CN105950781B - The complete genome sequence and its detection method of cucurbita pepo mosaic virus - Google Patents
The complete genome sequence and its detection method of cucurbita pepo mosaic virus Download PDFInfo
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Abstract
The present invention relates to the complete genome sequence of cucurbita pepo mosaic virus and its detection methods.The nucleotide sequence that the genome of the cucurbita pepo mosaic virus obtains after reverse transcription is as shown in SEQ ID NO:1.The specific primer and probe that can quickly detect cucurbita pepo mosaic virus are designed according to viral complete genome sequence, and fluorescent quantitative PCR detection method is established on this basis, from complicated pathogen environment in morbidity plant tissue and soil and it can carry in cotten aphid body of this virus and quickly and accurately detect cucurbita pepo mosaic virus.The detection kit constructed according to this method is easy to operate, specific good, high sensitivity, is of great significance to the early warning of cucurbita pepo mosaic disease epidemic situation, the cause of disease monitoring in epidemic-stricken area etc..
Description
Technical field
The present invention relates to microbiology and molecular Biological Detection fields, specifically, being related to cucurbita pepo mosaic virus
Complete genome sequence and its detection method.
Background technique
Summer squash virus is also known as mosaic disease, is an important disease of cucurbitaceae vegetable.According to investigating in recent years, either
Outdoor cropping or Protectorate cultivation have generation, and serious plot of falling ill can cause 30%~40% underproduction.Cucurbita pepo virus
Disease is caused by a variety of viral independent or Combined Infections.Usual Symptoms are 3 seed types such as floral leaf, shrinkage, mixing.Flower leaf type
It shows as occurring on blade yellowish green irregular mottled, complete stool can be caused to wilt.Shrinkage type shows as occurring along young leaves vein dark green
Color protuberance wrinkle or blade become smaller, and fern leaf, sliver occur, piebald occur in fruit face or generate rough knob, fruit
Mostly lopsided, diseased plant is withered when serious.Mixed type shows as two kinds of floral leaf, shrinkage mixing illnesss.
Cotten aphid (Aphis gossypii Glover) is a kind of common insect pests for being distributed widely in countries in the world, feeding and
Plant virus is propagated, agricultural production is caused to seriously affect.The cotten aphid transmitted virus disease class registered includes that small cucurbita pepo is yellow
The various plants such as mosaic virus (Zucchini yellow mosaic virus) virus.Curcurbitaceae is melon for the main of cotten aphid
Host is lived abroad, it is extremely serious as harm caused by the malicious medium of virus biography that this allows for cotten aphid.Cotten aphid is that Summer squash virus passes
The main path broadcast, detects cotten aphid as early as possible and carries virus, has important application value to the prevention and treatment of virosis.
To Summer squash virus Testing and appraisal, mainly there are biological method, electron microscopy, serological method and molecule raw at present
Object method.Wherein serological method has the characteristics that specificity is good, easy to operate, detection cycle is short, but in accuracy and spirit
There are also certain defects in terms of sensitivity；Molecular biology method has the characteristics that high specificity, high sensitivity, can make up for it serum
The shortcomings that method.Molecular biology method needs the nucleic acid sequence information according to virus.Cucurbita pepo mosaic virus gene information
Acquisition for exploitation cotten aphid provide solid foundation with the exploitation of virus detection method and cucurbita pepo mosaic virus molecular diagnostic techniques.
Summary of the invention
The object of the present invention is to provide a kind of new cucurbita pepo mosaic virus and its complete genome sequence.
It is a further object of the present invention to provide the detection methods of above-mentioned cucurbita pepo mosaic virus.
In order to achieve the object of the present invention, cucurbita pepo mosaic virus of the invention, the viral genome obtain after reverse transcription
Nucleotide sequence as shown in SEQ ID NO:1.Containing 3 reading frames, ORF1, ORF2 and ORF3 size be respectively 7140bp,
1416bp and 603bp (Fig. 1).
The present invention also provides the complete genome sequences of the virus.The virus belongs to normal chain ssRNA virus, and no DNA stage belongs to
In Densovirus (Densovirus) viroid, cucurbita pepo of catching an illness shows as mixed type symptom.
The present invention also provides the Specific PCR primers for detecting the cucurbita pepo mosaic virus, primer sequence is 15~
45, preferably 18-24 continuous nucleotide, the primer detect amplified production for sample after reverse transcription PCR.Forward primer
Meeting Tm value with reverse primer is 55-65 DEG C, and the two Tm value difference is no more than 2 DEG C, G/C content 40%-60%.
The present invention also provides the specific probe for detecting the cucurbita pepo mosaic virus, probe sequence is 15-45,
It is preferred that 20-30 continuous nucleotide, the probe is detected for RNA or DNA sample.
The probe can be used cooperatively with above-mentioned primer.The Tm value of probe is 65-70 DEG C, usually 5- higher than primer Tm
10 DEG C (at least 5 DEG C high), G/C content 40%-70%, 5 ' ends of probe should be avoided using guanine.
The present invention also provides the specific primer and probe for cucurbita pepo mosaic virus described in fluorescence quantitative PCR detection,
Include:
Primer-F:5 '-TTAGGAACGATTCCATTCTT-3 '
Primer-R:5 '-TTCGATCTCACTTATGTAGC-3 '
Probe:5 '-CTCGTCAAGCACACTAAGGCA-3 '
Wherein, the end of probe 5 ' has fluorophor, and 3 ' ends have fluorescent quenching group.
The present invention also provides the methods for detecting the cucurbita pepo mosaic virus based on fluorescent quantitative PCR technique, i.e., using upper
It states primers F, R and probe and carries out fluorescence quantitative PCR detection.
Specifically, it the described method comprises the following steps:
1) total serum IgE in sample is extracted, reverse transcription synthesizes cDNA；
2) using the cDNA of step 1) as template, fluorescent quantitative PCR reaction is carried out；
3) amplified production is analyzed.
Wherein, quantitative fluorescent PCR reaction system are as follows:
Quantitative fluorescent PCR response procedures are as follows: 94 DEG C of 3min；Then 98 DEG C of 10s, 68 DEG C of 60s, 40 circulations, and the 2nd
Fluorescence is collected when step.
The present invention further provides the detection kits containing the primer and/or probe.
The invention discloses the complete genome sequences of cucurbita pepo mosaic virus, and design and can quickly examine according to complete genome sequence
The specific primer and probe of the virus are surveyed, and establishes fluorescent quantitative PCR detection method on this basis, it can be from morbidity plant
It complicated pathogen environment and carries in cotten aphid body of this virus in tissue and soil and quickly and accurately detects cucurbita pepo flower
Mosaic virus.The detection kit constructed according to this method is easy to operate, specific good, high sensitivity, to cucurbita pepo mosaic disease epidemic disease
The early warnings of feelings, the cause of disease monitoring in epidemic-stricken area etc. are of great significance.
Detailed description of the invention
Fig. 1 is 3 reading frames contained in cucurbita pepo mosaic virus gene group sequence of the present invention.
Fig. 2 is the systematic evolution tree of the cucurbita pepo mosaic virus constructed in the embodiment of the present invention 1.
Fig. 3 is fluorescent quantitative PCR situation in the embodiment of the present invention 2, wherein concentration 1-8 respectively represent 1,10,100,
200,400,800,1600 and 3200 times of diluted cDNA samples.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW,
Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
The acquisition of 1 cucurbita pepo mosaic virus complete genome sequence of embodiment
Cotten aphid is picked up from June 12nd, 2011 in the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute, experimental plot cotton field, east (36 ° of 5'
34.8 " N, 114 ° of 31'47.19 " E).Aptery adult aphid is selected after acquisition, is placed in quick-frozen in liquid nitrogen, carries out Total RNAs extraction immediately.Benefit
Express spectra sequencing is carried out with the Solexa high-flux sequence platform construction Paired-End fragment library of Illumina.It is original to measuring
Data carry out quality control, and step is to remove low quality segment using slip window sampling, cut off sequence containing N section in original series
Column.Then Denovo splicing is carried out using software abyss1.3.0, obtains Unigenes.Using Blastn by Unigenes with
(E-value < 10-5) is compared in GenBank amplifying nucleic acid database NT (http://www.ncbi.nlm.nih.gov/), together
When Unigenes is compared with GenBank amplifying nucleic acid database NR using BlastX tool, selecting has homologous pass with virus
The sequence of system is analyzed.
As a result the virus genome sequence that a size is 9235bp is obtained, without 3 ' end polyA tails.The genome sequence
Column contain 3 reading frames, and ORF1, ORF2 and ORF3 size are respectively 7140bp, 1416bp and 603bp (Fig. 1).It is connect by friction
Kind test proves that the virus is a kind of cucurbita pepo mosaic virus.
The amino acid sequence (SEQ ID NO:2) encoded using viral genome ORF1 of the present invention, and in GenBank data
Library (www.ncbi.nlm.nih.gov/genbank/) search with the higher virus amino acid sequences of its homology, obtain 8 kinds with
The higher virus amino acid sequences of its similarity use MEGA6.0 software building systematic evolution tree.The results show that by viral base
The amino acid sequence derived by group sequence and Loreto virus, Negev virus etc. are got together, and with reported plant virus
There is a certain distance (Fig. 2).
2 fluorescence quantitative PCR detection of embodiment
1, the design and synthesis of specific primer and probe:
The nucleotide sequence (SEQ ID NO:1) obtained after reverse transcription according to cucurbita pepo mosaic virus gene group uses
Beacon Designer7.7 software design detects specific primer and the spy of the virus for quantitative fluorescent PCR (Taqman)
Needle, comprising:
Primer-F:5 '-TTAGGAACGATTCCATTCTT-3 '
Primer-R:5 '-TTCGATCTCACTTATGTAGC-3 '
Probe:5 '-CTCGTCAAGCACACTAAGGCA-3 ', 5 ' flag F AM fluorescence, 3 ' label TAMRA.
The synthesis of primer and probe is completed by Shanghai Ying Jun Bioisystech Co., Ltd.
2, the extraction of viral RNA
It will acquire from the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute experimental plot morbidity cucurbita pepo blade on June 18th, 2014
With parasitic cotten aphid thereon, takes back laboratory and liquid nitrogen progress tissue grinder is added, sterile water is then added with volume ratio 1:50.
In 4 DEG C, 6000rpm is centrifuged 30min, takes supernatant.Supernatant is crossed into 0.45 μM of filter membrane, then extracts the total serum IgE in supernatant.RNA's
Extraction illustrates to carry out according to the RNA gents Total RNA Isolation System Kit of Promega company.
3, reverse transcription synthesizes cDNA
Using Promega company reverse transcription reagent box (article No.: A3500), 5 μ g total serum IgE reverse transcriptions is taken to obtain the of cDNA
One chain, reaction system are as follows: 10 μ L of total serum IgE (5 μ g), Oligo (dT) (20 μm of ol/L) 1.5 μ L, 10 × buffer, 2.5 μ L,
2 μ L of dNTP mix (2.5mmol/L), 8 μ L of aqua sterilisa.)
By mixture of the above-mentioned reaction system containing each ingredient after 42 DEG C of water-bath 1min, 1 μ L (200U) is added thereto
SuperScriptTMII RT, is gently mixed, 42 DEG C of heat preservation 50min；70 DEG C of water-bath 15min, terminate the reaction；Obtain cDNA the
One chain.
4, fluorescent quantitative PCR is reacted
The cDNA sample of preparation is divided into 8 groups, is diluted respectively by following multiple: 1,10,100,200,400,800,1600
With 3200.Then it is formulated as follows reaction system.
Quantitative fluorescent PCR reaction system are as follows:
With 4 instrument of Mastercycler ep realplex of Ependorf company, carried out according to following reaction condition real
When quantitative fluorescent PCR: 94 DEG C of 3min；Then 98 DEG C of 10s, 68 DEG C of 60s, 40 circulations, when step 2, collect fluorescence.Reaction terminates
The C of every group of sample is collected afterwardsTValue.
(Fig. 3) as the result is shown, the kit can detect the sample of 1600 times of dilution.With CTValue is Y-axis, and sample concentration is
X constructs standard curve, according to formula E=(10- 1/ slope- 1) amplification efficiency × 100% is calculated, obtaining amplification efficiency is 99.5%,
High sensitivity can better meet production needs.
Using above-mentioned quantitative fluorescent PCR reaction system, the sample that can catch an illness to different cucurbita pepo mosaic virus is quantitatively divided
Son detection, using this method can Accurate Prediction crop field cucurbita pepo mosaic virus growth and decline state and epidemic situation development trend, convenient for timely
It determines disease best prevention and control period, achievees the purpose that efficiently to administer cucurbita pepo mosaic disease.
3 field cotten aphid of embodiment carries the detection of cucurbita pepo mosaic virus
In order to detect the case where field cotten aphid carries cucurbita pepo mosaic virus, acquired respectively in Augusts, 2014 and September respectively
Yellow River basin Earthquake of Anyang station in Henan county cucurbita pepo field, cotton field, cucumber field cotten aphid, per period acquires 50 everyly, and single head extracts total
RNA is illustrated to carry out by the RNA gents Total RNA Isolation System Kit of Promega company.By embodiment 3
Method carry out fluorescence quantitative PCR detection.
The results show that August part there are 18 carrying cucurbita pepo mosaic virus in cucurbita pepo field acquisition cotten aphid, acquired in cotton field
There are 5 carrying cucurbita pepo mosaic virus in cotten aphid, there are 9 carrying cucurbita pepo mosaic virus in cucumber field acquisition cotten aphid.August part
There are 16 carrying cucurbita pepo mosaic virus in cucurbita pepo field acquisition cotten aphid, there are 3 carrying cucurbita pepos in cotton field acquisition cotten aphid
Mosaic virus has 11 carrying cucurbita pepo mosaic virus in cucumber field acquisition cotten aphid.It can be seen that cucurbita pepo mosaic virus is a kind of
The higher virus of cotten aphid carrying rate.
4 cucurbita pepo mosaic virus inoculation test of embodiment
For the relationship for further appreciating that determining cucurbita pepo mosaic virus and Summer squash virus, tested by frictional inoculation,
Healthy cucurbita pepo seedling is inoculated with, every 12 plants of processing, 3 repetitions, while doing clear water friction control.Observe feelings of falling ill after being inoculated with
Condition, and detect virus there are situations.Fluorescence quantitative PCR detection is carried out as described in Example 3.
(table 1) as the result is shown falls ill in cucurbita pepo plant after inoculation, all detects depositing for cucurbita pepo mosaic virus
?.
1 frictional inoculation test result of table
Further study showed that not in other types of the non-cotten aphid such as radish aphid, green bugs, grain aphid, brevicoryne brassicae
The presence that cucurbita pepo mosaic virus is detected in aphid illustrates that cucurbita pepo mosaic virus is the single-minded viral species for infecting cotten aphid.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1. being used for the specific primer and probe of fluorescence quantitative PCR detection cucurbita pepo mosaic virus, comprising:
Primer-F:5 '-TTAGGAACGATTCCATTCTT-3 '
Primer-R:5 '-TTCGATCTCACTTATGTAGC-3 '
Probe:5 '-CTCGTCAAGCACACTAAGGCA-3 '
Wherein, the end of probe 5 ' has fluorophor, and 3 ' ends have fluorescent quenching group；
The nucleotide sequence that the genome of the cucurbita pepo mosaic virus obtains after reverse transcription is as shown in SEQ ID NO:1.
2. the method based on fluorescent quantitative PCR technique detection cucurbita pepo mosaic virus, which is characterized in that utilize claim 1 institute
It states primer and probe and carries out fluorescence quantitative PCR detection；
Wherein, the definition of the cucurbita pepo mosaic virus is the same as described in claim 1.
3. according to the method described in claim 2, characterized by comprising the following steps:
1) total serum IgE in sample is extracted, reverse transcription synthesizes cDNA；
2) using the cDNA of step 1) as template, fluorescent quantitative PCR reaction is carried out；
3) amplified production is analyzed；
Wherein, quantitative fluorescent PCR reaction system are as follows:
Quantitative fluorescent PCR response procedures are as follows: 94 DEG C of 3min；Then 98 DEG C of 10s, 68 DEG C of 60s, 40 circulations, and in step 2
Collect fluorescence.
4. the detection kit containing primer and probe described in claim 1.
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| CN104232660A (en) * | 2014-09-23 | 2014-12-24 | 中国农业科学院棉花研究所 | Whole-genome sequence of cotton aphid virus and detection, separation and application of whole-genome sequence |
| TW201514312A (en) * | 2013-10-08 | 2015-04-16 | Agricultural Res Inst | Primer kit and method for detecting if cucurbitaceae plant is infected by zucchini yellow mosaic virus |
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| TW201514312A (en) * | 2013-10-08 | 2015-04-16 | Agricultural Res Inst | Primer kit and method for detecting if cucurbitaceae plant is infected by zucchini yellow mosaic virus |
| CN104232660A (en) * | 2014-09-23 | 2014-12-24 | 中国农业科学院棉花研究所 | Whole-genome sequence of cotton aphid virus and detection, separation and application of whole-genome sequence |
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| AF127929.2;Lin,S.-S. 等;《GeneBank》;20011120;第4-7页 * |
| Lin,S.-S. 等.AF127929.2.《GeneBank》.2001,第4-7页. * |
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