CN104962662B - The apple rust fruit viral RT PCR detection techniques on basis are designated as within a kind of - Google Patents
The apple rust fruit viral RT PCR detection techniques on basis are designated as within a kind of Download PDFInfo
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- CN104962662B CN104962662B CN201510449388.8A CN201510449388A CN104962662B CN 104962662 B CN104962662 B CN 104962662B CN 201510449388 A CN201510449388 A CN 201510449388A CN 104962662 B CN104962662 B CN 104962662B
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Abstract
Become rusty fruit viral RT PCR detection techniques the present invention relates to the apple that basis is designated as within a kind of.Using with the cm cortical tissues of leaf bud apple annotinous branch top 0 ~ 5 as examination material, using RNA extraction improved method extraction total tissue RNAs, using total serum IgE as template, apple rust fruit virus specific primers and apple endogenous gene primer carry out RT PCR reactions, judge whether apple tissue carries apple rust fruit virus according to the presence or absence of amplified production in agarose gel electrophoresis and size.Criterion is：Endogenous gene specific fragment is not amplified represents the failure of an experiment；Amplify endogenous gene specific fragment and represent experiment effectively；Amplify endogenous gene specific fragment and apple rust fruit virus-specific fragment represents in apple tissue and takes apple band rust fruit virus；Endogenous gene specific fragment is amplified, apple rust fruit virus-specific fragment is not amplified and represents that not carry apple rust fruit in tissue viral.
Description
Technical field
Become rusty fruit virus RT-PCR detection technique the present invention relates to the apple that basis is designated as within a kind of.
Background technology
Apple rough skin disease also known as paint face's disease or cracked fruit, it is to endanger apple one of heavier non-latent virus, and
The important quarantine object of Chinese apple virus, is distributed widely in northeast, northwest, each apple producing region in North China, serious orchard of falling ill
Diseased plant rate is up to 30 more than %, there is the trend to expand；Under current technical merit, effectively preventing medicament is there is no, using de-
Malicious method breeds virus-free nursery stock and is presently the most effectively preventing means, accurate, sensitive, quick detection technique be fruit tree without
Poison the key link and basic guarantee of cultivation；But because virus is being set low in-vivo content, skewness, influenceed by extraneous factor
Greatly, easily there is false negative phenomenon in detection process in the features such as tissue is rich in polysaccharide, polyphenol, at present, existing ASSVd both at home and abroad
PCR detection report, but there is not yet within be designated as basis ASSVd RT-PCR detection techniques report；The present invention, which develops, to be contained
Havenad 5The apple rust fruit virus RT-PCR detection technique of internal standard gene, successfully overcomes false negative phenomenon, for the virus
Accurate detection, efficiently breeding for nontoxic nursery stock are laid the foundation, and theoretical foundation is provided for effective preventing and treating of Apple virus disease from now on.
The content of the invention
The present invention establish it is a kind of within be designated as basis apple rust fruit virus RT-PCR detection technique, the present invention adopted
Technical scheme：Apple tissue total serum IgE is extracted, being become rusty using apple, fruit is viral and a kind of apple endogenous gene is primer, passes through
The specific sequence of RT-PCR method amplification apple rust fruit virus and apple endogenous gene, it is false that apple endogenous gene plays exclusion
The effect of negative control, amplified production is observed finally by gel electrophoresis, judge whether the rust fruit containing apple is viral in tissue, this
The particular content of invention, i.e., within be designated as basis apple rust fruit virus RT-PCR detection technique specific method it is as follows：
（1）The extraction of apple tissue total serum IgE with leaf bud annotinous branch top 0 ~ 5cm cortical tissues, as examination material, to adopt
Removed with for the RNA extraction improved method extraction total serum IgEs rich in polysaccharide polyphenol tissue, constant volume in 20 μ L in RNase water, -80 DEG C of guarantors
Deposit standby；
（2）The amplification of virus-specific genes sequence is with step（1）The total serum IgE of acquisition is template, carries out RT-PCR and obtains
Amplified production.It is primer with ASSVdQCxin and nad 5, M-MLV reverse transcriptase carries out cDNA synthesis, and two pairs of primers are respectively：
Apple rust fruit virus primer：ASSVdQCxin-3'：C A C C A G T T C C G C T G T G G G T T C,
ASSVdQCxin-5'：G C C T A C A A G A A C G T A C G G T G T T G A G, the 2nd is endogenous to apple
Gene primer：nad 5-3′：C T C C A G T C A C C A A C A T T G G C A T A A,nad 5-5′：G
A T G C T T C T T G G G G C T T C T T G T T；
（3）Agarose gel electrophoresis detection takes 5 μ L pcr amplification products（Apple rust fruit virus-specific clip size
For 330 bp, apple endogenous genenad 5Specific fragment size is 181 bp）, 1.5% Ago-Gel is in 1 × TAE bufferings
Carry out electrophoretic analysis in liquid, 100V electrophoresis 40 minutes, after EB dyeing, observe result, having that it's too late according to electrophoretic band, size judges
Whether band is malicious；
The beneficial effect of patent of the present invention is, by designing apple rust fruit virus specific primers and can exclude false the moon
Property phenomenon apple endogenous gene primer, carry out the optimization of detection architecture and program, establish it is a kind of using internal standard based on
Apple rust fruit virus RT-PCR detection technique system, avoids the false negative result of Standard PCR, detection stability is good, specifically
Property it is strong, available for field sample and the quick detection of tissue-cultured seedling band poison situation, offer technology branch is bred for the nontoxic nursery stock of apple
Hold, laid the foundation for apple rust fruit virus disease prevention and control, reduce loss caused by disease.
Brief description of the drawings
Fig. 1 is the RT-PCR testing results that field fruit tree carries apple rust fruit virus situation.
Embodiment
, as examination material, to take 100 mg to organize, in liquid nitrogen with the cm cortical tissues of leaf bud apple annotinous branch top 0 ~ 5
In grind rapidly, add 0.5 mL extracts reagents（The μ L beta -mercaptoethanols of 400 μ L RNA extracts reagents+100）Extracted, finally
Add 20 μ L DEPC water and fully dissolve RNA.Then it is primer with specific primer ASSVdQCxin and nad 5, M-MLV is inverted
Record enzyme and carry out cDNA synthesis, using the cDNA of synthesis as template, prepare following PCR reaction systems：The μ L of cDNA templates 3.0,
ASSVdQCxin-3' （20 pmol/µL）1 μ L, ASSVdQCxin-5'（20 pmol/µL）1 μ L,nad 5-3' （20
pmol/µL）0.1 μ L,nad 5-5'（20 pmol/µL）The μ L of 0.1 μ L, 2 × ES Taq Mastermix 12.5, use
ddH2O supplies 25 μ L；PCR programs are：94 DEG C of 3 min of pre-degeneration；35 circulations：94 DEG C of 1 min of denaturation, 61.0 DEG C of annealing
45 s, 72 DEG C of 30 s of extension；72 DEG C extend 10 min eventually；5 μ L pcr amplification products are taken, 1.5% Ago-Gel is in 1 × TAE
Carry out electrophoretic analysis in buffer solution, 100V electrophoresis 40 minutes, after EB dyeing, observe result, according to the electrophoretic band size that has that it's too late
Judge whether band poison（ASSVd specific fragments size is 330 bp,nad 5Specific fragment size is 181 bp）.
Claims (1)
1. the apple rust fruit virus RT-PCR detection technique on basis is designated as within a kind of, it is characterised in that by extracting apple group
Total serum IgE is knitted, being become rusty using apple, fruit is viral and a kind of specific primer of apple endogenous gene, passes through RT-PCR method amplification rust
Whether fruit virus and apple endogenous gene specific sequence, size that gel electrophoresis observation amplified production has that it's too late, judge tissue
Carry apple rust fruit virus；
Comprise the following steps：
(1) extraction of apple tissue total serum IgE：Selection carry leaf bud annotinous branch top 0~5cm cortical tissues, using for
RNA extraction improved method extraction total serum IgEs rich in polysaccharide polyphenol tissue, constant volume are removed in RNase water in 20 μ L, and -80 DEG C save backup；
(2) amplification of virus-specific genes sequence：The total serum IgE obtained using step (1) uses M-MLV reverse transcriptase as template
CDNA is synthesized, uses apple rust fruit virus primer ASSVdQCxin-5' and ASSVdQCxin-3', apple endogenous gene primer
Nad 5-5 ' and nad 5-3 ' are expanded,
ASSVdQCxin-3'：C A C C A G T T C C G C T G T G G G T T C；
ASSVdQCxin-5'：G C C T A C A A G A A C G T A C G G T G T T G A G；
nad 5-3′：C T C C A G T C A C C A A C A T T G G C A T A A；
nad 5-5′：G A T G C T T C T T G G G G C T T C T T G T T；
Prepare following reaction system and enter performing PCR amplification：μ L, the 20pmol/ μ L of cDNA templates 3.0 ASSVdQCxin-3'1 μ L,
20pmol/ μ L ASSVdQCxin-5'1 μ L, 20pmol/ μ L nad 5-3'0.1 μ L, 20pmol/ μ L nad 5-5'0.1 μ
The μ L of L, 2 × ES Taq Mastermix 12.5, use ddH2O supplies 25 μ L；PCR programs are：94 DEG C of 3min of pre-degeneration；35 are followed
Ring：94 DEG C of denaturation 1min, 61.0 DEG C of annealing 45s, 72 DEG C of extension 30s；72 DEG C extend 10min eventually；
(3) agarose gel electrophoresis detection and criterion：5 μ L pcr amplification products are taken, 1.5% Ago-Gel is in 1 × TAE
Carry out electrophoretic analysis in buffer solution, 100V electrophoresis 40 minutes, after EB dyeing, observe result, according to the electrophoretic band size that has that it's too late
Judge whether band poison；Apple rust fruit virus-specific fragment is 330bp, and the specific fragments of apple endogenous gene nad 5 are
181bp；Criterion is：Endogenous gene specific fragment is not amplified represents the failure of an experiment；It is special to amplify endogenous gene
Property fragment to represent experiment effective；Amplify endogenous gene specific fragment and rust fruit virus-specific fragment represents apple
With rust fruit virus in tissue；Endogenous gene specific fragment is amplified, does not expand rust fruit virus-specific fragment i.e.
Represent in tissue without rust fruit virus.
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| CN201510449388.8A CN104962662B (en) | 2015-07-29 | 2015-07-29 | The apple rust fruit viral RT PCR detection techniques on basis are designated as within a kind of |
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| CN201510449388.8A CN104962662B (en) | 2015-07-29 | 2015-07-29 | The apple rust fruit viral RT PCR detection techniques on basis are designated as within a kind of |
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| Publication Number | Publication Date |
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| CN104962662A CN104962662A (en) | 2015-10-07 |
| CN104962662B true CN104962662B (en) | 2017-12-01 |
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| CN106957915B (en) * | 2017-04-13 | 2020-09-29 | 河北农业大学 | Primer and kit for detecting apple tree/pear tree rot pathogen and quantitative detection method |
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