf disease is newfound a kind of paddy rice virus disease in rice district, China south in recent years, and the whole nation took place 3,000,000 mu in 2009, rose to 1,900 ten thousand mu rapidly in 2010 by south China agricultural university reported first in 2008, produced to paddy rice and brought massive losses.Present result of study show white backed planthopper ( Sogatella furcifera) be the main vector in field of this virus disease, so the population quantity of white backed planthopper and to be with malicious rate be the pathogenetic important indicator of prediction southern rice black-streaked dwarf.

The difficult point that single worm band poison detects is that the volume of a plant hopper is very little, and the content of virus is considerably less, extracts virus in the single head insect body to be used further to detect very difficulty.Detect at present that (southern rice black streaked dwarf virus, detection technique SRBSDV) mainly is divided into two kinds of serological technique and molecular detection technologies about southern rice black-streaked dwarf virus in the plant hopper body.Research units such as Zhejiang University river utilize monoclonal antibody technique to carry out the detection of southern black-streaked dwarf virus; But this technology can not distinguish the very close with it ordinary rice black streaked dwarf virus (RBSDV) of sibship (rice black streaked dwarf virus, RBSDV).South China agricultural university has reported the method with a step dual RT-PCR detection technique southern rice black-streaked dwarf virus; This method utilizes 2 pairs of gene-specific primers to carry out reverse transcription and amplification; But 2 pairs of primers have not only increased the detection cost, and can't accurately determine the end when an amplified band occurring and whether be with poison.Therefore be necessary to set up a kind of can be accurately in the single head white backed planthopper body technology of detection southern rice black-streaked dwarf virus.

Summary of the invention

Technical problem to be solved by this invention provides a kind of PCR primer and method thereof that detects southern rice black-streaked dwarf virus in the plant hopper body; Utilize primer according to the invention and method can be accurately, rapid detection goes out the intravital southern rice black-streaked dwarf virus of plant hopper; Southern rice black-streaked dwarf virus and ordinary rice black streaked dwarf virus (RBSDV) are made a distinction accurately, particularly can in single head white backed planthopper body, detect southern rice black-streaked dwarf virus exactly.

Technical scheme provided by the invention is: a kind of primer that detects southern rice black-streaked dwarf virus in the plant hopper body, and its forward primer nucleotide sequence is shown in SEQ ID No.1, and the reverse primer nucleotide sequence is shown in SEQ ID No.2.

Simultaneously, the present invention also provides the method for southern rice black-streaked dwarf virus in a kind of detection plant hopper body, and this method is: extracting the total RNA of testing sample is template, and reverse transcription obtains cDNA; With described cDNA is template, utilizes described primer to carry out pcr amplification, gets amplified production then and detects, if the amplified production size is about 338bp, then has southern rice black-streaked dwarf virus in the testing sample.

In the above-mentioned method, said total RNA obtains to adopt random primer in the cDNA process for the template reverse transcription, and described random primer is the random primer of 6 bases, and for example giving birth to worker's biotechnology Shanghai ltd PIN is the product of B0043.

The present invention has following beneficial effect:

Compared with prior art; Advantage of the present invention is: use detection method of the present invention; Only just can southern rice black-streaked dwarf virus and ordinary rice black streaked dwarf virus (RBSDV) be made a distinction accurately with a pair of primer of the present invention; Particularly can detect the intravital southern rice black-streaked dwarf virus of single head plant hopper, and detected result is accurate, highly sensitive.The inventive method uses random primer to carry out reverse transcription, and the abundance of transcription product is big, makes band brighter clearer.Primer of the present invention and method can be applicable on disease screening and the prediction, utilize the inventive method can get rid of the interference of black streaked dwarf virus of rice, and Rapid identification goes out in the single head plant hopper body whether carry southern rice black-streaked dwarf virus.

Description of drawings

Fig. 1 is the agarose gel electrophoresis figure of primer amplification of the present invention, and article one swimming band is Marker among the figure from left to right, and second swimming lane is the SRBSDV positive control, and the 3rd swimming lane is RBSDV, all the other single worms for detecting.

Embodiment

Come further to illustrate the present invention through the detailed description of embodiment below, but be not limitation of the present invention, only make example description.

One, primer design:

The conservative property that the inventor utilizes viral nucleotide sequences to evolve; From NCBI search and download southern rice black-streaked dwarf virus S5 sequence; Utilization DNAssist software is compared to these sequences; The southern rice black-streaked dwarf virus nucleotide sequence of finding out different investigators report is common and primer candidate regions that other virus sequence is all different is with it designed a pair of Auele Specific Primer, and primer sequence is following

Forward primer: 5 '-TACGCAATACCTCATACCTG-3 ' (SEQ ID No.1),

Reverse primer: 5 '-AAGACTTGTTTCTGCTGGTT-3 ' (SEQ ID No.2).

Above-mentioned primer is synthetic by precious biotechnology (Dalian) ltd, and the PCR product of expection is 338bp.

Two, the RT-PCR process of virus:

(1) extract total RNA, method is following:

1. get the single head insect in centrifuge tube, liquid nitrogen grinding;

2. the Trizol reagent that adds 100 μ L;

3. add 500mL chloroform-primary isoamyl alcohol (24:1) concuss and shake up 15s;

4. ice bath 10min, the centrifugal 15min of 12000r/min gets supernatant in another new centrifuge tube;

5. add the slight mixing of isopyknic chloroform-primary isoamyl alcohol (24:1), get supernatant in another new centrifuge tube;

6. add the Virahol of 100 μ L, put upside down mixing;

7. room temperature is placed 10min, and the centrifugal 10min of 12000r/min abandons supernatant;

8. add 80% washing with alcohol deposition, the centrifugal 5min of 7500r/min abandons supernatant;

9. add the absolute ethanol washing deposition, the centrifugal 5min of 7500r/min abandons supernatant;

10. after room temperature settle and the thorough drying; Be dissolved in the 10 μ LDEPC water.

(2) with above-mentioned designed primer the total RNA that extracts is carried out the RT-PCR amplification:

In 0.2 mL PCR reaction tubes, add following reagent and carry out reverse transcription: template ribonucleic acid 3 μ L; 5 times of first synthetic damping fluid 4 μ L of chain, dNTPs (10mM) 2 μ L, (give birth to worker's biotechnology Shanghai ltd PIN is the product of B0043 to random primer; For example: (25 μ M) 0.8 μ L batch Lot ID:11-25); The sterilized water 9.2 μ L of no RNA enzyme, 65 ℃ of preparatory sex change 5 min place rapidly on ice behind the mixing; Add M-MLV ThermoScript II (200U/ μ L) 1 μ L then, 42 ℃ of water-bath 1 h, 4 ℃ of preservations are subsequent use.Pcr amplification reaction system 25 μ L, wherein, template cDNA 2 μ L; 10 * PCR Buffer, 2.5 μ L; DNTPs (10mM) 0.5 μ L, F end primer (SEQ ID No.1) (10 μ M) 1 μ L, R end primer (SEQ ID No.2) (10 μ M) 1 μ L; Sterilization deionized water 17.5 μ L, TaqDNA polysaccharase (2.5U/ μ L) 0.5 μ L.The reaction conditions of PCR is: 95 ℃ of preparatory sex change 5min, and 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 45s, 35 circulations, 72 ℃ are extended 10min.Adopt 1% agarose gel electrophoresis to detect amplification,, then have southern rice black-streaked dwarf virus in the testing sample if the amplified production size is about 338bp.

The white backed planthopper of field in 2011, Hunan Province being gathered with aforesaid method detects, and partial results sees also Fig. 1, amplifies the band of 338bp, consistent with expection.Explained that the present invention can accurately distinguish southern black-streaked dwarf virus and common black-streaked dwarf virus, and test strip is clear.

< 110>Agricultural University Of Hunan

< 120>the PCR primer and the method thereof of southern rice black-streaked dwarf virus in the detection plant hopper body

<160>?2

<210>?1

<211>?20

<212>?DNA

< 213>artificial sequence

<220>

< 223>description of artificial sequence: the sequence of synthetic

<400>?1

TACGCAATAC?CTCATACCTG 20

<210>?2

<211>?20

<212>?DNA

< 213>artificial sequence

<220>

< 223>description of artificial sequence: the sequence of synthetic

<400>?2

AAGACTTGTT?TCTGCTGGTT 20

Claims

1. primer that detects southern rice black-streaked dwarf virus in the plant hopper body, it is characterized in that: its forward primer nucleotide sequence is shown in SEQ ID No.1, and the reverse primer nucleotide sequence is shown in SEQ ID No.2.

2. method that detects southern rice black-streaked dwarf virus in the plant hopper body, it is characterized in that: extracting the total RNA of testing sample is template, reverse transcription obtains cDNA; With described cDNA is that template utilizes the described primer of claim 1 to carry out pcr amplification, gets amplified production then and detects, if the amplified production size is 338bp, then has southern rice black-streaked dwarf virus in the testing sample.

3. method as claimed in claim 2 is characterized in that: said total RNA obtains to adopt random primer in the cDNA process for the template reverse transcription.