CN102140541B - Method for synchronously detecting four viruses of carnation - Google Patents
Method for synchronously detecting four viruses of carnation Download PDFInfo
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- CN102140541B CN102140541B CN2011100386681A CN201110038668A CN102140541B CN 102140541 B CN102140541 B CN 102140541B CN 2011100386681 A CN2011100386681 A CN 2011100386681A CN 201110038668 A CN201110038668 A CN 201110038668A CN 102140541 B CN102140541 B CN 102140541B
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Abstract
The invention relates to a method for synchronously detecting four viruses of carnation, in particular to a method for synchronously detecting carnation mottle virus, latent virus, ringspot virus and necrotic spot virus. The invention discloses primers for synchronously detecting the four viruses, and the sequences of the primers are shown as SEQ ID No.1-8. The invention also discloses a method for synchronously detecting the four viruses, which comprises the following steps of: 1) extracting total RNA of the carnation; and 2) performing reverse transcription-multiplex polymerase chain reaction: performing reverse transcription of the RNA obtained in the step 1), performing synchronous amplification on a reverse transcription production by using four pairs of primers, and synchronously detecting the four viruses. The primers have good compatibility, and belts are clear and easy to identify; and by the method, the detection cost is reduced, the detection speed is improved, and the synchronization, high efficiency and sensitivity for detecting the viruses of the carnation are realized.
Description
Technical field
The invention belongs to the plant virus detection range, be specifically related to the synchronization detecting method of four kinds of viruses of a kind of oeillet.
Background technology
In the RT-PCR of Dianthovirus detection technique, and people such as Kong Baohua (Kong Baohua, Cai Hong etc. evaluation of carnation mottle virus and PT-PCR detect [J]. plant protection; 2002; 2,28 (1): 5-8), can dilute 10 from RNA according to the RNA sequences Design primer of carnation mottle virus CarMV
4The RNA crude extract in detect the virus of xiangshizhubanbo.People such as Zhao Shan (Zhao Shan; Wang Jihua; Wang Lihua, Yang Xiumei. the evaluation and the detection [J] of oeillet necrotic spot virus. southwestern agriculture journal, 2010; 1:103-106) can from the susceptible oeillet tissue of oeillet necrotic spot virus CNFV, detect CNFV virus through the design primer, susceptibility is measured the result and is shown that this Auele Specific Primer RT-PCR can be from diluting 10
6Detect virus among the total RNA of plant.
Constantly perfect through for many years; Round pcr has developed into a kind of current techique at present; The amplification information that multiple RT-PCR can provide conventional method to react for several times in a reaction simultaneously; This efficiently fast advantage that it is detected in promoting with virus-free seedling fast breeding plant virus is significant, do not appear in the newspapers as yet but utilize should technology to detect simultaneously in the multiple Dianthovirus.
The Dianthovirus disease is one type of important disease on the oeillet; Cause that the sick viral species of Dianthovirus is a lot; Kind surplus having reported 10 both at home and abroad, but it is generally acknowledged carnation mottle virus (Carnation mottle virus; CarMV), cryptovirus (Carnation latentvirus; CLV), (Carnation ring spot virus, CRSV) (Carnation necrotic fleck virus is in the world oeillet to be produced 4 kinds of maximum viruses of harm CNFV) to ring spot virus with necrotic spot virus.
Summary of the invention
In order to address the above problem, the object of the present invention is to provide primer, detection method and the detection kit thereof of the synchronous detection that is used for four kinds of viruses of oeillet.
One object of the present invention is to be provided for four kinds of viral synchronous detection primers of oeillet, comprising:
CarMV upstream primer: ACGCTCACCTCGCCAATTTGCTT
CarMV downstream primer: TCCGCAGTCCGCACATTCCTACT;
CRSV upstream primer: AATCCAAAATGGCGCTGGTGCCT
CRSV downstream primer: CATCAGTGAAACGCGACCGCTCA;
CLV upstream primer: TGTTGGGCTGTCGCACGTTACTG
CLV downstream primer: GGTGGGGATTTGGCGAACAGCAT;
CNFV upstream primer: TGGAGTGCGTCACCTTGAGTGGT
CNFV downstream primer: GTCGAAAGTGCCGCCTCCGAAAT.
Another object of the present invention is to provide oeillet four kinds of viral synchronization detecting methods, comprises the steps:
1) total RNA of extraction oeillet;
2) RT-MPCR: after the RNA reverse transcription that obtains in the step 1),, and carry out electrophoresis detection with the described primer amplification reverse transcription product of claim 1.
The present invention also provides the process for extracting of the total RNA of oeillet, and step is following:
1) takes by weighing 0.05～0.1g oeillet spire or petal to mortar; Under liquid nitrogen, be ground into superfine powder, powder be transferred to rapidly in the 1.5mL centrifuge tube of precooling, add behind 0.8～1.2mLTrizol with the abundant mixing of eddy mixer; Under room temperature, leave standstill 5min, make its abundant cracking;
2) 13000rpm, 4 ℃ of centrifugal 10～15min suct clearly, add isopyknic volume ratio and be 24: 1 chloroform: primary isoamyl alcohol, the abundant mixing of eddy mixer leaves standstill 2～3min;
3) 12000rpm, 4 ℃ of centrifugal 10～15min suct clearly extremely new centrifuge tube, add the Virahol of 0.7 times of volume, the abundant mixing of eddy mixer ,-70 ℃ leave standstill 10～30min;
4) 12000rpm, 4 ℃ of centrifugal 10～15min abandon supernatant, add 300～500 μ L, 75% ethanol and wash, gentle vibration centrifuge tube, the deposition that suspends, 7500rpm, 4 ℃ of centrifugal 5～10min abandon supernatant;
5) 300～500 μ L, 75% ethanol repeated washing once, room temperature is dried or vacuum-drying 5～10min, does not have the distilled water dissolving RNA sample of RNase with 50～100 μ L, and is subsequent use in-70 ℃ of preservations.
According to the present invention, the PCR reaction system of amplification oeillet reverse transcription product is:
The PCR response procedures is: 95 ℃ of preparatory sex change 3min; 94 ℃ of sex change 30s then, 53 ℃～60 ℃ annealing 30s, 72 ℃ are extended 30s, 32 circulations; 72 ℃ are extended 5min, at last in 4 ℃ of end.
Another object of the present invention is to provide the oeillet that contains said primer four kinds of viral synchronous detection test kits, and described detection kit also comprises ThermoScript II and reaction buffer, Taq archaeal dna polymerase and reaction buffer thereof, dNTPs, Oligo dT, RNase suppressor factor and do not have the distilled water of RNase.
The application of the test kit of the primer of described four kinds of viral synchronous detection provided by the invention or described four kinds of viral synchronous detection in four kinds of viral synchronous detection of oeillet.
Beneficial effect of the present invention:
(1) the special viral primer compatibility of four couple that the present invention designed is good, can simultaneously in a reaction system, obtain stronger amplification, and fragment length differs suitably, can be when guaranteeing normally to increase, reach that bands of a spectrum are clear, the purpose of easy differentiation.
(2) the present invention adopts improved Trizol method; Through traditional Trizol operation steps is optimized; Change the extracting of chloroform into chloroform: primary isoamyl alcohol, the interference that can remove insoluble protein, high molecular weight protein and DNA effectively is to obtain high purity, high quality and the good RNA of integrity degree.
(3) described PCR reaction system is doubled to 50 μ L by 25 μ L of routine, makes title product separately fully to increase.
(5) described method for detecting virus, fast stable, can accomplish in 7～8 hours.
(6) described method for detecting virus through multiple authentication, all can carry out its favorable repeatability.
(7) described method for detecting virus, each reactant becomes to obtain best low side numerical value in the optimization system, and total cost is cheap.
Description of drawings
Oeillet blade and petal RNA gel electrophoresis figure that Fig. 1 extracts through the Trizol method and the traditional T rizol method of improvement for the present invention.1,2 and 3,4 be respectively improvement the RNA that extracts of the blade that extracts of Trizol method and petal; 5,6 and 7,8 be respectively the blade of traditional T rizol method extraction and the RNA that petal extracts.
Fig. 2 is the quadruple PCR gel electrophoresis figure of different annealing temperature.Wherein, M:DNAMarker; 1～8:60 ℃～53 ℃
Fig. 3 is the compound RT-MPCR product gel of the quadruple electrophorogram of oeillet kind of the present invention ' Maas is special ' virus, M:DNAMarker 1:CarMV; 2:CRSV; 3:CLV; 4:CNFV; 5:CarMV+CRSV; 6:CarMV+CLV; 7:CarMV+CNFV; 8:CRSV+CLV; 9:CRSV+CNFV; 10:CLV+CNFV; 11:CarMV+CRSV+CLV; 12:CarMV+CRSV+CNFV; 13:CarMV+CLV+CNFV; 14:CRSV+CLV+CNFV; 15:CarMV+CRSV+CLV+CNFV.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
The compound RT-MPCR technology for detection of embodiment 1 quadruple oeillet CarMV, CNFV, CRSV, four kinds of viruses of CLV
1.1CarMV, the design of primers of CNFV, CRSV, CLV
Through with Primer 5.0 software design primers, filter out four pairs of special viral primers that compatibility is good, sequence is as shown in the table.
The primer sequence of table 1CarMV, CNFV, CRSV, CLV
1.2RNA extract
(1) spire or the petal that take by weighing 0.1g oeillet ' Maas special ' ' refined powder ' ' freedom ' (available from Yunnan Agriculatural Academy) are to mortar; Under liquid nitrogen, be ground into superfine powder; Powder is transferred to rapidly in the 1.5mL centrifuge tube of precooling; Add 1mL Trizol (Invitrogen) back with the abundant mixing of eddy mixer, under room temperature, leave standstill 5min, make its abundant cracking.
(2) 13000rpm, 4 ℃ of centrifugal 10min suct clearly, add the equal-volume chloroform: primary isoamyl alcohol (volume ratio 24: 1), the abundant mixing of eddy mixer leaves standstill 3min.
(3) 12000rpm, 4 ℃ of centrifugal 10min suct clearly extremely new centrifuge tube, add the Virahol of 0.7 volume, the abundant mixing of eddy mixer ,-70 ℃ leave standstill 10min.
(4) 12000rpm, 4 ℃ of centrifugal 10min abandon supernatant, and RNA is sunken to the pipe end.Add 300 μ L, 75% ethanol and wash, gentle vibration centrifuge tube, the deposition that suspends, 7500rpm, 4 ℃ of centrifugal 5min abandon supernatant.
(5) 300 μ L 75% ethanol repeated washing once, room temperature is dried or vacuum-drying 5～10min (the RNA sample is too not dry, otherwise is prone to decompose), does not have the ddH of RNase with 50 μ L
2O dissolving RNA sample, subsequent use in-70 ℃ of preservations.
The step of traditional T rizol method is the same basically, and different is adds Trizol and organize that centrifugal rotation speed is 12000rpm after the abundant cracking; The used reagent of extracting albumen is isopyknic chloroform; The amount of the Virahol that precipitated rna is used is 0.5 times of volume, does not also have-70 ℃ of steps that leave standstill.The blade of the oeillet of two kinds of method extractions and the RNA of petal are as shown in Figure 1.The result shows that the quality of the RNA that traditional T rizol extracts far is worse than the improved RNA process for extracting.
1.3 reverse transcription
The above-mentioned RNA that extracts with the Trizol method of improvement is carried out reverse transcription, system such as following table:
Table 2 reverse transcription (RT) reaction system
Add the ddH of total RNA, Oligo (dT) 18 and no RNase item by item according to tab sequential
2After O adds, 65 ℃ of water-bath 5min, ice baths immediately.And then add other reagent successively and be settled to 42 ℃ of water-bath 1h behind the 20 μ L.70 ℃ of water-bath 5min more at last.The entire operation process is carried out on ice bath, synthetic cDNA first chain.
1.4 the compound RT-PCR reaction of quadruple
The compound RT-PCR reaction system of table 3 quadruple
The PCR response procedures: 95 ℃ of preparatory sex change 3min, 94 ℃ of sex change 30sec then, 57 ℃ of annealing 30s, 72 ℃ are extended 30s, 32 circulations, 72 ℃ are extended 5min, at last in 4 ℃ of end.
The RT-MPCR system optimization that four kinds of viruses of embodiment 2 oeillets detect
In order to optimize RT-MPCR system of the present invention, be the basis with the RT-MPCR system of above-mentioned foundation, composition in the PCR system and concentration are studied.The fundamental principle of optimizing the PCR system is: obtain clear, the stable special product of target; Cost is lower; Reaction times is short etc.
The main factor that influences composite PCR comprises: pairing and competitive amplification, annealing temperature and primer concentration between the every pair of primer amplification clip size, primer design, primer.
2.1 the optimization of annealing temperature
Renaturation temperature (annealing temperature) is that the success or failure that influence the PCR test have very important influence.
This experiment has adopted Techne company's T C-512 grads PCR appearance that the annealing temperature of response procedures is groped.According to primer Tm value, be provided with 53,54,55,56,57,58,59,608 thermogrades and confirm optimum annealing temperature.
The result shows that annealing temperature is little to the expanding effect influence of four pairs of special primers, all can obtain expanding effect (Fig. 2) preferably at 55～58 ℃.
1.2 the optimization of reaction system
According to five factors, four horizontal L16 (4
5) orthogonal experiment is to other factors of influencing PCR reaction such as template concentrations, the amount of Taq archaeal dna polymerase, primer concentration, dNTPs concentration, Mg
2+Deng being optimized, specifically see table 4.
Table 4PCR reaction system design factor and water-glass
The result shows, carries out in the PCR reaction of CarMV, CNFV, CRSV, the compound RT-MPCR detection of four kinds of Dianthovirus quadruples of CLV, and the amount of cDNA template is 1.5 μ g; The Taq archaeal dna polymerase is 1.25U; DNTPs is 2.5mM and 3.0mM, and primer concentration is 0.8～1.2 μ M, Mg
2+Concentration can be got effect preferably during for 1.5mM.
Because the amount of primer is given birth to needed result for well with minimum primer volume production; Primer concentration higher meeting causing mispairing and non-specific amplification and increase dimeric chance; So the concentration of primer is selected 0.8 μ M, in like manner dNTPs also gets the minimum concentration 2.5mM that can increase.So it is following to get optimum reaction system:
The compound RT-PCR system of quadruple after table 5 is optimized
The PCR response procedures: 95 ℃ of preparatory sex change 3min, 94 ℃ of sex change 30sec then, 57 ℃ of annealing 30s, 72 ℃ are extended 30s, 32 circulations, 72 ℃ are extended 5min, at last in 4 ℃ of end.
Also carried out substance and two-fold, triple PCR amplification in the test respectively, response procedures is the same.
Can obtain expanding effect preferably through electrophoresis, as shown in Figure 3, explain that this suboptimization system can detect four kinds of viruses of oeillet effectively.
The MULTIPLE COMPOSITE RT-MPCR system that the present invention set up can be from diluting 10
6Obtain special amplified production in the RNA crude extract doubly, system is stable, and fast sensitive, whole process 7～8h can accomplish.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from know-why of the present invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (8)
1. four kinds of viral synchronous detection primers of oeillet comprise:
CarMV upstream primer: ACGCTCACCTCGCCAATTTGCTT
CarMV downstream primer: TCCGCAGTCCGCACATTCCTACT;
CRSV upstream primer: AATCCAAAATGGCGCTGGTGCCT
CRSV downstream primer: CATCAGTGAAACGCGACCGCTCA;
CLV upstream primer: TGTTGGGCTGTCGCACGTTACTG
CLV downstream primer: GGTGGGGATTTGGCGAACAGCAT;
CNFV upstream primer: TGGAGTGCGTCACCTTGAGTGGT
CNFV downstream primer: GTCGAAAGTGCCGCCTCCGAAAT.
2. four kinds of viral synchronization detecting methods of oeillet is characterized in that, comprise the steps:
1) total RNA of extraction oeillet;
2) RT-MPCR: after the RNA reverse transcription that obtains in the step 1),, and carry out electrophoresis detection with the described primer amplification reverse transcription product of claim 1.
3. detection method according to claim 2 is characterized in that, total RNA of described extraction oeillet carries out as follows:
1) takes by weighing 0.05 ~ 0.1g oeillet spire or petal to mortar; Under liquid nitrogen, be ground into superfine powder, powder be transferred to rapidly in the 1.5mL centrifuge tube of precooling, add behind 0.8 ~ 1.2mLTrizol with the abundant mixing of eddy mixer; Under room temperature, leave standstill 5min, make its abundant cracking;
2) 13000rpm, 4 ℃ of centrifugal 10 ~ 15min suct clearly, add the chloroform that isopyknic volume ratio is 24:1: primary isoamyl alcohol, the abundant mixing of eddy mixer leaves standstill 2 ~ 3min;
3) 12000rpm, 4 ℃ of centrifugal 10 ~ 15min suct clearly extremely new centrifuge tube, add the Virahol of 0.7 times of volume, the abundant mixing of eddy mixer ,-70 ℃ leave standstill 10 ~ 30min;
4) 12000rpm, 4 ℃ of centrifugal 10 ~ 15min abandon supernatant, add 300 ~ 500 μ L, 75% ethanol and wash, gentle vibration centrifuge tube, the deposition that suspends, 7500rpm, 4 ℃ of centrifugal 5 ~ 10min abandon supernatant;
5) 300 ~ 500 μ L, 75% ethanol repeated washing once, room temperature is dried or vacuum-drying 5 ~ 10min, does not have the distilled water dissolving RNA sample of RNase with 50 ~ 100 μ L, and is subsequent use in-70 ℃ of preservations.
4. detection method according to claim 2 is characterized in that, the PCR reaction system of described amplification reverse transcription product is:
The PCR response procedures is: 95 ℃ of preparatory sex change 3min; 94 ℃ of sex change 30s then, 53 ℃ ~ 60 ℃ annealing 30s, 72 ℃ are extended 30s, 32 circulations; 72 ℃ are extended 5min, at last in 4 ℃ of end.
5. the four kinds of viral synchronous detection test kits of oeillet that contain the said primer of claim 1.
6. test kit according to claim 5 is characterized in that, also comprises ThermoScript II and reaction buffer thereof, Taq archaeal dna polymerase and reaction buffer thereof, dNTPs, OligodT, RNase suppressor factor and does not have the distilled water of RNase.
7. the application of the described primer of claim 1 in four kinds of viral synchronous detection of oeillet.
8. the application of the described test kit of claim 5 in four kinds of viral synchronous detection of oeillet.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100265000B1 (en) * | 1996-06-17 | 2000-09-01 | 이구택 | Oligomer set for diagnosis of carnation mottle virus infection and method for diagnosing virus infection using the same |
| CN1680444A (en) * | 2005-01-12 | 2005-10-12 | 浙江大学 | Monoclone antibody against virus of xiangshizhubanbo and its use |
| CN1952648A (en) * | 2005-10-19 | 2007-04-25 | 中华人民共和国北京出入境检验检疫局 | A gene chip, nucleotide sequence and reagent kit for detecting virus of leguminous plant |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100265000B1 (en) * | 1996-06-17 | 2000-09-01 | 이구택 | Oligomer set for diagnosis of carnation mottle virus infection and method for diagnosing virus infection using the same |
| CN1680444A (en) * | 2005-01-12 | 2005-10-12 | 浙江大学 | Monoclone antibody against virus of xiangshizhubanbo and its use |
| CN1952648A (en) * | 2005-10-19 | 2007-04-25 | 中华人民共和国北京出入境检验检疫局 | A gene chip, nucleotide sequence and reagent kit for detecting virus of leguminous plant |
Non-Patent Citations (4)
| Title |
|---|
| H. P. Singh et al.Characterization of an Indian isolate of Carnation mottle virus infecting carnations.《RESEARCH ARTICLES》.2005,第88卷(第4期),594-601. * |
| 孔宝华等.香石竹斑驳病毒的鉴定和RT-PCR检测.《植物保护》.2002,第28卷(第1期),5-8. * |
| 杨雷亮等.香石竹斑驳病毒属和潜隐病毒属的PCR检测方法研究.《检验检疫学刊》.2009,第19卷(第2期),1-3. * |
| 王舒藜等.改良Trizol法快速高效提取香石竹总RNA.《分子植物育种》.2010,第8卷(第5期),987-990. * |
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