CN106381346A - Potato virus M Real-time RT (reverse transcription)-PCR (polymerase chain reaction) detection specific primer and detection method - Google Patents
Potato virus M Real-time RT (reverse transcription)-PCR (polymerase chain reaction) detection specific primer and detection method Download PDFInfo
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Abstract
The invention discloses a potato virus M Real-time RT (reverse transcription)-PCR (polymerase chain reaction) detection specific primer and a detection method, relates to a detection primer and a detection method of potato virus M and aims at solving the problems of the raditional detection technology being unable to detect the potato virus M in a dormant tuber or strict in detection condition, and long in detection time. An upstream primer of the detection specific primer is shown as SEQ ID NO:1, and a downstream primer of the detection specific primer is shown as SEQ ID NO:2. The detection method comprises the following steps that 1, total RNA of a potato detection sample is extracted; 2, reverse transcription is carried out; 3, Real-time PCR is carried out; and 4, a dissolution curve of the sample is observed, and the sample contains a PVM virus if a specific peak of a Tm value occurs between 87.07 to 90.2 and does not contain the PVM virus if no specific peak occurs. According to the detection specific primer and the detection method, the detection sensitivity is higher than that of a RT-PCR method by one order of magnitude, the accuracy reaches 100 percent, and the detection specific primer and the detection method are used for detecting the potato virus M.
Description
Technical field
The present invention relates to a kind of detection primer of marmor angliae and detection method.
Background technology
China's potato planting area and total output occupy first place in the world, with the proposition of 2015 " Rhizoma Solani tuber osi staple food grain ",
Rhizoma Solani tuber osi will have bigger development space, be that China's Development of Potato Industry provides opportunities and challenges.But, China Ma Ling
Potato per unit area yield only has the 1/3 of the Potato Industry developed country such as Holland, and main cause is China's qualified production of seed stock rate and utilization rate
Low, during production of seed stock, virosiss are one of key factors of harm potato seed quality, therefore lift Rhizoma Solani tuber osi unit yield
Maximally effective prophylactico-therapeutic measuress are to promote virus-free seed potato, strengthen potato seed detection work, research and develop accurate, sensitive, efficient detection technique
To guarantee potato seed quality, there is very important realistic meaning.
Marmor angliae (potato virus M, PVM), for positive single strand RNA viruses, is not yet divided into suitable section, belongs to
Carnation Latent Virus In China belongs to, and virion is about 8500bp.The disease causing on Rhizoma Solani tuber osi is referred to as Rhizoma Solani tuber osi secondary wrinkle floral leaf
Mosaic disease between disease, Rhizoma Solani tuber osi volume mosaic viruss and Rhizoma Solani tuber osi arteries and veins, PVM infects symptom caused by potato plant with Rhizoma Solani tuber osi
Kind is different with PVM strain and there is great difference, be mainly shown as the floral leaf that hides, leaf-shrinkage, lobule deformity, plant top
The symptoms such as end curling, petiole and stem's necrosis.After PVM virulent strain department infects, leaf necrosis symptom of hanging down, class are produced on some kinds
Like PVY symptom, there is larger economic impact, the symptom of low virulent strain system harm is floral leaf between small vein, and leaflet tip slightly distorts, leaf
Edge is in wavy, and some leaf rolls of diseased plant top or blade face are matt.
In field, PVM passes through machinery and aphis propagation, and 9%～49%, host range is wide, Zheng Hong English in 2003 for the underproduction
Deng having isolated PVM virion from Herba Herminii.PVM since nineteen twenty-three reported first, in the U.S., Britain, France, lotus
All there is generation in the state such as blue, Russian, and wherein in Russia based on the strong strain of PVM, 1979 in China Heilungkiang, Inner Mongol horse
There is generation in bell potato producing region, and so far, Ningxia, Qinghai, Henan, Sichuan, Yunnan, Guangxi etc. save potato seed growing area all report
Road.
The ground such as the northern Heilungkiang of China, the Inner Mongol, Gansu, Ningxia are due to the advantage in its geographical position and weather, aphid
Evening generation phase, the production of suitable potato seed, it is responsible for the important task breeding seed potato, the potato seed being produced not only meets province's domestic demand
Ask, be also supplied with the commodity potato producing region such as Shandong, Guangdong, Yunnan simultaneously.Meanwhile, because these areas are bordered on Russia or ground
Reason is located proximate to, and PVM is the important disease that harm Russia Rhizoma Solani tuber osi produces, the potato plant of more than 90% infection PVM,
Also infected PVY virus, it is even more serious that both virus infects the harm causing simultaneously, 2014～2015 years, my unit simultaneously
Finding to hang down in virosiss fact-finding process, to be that PVM infects caused for the plant of leaf necrosis, and Russia is that China Rhizoma Solani tuber osi is introduced a fine variety
Emphasis country it is more likely that can during introducing a fine variety incoming China.Therefore, accurate detection technique is to ensure China's potato seed
The important measures of potato production safety.
2015, Zhang Wei etc. established PVM RT-PCR detection technique, established containing PVM in 5 interior weight RT-PCR simultaneously
Detection technique system.The virion in potato plant can be detected, but dormancy tuber can not be detected.Planting
Often use DAS-ELISA method in the world in detection work after the results of potato, need the potato tuberss harvesting application
Gibberellins carry out accelerating germination and then again after chamber planting, and when plant to be planted grows to 20 centimetres, collection blade is detected.The method needs
Want the equipment such as greenhouse, and the time is longer.Part potato seed needs to transport south plantation, conventional DAS-ELISA method to after harvesting
Time-consuming 1.5～2 months, the time is unable to reach customer requirement, and conventional RT-PCR method is restricted by its detection sensitivity, also no
Method accurately detects the virus in dormancy potato wedges.Real-time PCR is most advanced in the world at present and sensitivity highest
Detection technique, all appears in the newspapers in PVX, PVY, PVA, PLRV virus, but not yet has the report of PVM Real-time PCR detection technique
Road, its reason is probably that PVM strain differentiations are complicated, for ensureing the accuracy of detection, design of primers is had high demands.
Content of the invention
The present invention is to solve existing detection technique or marmor angliae in dormancy tuber cannot be detected, or detection
Condition is harsh, the problem of detection time length, provides marmor angliae Real-time RT-PCR detection special primer and detection side
Method.
Marmor angliae Real-time RT-PCR of the present invention detects that special primer is：
Forward primer：SEQ ID NO in 5 '-TGCTTTGAHTACGTKGAGAA-3 ', such as sequence table：Shown in 1.Entitled
PVM 9F, it is 7927-7946 that this sequence pair answers the position on PVM complete sequence；
Downstream primer：SEQ ID NO in 5 '-TGAGYTCDGGACCATTC-3 ', such as sequence table：Shown in 2.Entitled PVM
8R, it is 8096-8114 that this sequence pair answers the position on PVM complete sequence.
H in primer sequence represents base A, T or C, and K represents bases G or T, and Y represents base C or T, and D represents bases G, A
Or T.
The primer sequence of the present invention is used for marmor angliae EVA-Green Real-time RT-PCR and detects.
The present invention utilizes the method that above-mentioned primer pair detects marmor angliae, specially：
First, plant Total RNAs extraction
Take leaf sample or potato wedges tissue sample 100mg, application Trizol reagent extracts plant total serum IgE；
2nd, reverse transcription
Apply 6 nucleotide random primers and M-MLV reverse transcription, RNA reverse transcription is become cDNA；
3rd, application Eva-Green carries out Real-time PCR, reaction system μ L, 0.25mM containing 10 × buffer 2
MgCl2, 0.1mM dNTP, each 0.4 μM of upstream and downstream primer, Eva-Green 1 μ L, Taq enzyme 5U, cDNA2 μ L, with ultra-pure water mend to
20μL；Response procedures are：95 DEG C, 3min；95 DEG C, 25s, 49 DEG C, 30s, 72 DEG C, 30s；40 circulations, survey amplification for 60～90 DEG C
The melting curve of product；
4th, observe sample solubility curve, specific peak occur between Tm value 87.07-90.2, in sample, contain PVM virus,
Viral without then not containing PVM.
Beneficial effects of the present invention：
The PVM degenerate primer of application present invention design, establishes PVM EVA-Green Real-time PCR detection technique
System, can detect the separation strains of known PVM virus.Detection sensitivity high an order of magnitude than RT-PCR method, additionally, energy
The virion in potato tuberss is enough detected.The PVM separation strains of 7 provinces all can be detected, accuracy reaches
100%.
The good stability of primer detection of the present invention, high specificity are it is adaptable to large-scale promotion is it is achieved that Rhizoma Solani tuber osi dormancy block
The M efficient virus quick detection of stem.
Brief description
Fig. 1 is the RT-PCR testing result of primer PVM9F/8R and PVM7F/7R；
Fig. 2 is the RT-PCR testing result of primer PVM8F/8R and PVM2F/2R；
Fig. 3 is PVM RT-PCR sensitivity technique result；
Fig. 4 is PVM Real-time PCR sensitivity technique result；
Fig. 5 is PVM real-time PCR field sample detection effect.
Specific embodiment
Technical solution of the present invention is not limited to act specific embodiment set forth below, also includes between each specific embodiment
Combination in any.
Specific embodiment one：Present embodiment marmor angliae Real-time RT-PCR detects the upper of special primer
Trip primer such as SEQ ID NO in sequence table：Shown in 1, SEQ ID NO in downstream primer such as sequence table：Shown in 2；In primer sequence
H represent base A, T or C, K represents bases G or T, and Y represents base C or T, and D represents bases G, A or T.
Specific embodiment two：Present embodiment from unlike specific embodiment one：Described Real-time RT-
PCR detects that special primer amplifies the product of 188bp to marmor angliae PCR.Other identical with specific embodiment one.
Specific embodiment three：Present embodiment utilizes marmor angliae Real-time RT-PCR to detect special primer
The method of detection marmor angliae, specially：
First, extract the total serum IgE of Rhizoma Solani tuber osi detection sample；
2nd, reverse transcription：The RNA reverse transcription of extraction is become cDNA；
3rd, application Eva-Green carries out Real-time PCR, reaction system μ L, 0.25mM containing 10 × buffer 2
MgCl2, 0.1mM dNTP, each 0.4 μM of upstream and downstream primer, Eva-Green 1 μ L, Taq enzyme 5U, cDNA 2 μ L, is mended with ultra-pure water
To 20 μ L；
4th, observe sample solubility curve, specific peak occur between Tm value 87.07-90.2, in sample, contain PVM virus,
Viral without then not containing PVM.
Specific embodiment four：Present embodiment from unlike specific embodiment three：Described Real-time PCR's
Response procedures are：95 DEG C, 3min；95 DEG C, 25s, 49 DEG C, 30s, 72 DEG C, 30s；40 circulations, 60～90 DEG C of survey amplified productions
Melting curve.Other identical with specific embodiment three.
Specific embodiment five：Present embodiment from unlike specific embodiment three or four：Described detection sample is
Potato leaf or dormancy tuber.Other identical with specific embodiment three or four.
Below embodiments of the invention are elaborated, following examples are entered under premised on technical solution of the present invention
Row is implemented, and gives detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following realities
Apply example.
First, the design of marmor angliae primer：
1. from NCBI Gene Bank data base, first, download PVM sequence information, application software Bio-Edit is carried out
Local Blast data analysiss, find the conservative region of PVM difference separation strains.
2. application software Primer Premier 5.0 design PVM special primer, due to the homology between PVM each bar sequence
There is larger difference in property.Therefore, according to PVM sequence alignment result, representative PVM sequence gi72257063 of screening,
Gi35902731 and gi120431386, in the high region of homology, in application primer-design software Primer Premier 5.0
Design PVM degenerate primer, designs 4 groups of primers, primer details are shown in Table 1 altogether.Next this 4 groups of primers are carried out RT-PCR and
Real-timePCR amplification test.
Table 1 PVM primer sequence information
The marmor angliae EVA-Green Real-time RT-PCR detection special primer of the present invention, forward primer
Sequence is 5 '-TGCTTTGAHTACGTKGAGAA-3 ', entitled PVM 9F, and this sequence pair answers the position on PVM complete sequence to be
7927-7946；Downstream primer is 5 '-TGAGYTCDGGACCATTC-3 ', entitled PVM 8R, and this sequence pair answers PVM complete sequence
On position be 8096-8114, the purpose fragment length of this primer pair amplifies is 188bp；H in sequence represent base A, T or
C, K represent bases G or T, and Y represents base C or T, and D represents bases G, A or T.
2nd, application RT-PCR tests to 4 groups of primers in table 1
1. plant Total RNAs extraction
Application Trizol (Invitrogen, the U.S.) reagent extracts plant total serum IgE, and leaf sample and potato wedges tissue sample are equal
Take 100mg, operated to specifications.
2. reverse transcription
Apply 6 nucleotide random primers and M-MLV reverse transcription, to specifications RNA reverse transcription is become cDNA.
3.RT-PCR tests
4 groups of primers in application table 1, application TaqE (TAKARA) is entered performing PCR and is expanded to cDNA, (addition reaction system),
5 μ L PCR primer are taken to carry out 1% sepharose electrophoresis, EB dyes, gel imaging system is observed.Primer PVM9F/8R and PVM7F/7R
Testing result is shown in Fig. 1, and wherein line1～12 are PVM9F/8R amplification, and line13～24 are PVM7F/7R amplification；
Line12,24 be water comparison, line6,8,18,20 be negative control.Primer PVM8F/8R and PVM2F/2R testing result are shown in figure
2, line 1～12 be PVM8F/8R amplification, line 13～24 be PVM2F/2R amplification, line12,24 be water pair
According to line6,8,18,20 are negative control.
Result shows：4th group of primer PVM9F/8R expanding effect is optimal, can all expand 9 PVM positive
Out, second group of primer PVM7F/7R effect is taken second place, and second group of primer PVM2F/2R and the 3rd group of primer PVM8F/8R effect is not
Good.Therefore, next step, application Real-time PCR is carried out to the 4th group of primer PVM9F/8R and second group of primer PVM7F/7R
Real-time PCR tests, the preferable primer of one group of Detection results of selection.
3rd, application Real-time PCR tests to the 2 groups of primers filtering out
RNA extracts with cDNA reverse transcription ibid.Then, application Eva-Green carries out Real-time PCR, reaction system
Containing 10 × buffer 2 μ L, 0.25mM MgCl2, 0.1mM dNTP, each 0.4 μM of upstream and downstream primer, Eva-Green 1 μ L, Taq
Enzyme 5U, cDNA 2 μ L, is mended to 20 μ L with ultra-pure water.Response procedures are：95 DEG C, 3min；95 DEG C, 25s, 49 DEG C, 30s, 72 DEG C,
30s；40 circulations, the melting curve of 60～90 DEG C of survey amplified productions.
Result shows：The Tm value of the 4th group of primer PVM9F/8R amplified fragments has multiple peaks, between 87.07-89.64,
In the range of theoretical value, but because PVM separation strains sequence difference is big, therefore Tm value is variant.Meanwhile, leaf sample and potato wedges
Sample is all detected.The Tm value of second group of primer PVM7F/7R amplified fragments also has multiple peaks, respectively 87.34～
Between 91.05, also it is consistent with theoretical value.But the amplification efficiency of the 4th group of primer PVM9F/8R is higher than second group of primer PVM7F/
7R.And, second group of primer has nonspecific peak value, the judgement of impact result in the vicinity of main peak.Therefore, final choice is drawn
Thing PVM9F/8R is as PVM real-time PCR detection primer.
4th, PVM RT-PCR detection system and PVM Eva-Green Real-time PCR sensitivity
Apply the 4th group of primer PVM9F/8R, the PVM RT-PCR and Real-time PCR detection body system of foundation, to not
PVM plasmid with Concentraton gradient is detected.Compare the sensitivity of RT-PCR detection system and Real-time detection system.
1.PVM RT-PCR expands
The primer PVM9F/8R that application screening obtains carries out the RT-PCR amplification of PVM.Apply 6 nucleotide random primers and M-
MLV reverse transcriptase description, RNA reverse transcription is become cDNA.PCR reaction system μ L, 0.25mM MgCl containing 10 × buffer 22,
0.25mM dNTP, each 0.5 μM of upstream and downstream primer, Taq enzyme 1U, cDNA 1 μ L, is mended to 20 μ L with ultra-pure water.Response procedures are：95
DEG C, 3min；95 DEG C, 25s, 49 DEG C, 30s, 72 DEG C, 30s；40 circulations.
2.PCR product reclaims
5 μ L PCR primer are taken to carry out 1% sepharose electrophoresis, EB dyes, gel imaging system is observed, by the band of 188bp
Scale off, application PCR primer QIAquick Gel Extraction Kit reclaims PCR primer.
3. clone and be sequenced
By pEASY-T5Zero Cloning Kit description, DNA product is connected in pEASY-T5Zero carrier, conversion
To in Trans-T1 competent cell, application PCR method identification positive colony, is sent to raw work biological engineering (Shanghai) share limited
Company is sequenced.Sequencing result shows：PVM purpose fragment successful conversion is in Trans-T1 competent cell.Next step is by plasmid
Extract, calculate copy number, for sensitivity test.
4. plasmid extraction and copy number calculate
The application little extraction reagent kit of plasmid, extracts plasmid to specifications, and application ultraviolet spectrophotometer tests the dense of plasmid
Degree, and the copy number of plasmid is calculated according to below equation.
Plasmid copy number computing formula：
(6.02×1023Copy number/mole) × (concentration ng/ μ L × 10-9)/DNA length × 660)=copy/μ L
Wherein 6.02 × 1023For Avogadro constant number, dsDNA=base number × 660 dalton/base, in this research
Use pEASY-T5Zero cloning vehicle, its base logarithm is 3955bp+ purpose fragment base number.
PVM purpose fragment length is 188bp, and therefore, recombinant vector length is 4143bp, applies spectrophotometric determination
The concentration of PVM plasmid is 11.05ng/ μ L, according to above-mentioned formula, calculates PVM2.43E+09.Plasmid is carried out continuous gradient dilute
Release, for measuring further
5. gradient dilution
PVM recombiant plasmid is done 10 times of continuous gradient dilutions and prepares standard substance, method is as follows：Draw 27 μ L no enzyme water respectively
In the PCR pipe of 10 200 μ L, it is respectively labeled as 1,2,3 ... 10, draw recombiant plasmid stock solution 3 μ L and be added in pipe 1, fill
Divide after mixing, more therefrom draw 3 μ L and be added in pipe 2, by that analogy, dilute 10 gradients, stock solution and gradient solution concentration altogether
Represented with copy number.The original concentration of PVM recombiant plasmid is：2.43×109Copy, therefore, PVM recombiant plasmid after dilution
Concentration is followed successively by：2.43×108Copy, 2.43 × 107Copy, 2.43 × 106Copy, 2.43 × 105Copy, 2.43 × 104Copy
Shellfish, 2.43 × 103Copy, 2.43 × 102Copy, 2.43 × 101Copy, 2.43 × 100Copy, 2.43 × 10-1Copy.
6.PVM RT-PCR sensitivity technique
The PVM recombiant plasmid of above-mentioned 10 concentration is carried out PVM RT-PCR amplification, plasmid copy number is followed successively by：2.43×
108Copy, 2.43 × 107Copy, 2.43 × 106Copy, 2.43 × 105Copy, 2.43 × 104Copy, 2.43 × 103Copy,
2.43×102Copy, 2.43 × 101Copy, 2.43 × 100Copy, 2.43 × 10-1Copy.Reaction system and response procedures are same
On, result shows：RT-PCR detection is minimum to be limited to 2.43 × 103Copy.PVM RT-PCR sensitivity technique result such as Fig. 3 institute
Show, wherein M is Marker, 1 is water, 2 is-CK, 3 is 2.43 × 108Copy, 4 is 2.43 × 107Copy, 5 is 2.43 × 106Copy
Shellfish, 6 is 2.43 × 105Copy, 7 is 2.43 × 104Copy, 8 is 2.43 × 103Copy, 9 is 2.43 × 102Copy, 10 are
2.43×101Copy, 11 is 2.43 × 100Copy, 12 is 2.43 × 10-1Copy.
7.PVM Real-time PCR testing result
The PVM recombiant plasmid of above-mentioned 8 concentration is carried out PVM Real-time PCR amplification, plasmid copy number is followed successively by：
2.43×106Copy, 2.43 × 105Copy, 2.43 × 104Copy, 2.43 × 103Copy, 2.43 × 102Copy, 2.43 × 101
Copy, 2.43 × 100Copy, 2.43 × 10-1Copy, 3 repetitions.Reaction system and response procedures are ibid.RNA extracts and cDNA
Reverse transcription ibid, Real-time PCR reaction system μ L, 0.25mM MgCl containing 10 × buffer 22, 0.1mM dNTP, up and down
The trip each 0.4uM of primer, Eva-Green 1 μ L, Taq enzyme 5U, cDNA 2 μ L, is mended to 20 μ L with ultra-pure water.Response procedures are：95
DEG C, 3min；95 DEG C, 25s, 49 DEG C, 30s, 72 DEG C, 30s；40 circulations, the melting curve of 60～90 DEG C of survey amplified productions.Result
Display：PVM Real-time PCR detection is minimum to be limited to 2.43 × 102Copy.Higher an order of magnitude than RT-PCR detection system.
PVM Real-time PCR sensitivity technique result is as shown in figure 4, wherein right side peak value is followed successively by from high in the end：2.43×106
Copy, 2.43 × 105Copy, 2.43 × 104Copy, 2.43 × 103Copy, 2.43 × 102Copy, 2.43 × 101Copy, 2.43
×100Copy, 2.43 × 10-1Copy.
5th, PVM Eva-Green Real-time PCR Detection results experimental verification
1. sample collecting：
Sample picks up from China northeast, northwest, the potato fields of southwestern 14 provinces, municipalities and autonomous regions, through ELISA primary dcreening operation result
For positive or p+ 25 parts of PVM field samples, for the checking of Real-time PCR experiment, verify the inspection of this reaction system
Survey effect, sample details are shown in Table 2 as follows.
Table 2 PVM sample message
Note：* it is potato wedges sample
2. plant Total RNAs extraction
Application Trizol (Invitrogen, the U.S.) reagent extracts plant total serum IgE, and leaf sample and potato wedges tissue sample are equal
Take 100mg, operated to specifications.
3. reverse transcription
Apply 6 nucleotide random primers and M-MLV reverse transcription, to specifications RNA reverse transcription is become cDNA.
4.PVM Eva-Green Real-time PCR experiment is verified
Reaction system μ L, 0.25mM MgCl containing 10 × buffer 22, 0.1mM dNTP, each 0.4 μM of upstream and downstream primer,
Eva-Green 1 μ L, Taq enzyme 5U, cDNA 2 μ L, is mended to 20 μ L with ultra-pure water.Response procedures are：95 DEG C, 3min；95 DEG C,
25s, 49 DEG C, 30s, 72 DEG C, 30s；40 circulations, the melting curve of 60～90 DEG C of survey amplified productions.
PVM real-time PCR field sample detection effect is as shown in Figure 5.PVM Real-time PCR testing result
Display, 25 parts of samples picking up from 7 provinces of China all can detect, and accuracy rate is 100%.Illustrate that this detection primer is permissible
The marmor angliae of China's zones of different is detected.Therefore, this group primer can be grasped and planted with actual response potato seed quality in time
The quality of potato, for producing service.
Sequence table
<110>Plant De-toxin Nursery Stock Inst., Heilongjiang Prov. Academy of Agriculture Sc
<120>Marmor angliae Real-time RT-PCR detection special primer and detection method
<160> 7
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer PVM 9F
<400> 1
tgctttgahtacgtkgagaa 20
<210> 2
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>Primer PVM 8R
<400> 2
tgagytcdggaccattc 17
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>Primer PVM 2F
<400> 3
acdaatccktacaacagg 18
<210> 4
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>Primer PVM 2R
<400> 4
atcachacytgctgcac 17
<210> 5
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>Primer PVM 7F
<400> 5
agrgtvtgyaggctgta 17
<210> 6
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>Primer PVM 7R
<400> 6
ttctcmacgtadtcaaag 18
<210> 7
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>Primer PVM 8F
<400> 7
ggrgchaatcgcaayca 17
Claims (5)
1. marmor angliae Real-time RT-PCR detect special primer it is characterised in that described detection special primer upper
Trip primer such as SEQ ID NO in sequence table：Shown in 1, SEQ ID NO in downstream primer such as sequence table：Shown in 2；In primer sequence
H represent base A, T or C, K represents bases G or T, and Y represents base C or T, and D represents bases G, A or T.
2. according to claim 1 marmor angliae Real-time RT-PCR detection special primer it is characterised in that described
Real-time RT-PCR detects that special primer amplifies the product of 188bp to marmor angliae PCR.
3. utilize marmor angliae Real-time RT-PCR detection special primer detection marmor angliae described in claim 1
Method it is characterised in that the method comprises the following steps：
First, extract the total serum IgE of Rhizoma Solani tuber osi detection sample；
2nd, reverse transcription：The RNA reverse transcription of extraction is become cDNA；
3rd, carry out Real-time PCR, reaction system μ L, 0.25mM MgCl containing 10 × buffer 22, 0.1mM dNTP, up and down
Each 0.4 μM of primer of trip, Eva-Green 1 μ L, Taq enzyme 5U, cDNA 2 μ L, is mended to 20 μ L with ultra-pure water；
4th, observe sample solubility curve, specific peak occurs between Tm value 87.07-90.2, in sample, contain PVM virus, if
Then do not contain PVM virus.
4. detect the method for marmor angliae it is characterised in that described Real-time PCR's is anti-according to claim 3
The program is answered to be：95 DEG C, 3min；95 DEG C, 25s, 49 DEG C, 30s, 72 DEG C, 30s；40 circulations, survey the molten of amplified productions for 60～90 DEG C
Solution curve.
5. detect the method for marmor angliae according to claim 3 it is characterised in that described detection sample is leaf of potato
Piece or dormancy tuber.
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| KR20050079651A (en) * | 2004-02-05 | 2005-08-11 | 대한민국(관리부서:농촌진흥청) | Specific primers for detection of potato virus m |
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| KR20050079651A (en) * | 2004-02-05 | 2005-08-11 | 대한민국(관리부서:농촌진흥청) | Specific primers for detection of potato virus m |
Non-Patent Citations (1)
| Title |
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| HUIMIN XU, ET AL.: "Genomic variability in Potato virus M and the development of RT-PCR and RFLP procedures for the detection of this virus in seed potatoes", 《VIROLOGY JOURNAL》 * |
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| CN107227379A (en) * | 2017-06-21 | 2017-10-03 | 陈定虎 | Primer, kit and the authentication method of marmor angliae identification |
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