CN101210271A - Potato virus and viroid detecting kit and application thereof - Google Patents
Potato virus and viroid detecting kit and application thereof Download PDFInfo
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Abstract
The invention discloses a RT-PCR test kit for combined detection of potato virus and viroid and an application thereof. The inventive test kit performs combined reverse transcription and polymerase chain reaction with specific a primer composition, and can be used for rapid, accurate, sensitive and synchronous detection of potato virus X, potato virus Y, potato virus A, potato virus S, potato leaf roll virus and potato spindle tuber viroid. The inventive test kit introduces mRNA from potato gene as an internal control for monitoring the combined detection process of potato virus and viroid, solves the false negative problem during combined RT-PCR detection of potato virus and viroid, and uses a positive control without infection activity for monitoring kit reagent quality. The inventive test kit has the advantages of simple and rapid operation and low cost; and can be widely used for in door detection and field detection of virus-free potato seed and seedling, identification and screening of antiviral materials, and plant inspection and quarantine.
Description
Technical field
The present invention relates to a kind of test kit, relate in particular to the compound RT-PCR detection kit of a kind of potato virus and viroid, the invention still further relates to the preparation method and the utilisation technology working specification of this test kit, belong to plant virus check, quarantine field.
Background technology
Potato (Solanum tuberosum L.) is the higher cash crop of China's using value, and cultivated area ranks first in the world, and area has reached 6,600 ten thousand mu, 5609.6 ten thousand tons of ultimate productions.Potato suitable cultivation zone extensively, but virus disease has had a strong impact on the yield and quality of potato, cause that in China the main virus of potato degeneration has corium solani (Potato leafroll virus, PLRV), potato virus X (Potato virusX, PVX), marmor upsilon (Potato virus Y, PVY), potato virus S (Potato virus S, PVS), marmor solani (Potato virus A, PVA) and potato spindle tuber viroid (Potato spindle tuberviroid, PSTVd), and majority be multiplicity of infection.
Potato has been listed in one of China 7 big crops, also be most important crop varieties of western part of China and main column support type industry, growth momentum is good, but China's potato per unit area yield has only 1/3 of the average per unit area yield of developed country at present, and potato virus is to cause low, the ropy major cause of potato yield.Generally adopt virus-free seed potato to prevent and treat the harm of virus disease on producing at present, for guaranteeing the detoxification quality of potato primary stock, original seed, foundation is quick, accurate, sensitive, potato virus viroid detection technique is the key technique that seed potato is produced efficiently, also is pressing for of Potato Industry benign development.
It mainly is the ELISA detection kit that academy of agricultural sciences, Shandong and international potato research centre provide that domestic potato virus detects, this kind of enzyme connection immunoassay technology depends on antiserum(antisera), the most dependence on import of serum, the price height, the needs that are not suitable for producing, and the low and poor accuracy of the sensitivity of immunology detection technology, application has bigger limitation on producing, and has greatly limited China's seed potato and has produced popularizing of virus detection and application; ELISA can accurately not detect PLRV and the PVY virus in the dormancy stem tuber simultaneously, needs breaking dormancy before detection; In addition, for not having immunogenic PSTVd viroid, can't use elisa technique to detect at all.Therefore press for a kind of comprehensive, quick, easy, sensitive, cheap detection technique on producing.
Polysaccharase connection formula reaction (Polymerase chain reaction, PCR) detect the characteristics that have highly sensitive, high specificity and reach good stability accurately and reliably of comparing with ELISA, the revolution that is considered to virus detection techniques is used on many plant viruses.Common potato virus is single strand RNA virus mostly, needs will to carry out pcr amplification after the viral RNA reverse transcription more earlier, i.e. inverse transcription polymerase chain reaction (Reverse-transcriptionPolymerase Chain Reaction, RT-PCR) technology.Along with the continuous development of Protocols in Molecular Biology, in recent years by the single RT-PCR technical development that once can only detect a kind of virus to can synchronous detection two kinds of double PCRs (d-RT-PCR) to multiple virus, compound RT-PCR (m-RT-PCR) detect.Compound RT-PCR detection technique can detect multiple virus and viroid simultaneously, when improving the accuracy and sensitivity that detects greatly, also greatly reduce detection cost, shortened detection time, become direction and main flow that potato virus viroid RT-PCR detects.Canada scientist Nie etc. once inquired into the compound RT-PCR that carries out main virus of potato and viroid with random primer and detects and study, and health etc. has also been carried out relevant research among the domestic king.But they do not propose the way that effectively solves to the problem of the false negative result that exists in the viral testing process, fail to make up 5 kinds of viruses and a kind of viroid that a kind of test kit detects potato simultaneously.5 kinds of viruses and a kind of viroid RT-PCR detection simultaneously and rapidly of research potato, solve the problem that is prone to false negative result in the RT-PCR testing process, solve the problem that detects potato virus and viroid simultaneously, solve toxicity-removing white potato restriction on industry development problem and all have great importance.
Summary of the invention
Technical problem to be solved by this invention is to overcome existing detection method can not detect potato virus and viroid simultaneously, detects the cost height, the problem that the time is long; Overcome the false negative problem in the RT-PCR detection, a kind of new potato virus and the compound RT-PCR detection kit of viroid are provided, detect potato virus and viroid simultaneously, and quick, easy, sensitive, cheap.
Technical problem to be solved by this invention is achieved through the following technical solutions:
The compound RT-PCR detection kit of a kind of potato virus and viroid, form by component I and component I I, component I is made up of the necessary reagent place that with potato virus and viroid mRNA reverse transcription is cDNA the 1st chain, comprise: reversed transcriptive enzyme, RNA enzyme inhibitors, the dNTP mixture, the complementary primer of cDNA the 1st chain of detected object, cDNA the 1st chain reaction damping fluid; Component I I comprises: PCR reaction buffer, dNTP mixture, MgCl by being to touch the necessary reagent place that plate carries out pcr amplification to form with cDNA the 1st chain
2, Taq archaeal dna polymerase, composite PCR combination of primers etc.;
The mixture that the primer of synthetic cDNA the 1st chain of described detected object is made up of according to arbitrary proportion the complementary primer in 7 pairs of primers: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14.
Described PCR composite primer combination is made up of following 7 pairs of primers: SEQ ID NO:1, SEQ IDNO:2 (primer is to 1); SEQ ID NO:3, SEQ ID NO:4 (primer is to 2) SEQ ID NO:5, SEQID NO:6 (primer is to 3); SEQ ID NO:7, SEQ ID NO:8 (primer is to 4); SEQ ID NO:9, SEQ ID NO:10 (primer is to 5); SEQ ID NO:11, SEQ ID NO:12 (primer is to 6) and SEQ IDNO:13, SEQ ID NO:14 (primer is to 7).
Preferably, above-mentioned 7 pairs of primers are formed according to following ratio: primer is to 1: primer is to 2: primer is to 3: primer is to 4: primer is to 5: primer is to 6: primer is to 7=1: 1: 1: 1: 1: 1: 0.5.
Wherein, also can comprise the deionized water that has or not the RNA enzyme in the component I; The mixture that also can include corium solani (PLRV), potato virus X (PVX), marmor upsilon (PVY), potato virus S (PVS) A, marmor solani (PVA) and potato spindle tuber viroid (PSTVd) among the component I I, be positive control sample, this positive control does not have the activity of infecting.
In the mentioned reagent box, the PCR primer can not be from the purchase of biological reagent company obtains among the primer of synthetic cDNA the 1st chain and the component I I in component I, remaining reagent or enzyme can both be bought from domestic and international common biological reagent company and obtain, and wherein said reversed transcriptive enzyme is preferably the M-MLV reversed transcriptive enzyme.
In the component I among the primer of synthetic cDNA the 1st chain and the component I I PCR primer can according to the nucleotide sequence of disclosed each primer in the sequence table with its synthetic after, again it is mixed according to aforementioned proportion, being mixed with concentration is the Nucleotide mixed solution of 50 μ M.
The amount of each component can be according to practical situation (for example: the quantity of sample to be checked) decide among component I and the component I I.For example, the amount of each component can be that the consumption of finishing 1 cDNA the 1st chain synthesis reaction also can be a consumption of finishing repeatedly cDNA the 1st chain synthesis reaction in the component I, equally, the amount of each component can be that the consumption of finishing 1 PCR reaction also can be a consumption of finishing repeatedly the PCR reaction among the component I I, and these all are that those skilled in the art is easy to grasp or design.
As a kind of reference, the compound RT-PCR detection kit of potato virus of the present invention and viroid, in the component I among the amount of each reagent and the component I I amount of each reagent can assemble according to following consumption:
Component I: the primer 0.5-5000 μ L of synthetic cDNA the 1st chain of 50 μ M
5 * cDNA the 1st chain reaction damping fluid 1-5000 μ L
40U μ L
-1RNA enzyme inhibitors 0.5-5000 μ L
10mmol·L
-1 dNTPs 0.2-5000μL
200U μ L
-1M-MLV ThermoScript II 0.5-5000 μ L
Component I I 10 * PCR reaction buffer 1-5000 μ L
25mM MgCl2 0.1-5000μL，
10mM dNTPs 0.1-5000μL，
50 μ M PCR primer 0.1-5000 μ L
Taq archaeal dna polymerase 0.1-5000 μ L.
Positive control 1-5000 μ L
After the independent packing of above-mentioned all ingredients, be positioned in the test kit, be kept in-20 ℃ of refrigerators, promptly get test kit of the present invention.Wherein, all ingredients in the component I is wanted strict deactivation RNA enzyme, and the container that is used for packing component I all ingredients also will accomplish not have the pollution of RNA enzyme.
The using method of test kit of the present invention, can carry out with reference to following working specification:
1. synthetic cDNA the 1st chain:
Cause of disease sample RNA to be measured (10-100ng/ μ L) 5 μ L
The primer 1 μ L of 50 μ M cDNA the 1st chain
88℃，10min，0℃5min；
Add following reagent again:
5 * cDNA the 1st chain reaction damping fluid, 6 μ L
RNA enzyme inhibitors (40U μ L
-1) 1 μ L
dNTPs(10mmol·L-1) 2μL
ThermoScript II (200U μ L-1) 1.5 μ L
The deionized water of adding no RNA enzyme is up to reacting cumulative volume to 30 μ L;
Reaction conditions: 37 ℃ of insulation 10min behind the mixing, 42 ℃ of reaction 1.5h, promptly synthetic cDNA the 1st chain;
2. carry out composite PCR reaction (positive control is set simultaneously) by following component:
CDNA the 1st chain product 5 μ L,
10 * PCR reaction buffer, 5 μ L,
25mM MgCl2 1μL，
10mM dNTPs 1μL，
Taq archaeal dna polymerase 1.4 μ L,
Add the sterilization distilled water and supply 50 μ L
The pcr amplification condition:
94℃ 3min；
94 ℃ of 50s, 54 ℃ of 30s, 72 ℃ of 1.0min, 35 circulations;
72℃ 10min。
3.5% polyacrylamide gel electrophoresis and decision method:
Reaction is got 10 μ L PCR products through 5% polyacrylamide gel electrophoresis observations after finishing, and positive control each all occurs and detects band.During test sample,, then be healthy sample if N2 190bp fragment person only occurs; If the DNA of no 190bp takes out of existing, then is the false negative sample, need proceed to detect and determine; If 190bp fragment and other fragments occur simultaneously, then are susceptible sample, wherein the amplified band of PVX is 520bp, and the amplified band of PVY is 420bp, and PVA is 410bp, and PSTVd is 360bp, and PVS is 310bp, and PLRV is 270bp.
Used primer is that this studies special design in the RT-PCR detection kit of the present invention, can detect the not homophyletic system and the differentiation easily of virus, do not have the phase mutual interference between them,, increased the sensitivity that detects because the design of primers aspect has reduced the length of amplified fragments.
The present invention at first starts with from potato virus and viroid gene order clone and homology analysis, at first set up the single RT-PCR detection technique of these cause of diseases, and the PCR product cloned and sequence homology analysis, obtain not have and infect active positive control, for compound RT-PCR special primer in the design test kit of the present invention provides sequence information, designed primer specificity is strong, and fully take into account the genome mutation of virus strain, from the most conservative sequence section design special primer of genome, and not mutual complementary disturbance reponse between the primer of different virus, can detect the not homophyletic system of virus and viroid, there be not complementary the interference between the primer, and the primer amplification stripe size is distinguished easily, but multiple 5 kinds of main viruses and the a kind of viroid of infecting potato of synchronous detection.
Existing detection technique system aspect does not have effectively to solve the problem of the false negative result that exists in the compound RT-PCR testing process.The mRNA that has introduced the potato gene group in the RT-PCR detection kit of the present invention monitors whole potato virus viroid RT-PCR process as internal reference, has fundamentally solved the problem that is prone to false negative result in the detection of potato virus and viroid.
This test kit is the RT-PCR detection kit of RNA that is used to detect the virus of PLRV, PVY, PVX, PVS, PVA and PSTVd, can detect these viruses and viroid simultaneously and rapidly, adopt two one step RT-PCR methods, be used for qualitative detection PLRV, PVY, PVX, PVS, PVA and PSTVd.This test kit has added not have and has infected the internal reference RNA of active positive control and plant, is used for internal reference experiment and positive control test, can prevent false positive and false-negative appearance.
Test kit of the present invention has characteristics with low cost, compare with the enzyme connection adsorption measurement (ELISA) that China uses at present, not only improved potato detection technique level greatly, the sensitivity of Jian Ceing has simultaneously obtained increasing substantially, agents useful for same is except the M-MLV ThermoScript II is imported product, remaining reagent is domestic reagent, has saved cost and fund greatly, for the detection of China's potato virus, viroid provide fast, accurately, sensitive, high-efficiency method.
Utilize the detection of a large amount of samples of test kit of the present invention and detoxification test tube plantlet susceptible naturally, prove fully and can carry out quick test the potato virus viroid to the field; Simultaneously potato primary stock, original seed and seedling are carried out detoxification check, can specific detection go out the kind of main virus.Compare with traditional ELISA and two-dimensional electrophoresis method, this technology has higher stability, sensitivity and accuracy as a result.Detection kit of the present invention is easy and simple to handle, quick, cost is low, and indoor detection, the production field that can be widely used in toxicity-removing white potato potato seed and seedling detected, the aspects such as evaluation, screening and plant inspection quarantine of antitoxin viral material.
Description of drawings
The specificity of Fig. 1 internal reference primer N2 is specially changed the RNA amplified production
1:DNsae handles back RT-PCR; 2:DNsae handles back PCR; 3: the direct RT-PCR of sample; 4: the direct PCR of sample.
Figure 25 % native polyacrylamide gel electrophoresis detected result
1,3,5,10: blank; The 2:PLRV product; The 4:PVY product; 6: be two amplified productions of PLRV and PVY; 7:200bp DNA marker; Two amplified productions of 8:N2 and PLRV; Single amplified production of two amplified production .11:N2 of 9:PVY and N2
Fig. 3 is that the internal reference dual RT-PCR detects PLRV and PVY result with the N2 subunit gene
M:PCR marker (994bp, 697bp, 515bp, 377bp, 237bp); 1: blank; 2: normal healthy controls; 3-5: land for growing field crops potato sample.
Figure 45 % native polyacrylamide gel electrophoresis detected result
Two amplified productions of 1:N2 and PSTVd; The single amplified production of 2:PSTVd; 3: blank; M:DNAMarker.
The compound RT-PCR test kit of Fig. 5 the present invention test section sample result
1-7 number: the different sample detection results of collection, see the material part.The M:DNA molecular weight standard; J: normal healthy controls; K: blank.
The compound RT-PCR test kit of Fig. 6 the present invention test section sample result
8-19 number: the different sample detection results of collection, see the material part.The M:DNA molecular weight standard; J: normal healthy controls; K: blank; Z: biased sample multiple RT-PCR product
The test of Fig. 7 multiple RT-PCR detection sensitivity
The M:DNA molecular weight standard; K: blank; J: normal healthy controls; 1-6: extent of dilution test (1 *, 10 *, 100 *, 200 *, 300 *, 400 *).
Embodiment
Further describe the present invention by the following examples, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
Test materials
1, the susceptible sample of potato: gather in the susceptible sample segment of potato area from the dam, Zhangjiakou, and a part of in addition sample is that Hebei Hui Gu Science and Technology Ltd. carries out obtaining when the toxicity-removing white potato seedling detects.These samples are through the single RT-PCR technology for detection of potato virus and viroid, and the kind of its band poison is through determining (table 1).Wherein sample is for No. 2 the tobacco leaf of multiplicity of infection PVA, PVS and PVX, is so kind as to give by professor Wang Zhongkang of University Of Chongqing.
Table 1 different potato sample infective virus and viroid situation
| Sample number into spectrum | The position of drawing materials | The infective virus kind | Gather | Kind | |
| 1 | Blade | PLRV，PVX，PVY，PVS | Zhangjiakou | Open potato No. 7 | |
| 2 | Tobacco leaf | PVX，PVS， | Chongqing | Unknown | |
| 3 | Stem tuber | PSTVd | Zhangjiakou | The |
|
| 4 | Blade | PLRV，PVX，PVY | | Xia Bodi | |
| 5 | Stem tuber | PVX，PVY | Zhangjiakou | Da Bai early | |
| 6 | Detoxic seedling | PVX | Zhangjiakou | Da Bai early | |
| 7 | Blade | PLRV | Zhangjiakou | The |
|
| 8 | Blade | PLRV，PVS，PSTVd | Zhangjiakou | The |
|
| 9 | Detoxic seedling | PVY | Zhangjiakou | |
|
| 10 | Stem tuber | PLRV， | Zhangjiakou | Favorita | |
| 11 | Stem tuber | PLRV，PVX，PVY | Zhangjiakou | Xia Bodi | |
| 12 | Stem tuber | PLRV， | Zhangjiakou | Favorita | |
| 13 | Blade | PLRV，PVY，PVS，PSTVd | Zhangjiakou | Guyuan farmers' |
|
| 14 | Blade | PLRV，PVX，PVY，PVS | Zhangjiakou | Open potato No. 7 | |
| 15 | Blade | Do not have | Zhangjiakou | Open potato 90-2-10 | |
| 16 | Stem tuber | PVX，PSTVd | Zhangjiakou | Restrain new No. 1 |
| 17 | Detoxic seedling | Do not have | Zhangjiakou | No. 2, |
|
| 18 | Stem tuber | PLRV， | Zhangjiakou | Favorita | |
| 19 | Stem tuber | PLRV，PVY | Shijiazhuang | Rheum officinale early | |
| 20 | Stem tuber | PLRV | Zhangjiakou | Potato is opened No. 3 in the Ji | |
| 21 | Blade | PLRV，PVS，PVA | Zhangjiakou | Guyuan farmers' kind | |
| 22 | Blade | PLRV | Zhangjiakou | Guyuan farmers' kind | |
| 23 | Detoxic seedling | Do not have | Shijiazhuang | Rheum officinale early | |
| 24 | Blade | PLRV，PVX，PVY | Zhangjiakou | Shachen City farmers' kind | |
| 25 | Stem tuber | PLRV，PVX，PVY | Zhangjiakou | Shachen City farmers' kind | |
| 26 | The detoxification stem tuber | Do not have | Shijiazhuang | Rheum officinale early | |
| 27 | Blade | PLRV，PVY | Shijiazhuang | Rheum officinale early | |
| 28 | Blade | PLRV，PVX，PVS | Zhangjiakou | Shachen City farmers' kind | |
| 29 | Blade | PLRV，PVX | Zhangjiakou | Shachen City farmers' kind |
2, agents useful for same: RNA enzyme inhibitors (Rnasin) is available from precious biotech firm, and the M-MLV ThermoScript II is available from Progmega company, and Taq archaeal dna polymerase, dNTP etc. are available from Beijing ancient cooking vessel state Bioisystech Co., Ltd.Other various chemical reagent are available from Beijing chemical reagents corporation.
3, primer sequence:
The compound RT-PCR of table 2 potato virus and viroid detect used primer to and the amplification size
| Virus and viroid | Primer sequence number | The primer amplification district | The amplification size |
| PLRV | SEQ ID NO:1 and SEQ ID NO:2 | CP | 270 |
| PVY | SEQ ID NO:3 and SEQ ID NO:4 | CP | 429 |
| PVX | SEQ ID NO:5 and SEQ ID NO:6 | CP | 527 |
| PVS | SEQ ID NO:7 and SEQ ID NO:8 | CP | 314 |
| PVA | SEQ ID NO:9 and SEQ ID NO:10 | CP | 415 |
| PSTVd | SEQ ID NO:11 and | Comp lete gene | 360 |
| SEQID NO：12 | |||
| N2 | SEQ ID NO:13 and SEQ ID NO:14 | Nad2gene | 192 |
The specificity test of test example 1 internal reference primer
N2 primer (SEQ IDNO:13 and the SEQ ID NO:14) performance of design is to the specific amplification of specialization of RNA, after carrying out reverse transcription after the total RNA of the sample that extracts handles through DNase, pcr amplification goes out a special band, and does not have any band without the step pcr amplification of reverse transcription.The NAD2 gene primer amplifies the special band (Fig. 1) of 190bp.
The present invention is incorporated into internal reference in the RT-PCR detection kit, the N2 gene of higher plant extensively is present in the organs such as root, stem, leaf, flower, fruit and seed of plant, and this gene has very high conservative property in higher plant, utilize the N2 primer that three lives cigarette, common cigarette, Nicotiana glutinosa, tomato, eggplant and capsicum etc. have been carried out the RT-PCR test, the result also can be special amplify the purpose fragment, this explanation N2 gene has certain conservative property in plant, can be used as the internal reference that plant RNA virus detects.
The double check of test example 2 PLRV and PVY
To multiplicity of infection viral sample (the table 1: sample label 10) carried out double check of PLRV and PVY, PAGE electrophoresis through 5% shows (Fig. 2), N2 special primer (SEQ ID NO:13 and SEQ IDNO:14) can amplify the specific fragment of a treaty 190bp, PLRV special primer (SEQ ID NO:1 and SEQID NO:2) can amplify the specific fragment of a treaty 310bp, the special primer of PVY (SEQ ID NO:3 and SEQ ID NO:4) then amplifies the fragment of about 420bp, and this is big or small consistent with Design Theory.
When carrying out compound RT-PCR, carried out composite amplification with combinations such as N2 primer and PLRV composite primer, N2 and PVY composite primer, PVY and PLRV composite primer respectively, the result all amplifies purpose band (Fig. 3).
Test example 3 internal reference RT-PCR detect PSTVd
Sample (the table 1 of stem tuber will be extracted, sample label 6) total RNA carries out dual RT-PCR, PAGE electrophoresis through 5% shows, can amplify the specific nucleotide fragment of a treaty 190bp with the N2 special primer, and the PSTVd special primer can amplify the specific nucleotide fragment of a treaty 360bp.When carrying out dual RT-PCR, the result amplifies the band of two clauses and subclauses, as shown in Figure 4.
The compound RT-PCR test kit of test example 4 the present invention detects potato virus and viroid
One, potato infective virus material: collected potato infective virus material detects through ELISA and single RT-PCR, confirms the kind (table 1) of infective virus.
Two, detection kit: the compound RT-PCR test kit of the present invention
Three, detection method
1. extract high-quality total RNA (for example: the RNA of each biotech company extracts test kit, and perhaps modified CTAB method is extracted RNA etc.) of sample according to art methods.
2. total RNA 5 μ L of sample, the complementary primer 1 μ L of cDNA the 1st chain of 20 μ mol/L.88 ℃ of sex change 10min, cooled on ice 3-5min; And then add following reagent: 20 μ mol/L RNasin, 1 μ L, 5 * buffer, 6 μ L, dNTPs (10mmolL-1) 2 μ L, M-MLV ThermoScript II 1.5 μ L (200U μ L-1), DEPC water is supplied 30 μ L, 37 ℃ of insulation 10min behind the mixing, 42 ℃ of reaction 1.5h, promptly synthetic cDNA first chain.
3, PCR multiplex amplification: (1) 10 * PCR buffer 5 μ L, 25mM MgCl
21 μ L, 10mM dNTPs1 μ L, the PCR composite primer makes up 1 μ L, and Taq archaeal dna polymerase 1.4 μ L add the sterilization distilled water and supply 50 μ L, add a mineral oil, and 88 ℃ of sex change are after 5～10 minutes; (2) add cDNA the 1st chain product 5 μ L, carry out the PCR cyclic amplification then;
Pcr amplification condition: 94 ℃ of 3min; 94 ℃ of 50s, 54 ℃ of 30s, 72 ℃ of 1.0min, 35 circulations; 72 ℃ of 10min;
4. polyacrylamide gel electrophoresis is analyzed the composite PCR amplified production, and silver dyes polyacrylamide gel;
5. electrophoresis result analysis:, then be healthy sample if 190bp fragment person only occurs; If the DNA of no 190bp takes out of existing, then is the false negative sample, need proceed to detect and determine; If 190bp fragment and other fragments occur simultaneously, then are susceptible sample, wherein the amplified band of PVX is 520bp, and the amplified band of PVY is 420bp, and PVA is 410bp, and PSTVd is 360bp, and PVS is 310bp, and PLRV is 270bp.
Four, detected result:
Detected result is seen Fig. 5 and Fig. 6.
Detected result shows, with test kit test sample of the present invention, and virus that can detect sample accurately and infected and the kind of viroid.The specificity of the designed primer of the present invention is very strong, only the special band to virus increases in compound RT-PCR detects, and the primer between different virus and the viroid does not have interference, and the special band of each cause of disease all can increase out, and the special band of internal reference also can increase out clearly.The gene fragment that will the increase size of the band of amplification and size and desired design is consistent, and the segmental intensity that different virus increases is different, and this may be that degree is different to be caused because virus is matched with the gene order of virus at the different or viral primer sequence of the intravital concentration of host.
With the fragment cloning that amplified and measure sequence, the compound RT-PCR product that the order-checking proof is increased is the specific fragment of virus and viroid, the fragment of the 190bp of N2 primer amplification is the sophisticated mRNA part fragment of the gene of potato own, and is consistent with desired design sequence of interval size.
The sensitivity test of the multiple detection of test example 5 test kits of the present invention
In order to determine to detect the sensitivity of potato virus and viroid based on the multiple RT-PCR kit of the present invention of internal reference, to having infected PLRV, PVS, the sample (table 1) of PVY has carried out sensitivity test, the sample RNA (100ng) that extracts is diluted 1 respectively *, 10 *, 100 *, 200 *, 300 *, 400 * the gradient standard, carry out multiple RT-PCR with test kit of the present invention and detect, the result shows under 200 * (0.3-0.5ng) extent of dilution all bands still visible (Fig. 7).But the segmental intensity of different virus amplification is different, and this may be because the initial concentration of sample RNA is different or virus matches with the gene order of virus at the different or viral primer sequence of the intravital concentration of host that degree is different to be caused.
Sequence table
＜110〉Hebei Academy of Agriculture and Forestry Science, Economic Crops Institute
＜120〉potato virus and viroid detecting kit and application thereof
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<213>Solanum tuberosum L
<400>14
taagatcata gaagcaatgc tgc 23
Claims (8)
1. the compound RT-PCR detection kit of potato virus and viroid is made up of component I and component I I; The independent packing of each reagent among component I and the component I I, component I is made up of the essential reagent place that with potato virus or viroid RNA reverse transcription is cDNA the 1st chain, comprising: reversed transcriptive enzyme, RNA enzyme inhibitors, dNTP mixture, the complementary primer of cDNA the 1st chain of detected object, reaction buffer; Component I I is made up of the essential reagent place that carries out pcr amplification, comprising: PCR reaction buffer, dNTP mixture, MgCl
2, Taq archaeal dna polymerase, the combination of PCR composite primer;
It is characterized in that: the complementary primer of cDNA the 1st chain of described detected object is by following 7 mixtures that primer is formed according to arbitrary proportion: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14;
Described PCR composite primer combination is made up of following 7 pairs of primers: SEQ ID NO:1, SEQ IDNO:2 (primer is to 1); SEQ ID NO:3, SEQ ID NO:4 (primer is to 2); SEQ ID NO:5, SEQID NO:6 (primer is to 3); SEQ ID NO:7, SEQ ID NO:8 (primer is to 4); SEQ ID NO:9, SEQ IDNO:10 (primer is to 5); SEQ IDNO:11, SEQ IDNO:12 (primer is to 6); SEQ IDNO:13, SEQ ID NO:14 (primer is to 7).
2. according to the RT-PCR test kit of claim 1, it is characterized in that: the proportion of composing of 7 pairs of primers is in the combination of described PCR composite primer: primer is to 1: primer is to 2: primer is to 3: primer is to 4: primer is to 5: primer is to 6: primer is to 7=1: 1: 2: 1: 2: 2: 0.5.
3. according to the RT-PCR test kit of claim 1, it is characterized in that: the consisting of of component I: the primer 0.5-5000 μ L of synthetic cDNA the 1st chain of 50 μ M, reaction buffer 1-5000 μ L, RNA enzyme inhibitors 0.5-5000 μ L, 10mmolL
-1DNTPs 0.2-5000 μ L, 200 U μ L
-1M-MLV ThermoScript II 0.5-5000 μ L; Component I I consists of: PCR reaction buffer 1-5000 μ L, 25mM MgCl
20.1-5000 μ L, 10mM dNTPs 0.1-5000 μ L, 50 μ M PCR composite primer combination 0.1-5000 μ L, TaqDNA polysaccharase 0.1-5000 μ L.
4. according to the RT-PCR test kit of claim 1, it is characterized in that: the consisting of of component I: the primer 0.5-5000 μ L of synthetic cDNA the 1st chain of 50 μ M, reaction buffer 1-5000 μ L, 40U μ L
-1RNA enzyme inhibitors 0.5-5000 μ L, 10mmolL
-1DNTPs 0.2-5000 μ L, 200U μ L
-1M-MLV ThermoScript II 0.5-5000 μ L; Component I I consists of: PCR reaction buffer 1-5000 μ L, 25mM MgCl
20.1-5000 μ L, 10mM dNTPs 0.1-5000 μ L, 50 μ M PCR composite primer combination 0.1-5000 μ L, Taq archaeal dna polymerase 0.1-5000 μ L.
5. according to the RT-PCR test kit of claim 1, it is characterized in that: the deionized water that also comprises no RNA enzyme in the component I; The nothing that also includes corium solani, potato virus X, marmor upsilon, potato virus S, marmor solani and potato spindle tuber viroid mixture among the component I I infects active positive control.
6. the application of the RT-PCR test kit of claim 1 in detecting corium solani, potato virus X, marmor upsilon, potato virus S, marmor solani and potato spindle tuber viroid comprises: (1) uses potato sample RNA to be checked the reagent of component 1 according to synthetic cDNA the 1st chain of the reaction conditions reverse transcription of routine; (2) cDNA the 1st chain is carried out pcr amplification with the reagent of component I I according to the reaction conditions of routine; (3) result judges: pcr amplification product is carried out 5% polyacrylamide gel electrophoresis; If sample has only amplified 190 bp fragments, then be healthy sample; Not taking out of now if sample has the DNA of 190 bp, then is the false negative sample, need proceed to detect and determine; If in the sample amplification product, 190 bp fragments and other fragment occur simultaneously, it then is susceptible sample, wherein the amplified band of potato virus X is 520 bp, the amplified band of marmor upsilon is 420 bp, and the amplified band of marmor solani is 410 bp, and the amplified band of potato spindle tuber viroid is 360 bp, the amplified band of potato virus S is 310 bp, and the amplified band of corium solani is 270 bp.
7. according to the application of claim 6, it is characterized in that the reverse transcription described in the step (1) is to synthesize cDNA the 1st chain according to following reaction conditions: (1) at first adds the complementary primer 1 μ L of cDNA the 1st chain of the detected object of total RNA 5 μ L of sample and 20 μ mol/L; 88 ℃ of sex change 10min, cooled on ice 3-5min; (2) add following reagent again: 20 μ mol/L RNA enzyme inhibitorss, 1 μ L, 5 * reaction buffer, 6 μ L, dNTPs2 μ L, M-MLV ThermoScript II 1.5 μ L, DEPC water is supplied 30 μ L, 37 ℃ of insulation 10min behind the mixing, 42 ℃ of reaction 1.5h.
8. according to the application of claim 6, it is characterized in that the pcr amplification condition described in the step (2) is as follows: (1) 10 * PCR reaction buffer 5 μ L, 25mM MgCl
21 μ L, 10mM dNTPs 1 μ L, the PCR composite primer makes up 1 μ L, and Taq archaeal dna polymerase 1.4 μ L add the sterilization distilled water and supply 50 μ L, add a mineral oil, 88 ℃ of sex change 5～10 minutes; (2) add cDNA the 1st chain product 5 μ L, carry out the PCR cyclic amplification; Pcr amplification condition: 94 ℃ of 3min; 94 ℃ of 50s, 54 ℃ of 30s, 72 ℃ of 1.0min, 35 circulations; 72 ℃ of 10min.
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| CNA200610156123XA CN101210271A (en) | 2006-12-27 | 2006-12-27 | Potato virus and viroid detecting kit and application thereof |
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