CN100368567C - Two step method multiple RT-PCR solidified detection reagent kit for potato virus disease and its detecting method - Google Patents
Two step method multiple RT-PCR solidified detection reagent kit for potato virus disease and its detecting method Download PDFInfo
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Abstract
The present invention relates to a two-step method and a detection kit. The two-step method is used for synchronously detecting potato viruses Y and potato leaf roll viruses in a duplex RT-PCR mode and synchronously detecting potato viruses X, potato viruses A and potato viruses S in a ternary RT-PCR mode. The method of the present invention is established for fast and synchronously detecting two or three kinds of viruses specially aiming at five kinds of potato viruses with large influence on potato industry, the detection time is only 4h, and the method of the present invention has the advantages of high sensitivity, strong specificity, high stability and high reliability. The method of the present invention overcomes the defects that a conventional diagnostic method for detecting potato virus diseases according to the semeiology in the prior art has high misdetection rate, a serological method has the difficulty in detecting viruses in dormancy potatoes and detecting phloem viruses of potato leaf roll viruses with low virus content, etc. The method of the present invention is suitable for the early diagnosis of various virus diseases of potato seedlings and potato seeds, and has the important effect on monitoring the potato seed quality and preventing the virus diseases.
Description
Technical field
The present invention relates to a kind of Agricultural biotechnologies, specifically, relate to the multiple potato virus of a kind of synchronous amplification simply, immobilization kit for detecting nucleic acid fast, be suitable for departments such as plant protection, agricultural product quality technical supervision and use.
Background technology
Potato is global high-yield crop, and the cultivated area and the output of China all rank first in the world.Potato virus disease is important restraining factors during potato produces, and is the major cause that causes the potato deterioration of strains, has had a strong impact on output and the quality of potato.In China, several main virus of harm potato and viroid are potato virus X (being called for short PVX), marmor upsilon (being called for short PVY), corium solani (being called for short PLRV), marmor solani (being called for short PVA), potato virus S (being called for short PVS), and wherein the most serious virus of harm is PLRV, PVY.During potato produced, several different potato virus diseases often mixed generation in different areas.Potato virus disease does not usually have symptom or symptom to mix in early days, have only when virus disease certain symptom takes place just to show when serious, the symptom of various virus performances is difficult to again distinguish, and accurately identify the viral species that potato infects, and light causes erroneous judgement easily with observation of symptoms.China potato generally adopts the potato seed detoxification technology to prevent the harm of virus disease on producing at present, detoxification quality in order to ensure potato primary stock, original seed, accurately identify potato seed virus disease kind, be badly in need of a kind of more sensitive and method of inspection accurately in the seed potato production.Traditional biological detection method requires high to professional technique, and wastes time and energy; General immunological method is difficult to the few phloem virus of detection level as the virus in PLRV and the dormancy potato seed.Advantages such as that the polymerase chain reaction of detecting based on nucleic acid molecule (polymerase chain reaction is hereinafter to be referred as PCR) has is highly sensitive, high specificity, accuracy are good demonstrate powerful advantage day by day in the detection of virus.Because common potato virus mostly is strand justice RNA (ssRNA) virus, so needs in the actual detected will to carry out pcr amplification after the viral RNA reverse transcription more earlier.Traditional biological detection method requires high to professional technique, and wastes time and energy; General immunological method is difficult to the few phloem virus of detection level as the virus in PLRV and the dormancy potato seed.Detect round pcr based on nucleic acid molecule and have advantages such as highly sensitive, high specificity, accuracy are good, in the detection of virus, demonstrate powerful advantage day by day.
The present invention is directed to the field often is that 2～3 kinds of potato viruses cause compound infecting, with conventional reverse transcription polymerase chain
Formula reaction (hereinafter to be referred as RT-PCR) detects needs multi-pass operations, detects the cost height; And single stage method RT-PCR method detects multiple potato virus, and the disadvantage that the vying each other property restraining effect between the multiple primer of adding causes detection sensitivity to reduce adopts two-step approach dual RT-PCR synchronous detection marmor upsilon, corium solani; Triple RT-PCR synchronous detection potato virus Xs, marmor solani, potato virus S can be guaranteed detecting delicately simultaneously and rapidly of micro-potato virus nucleic acid.Be applicable to early stage evaluation seed potato original silkworm egg and original seed detoxification efficiency, potato seed carries the Rapid identification of Virus Type.Utilize immobilization nucleic acid two-step approach RT-PCR test kit and supporting sample preparation, detection technique, be fit to the evaluation and the monitoring of multiple virus of the compound potato of infecting in field and biography virulent aphis worm carrier state, time saving and energy saving, can reduce the detection cost.
Find in potato planting areas such as Chongqing, Sichuan that by field investigation in 2 years and Molecular Detection potato virus disease is compound the infecting of 2-3 kind virus usually, presses for economy multiple detection method easily, but do not detect the PSTVd viroid.Potato virus is a strand justice RNA viruses (ssRNA), and utilizing nucleic acid molecule to detect needs at first the viral RNA reverse transcription to be cDNA, carries out pcr amplification (RT-PCR) again.Also there is certain limitation in the conventional potato virus nucleic acid molecule detection technique of having reported at aspects such as sample making technology, detected object, detection specificity and detection architecture optimizations.At present only external Rudra has discussed in the double RT-PCR reverse transcription reaction reagent concentration to the influence of PCR.At present potato virus molecule diagnosis kit domestic still do not have produce and there is poor specificity in a small amount of serology test kit of import, lack positive control, can not determine problems such as viral species, the quality detection technology of qualified seedling is difficult to guarantee, seriously restricts the application of virus-free seed potato and bring into play its yield potential.This research is intended optimizing by Auele Specific Primer screening, sample fast preparation method, steps such as the primer concentration ratio in multiple RT-PCR reverse transcription and the PCR reaction, reagent concentration, reaction condition optimization, set up a kind of molecular detection technology of multiple RT-PCR fast and accurately platform, in the hope of detecting multiple virus simultaneously, simplify procedures, reduce and detect cost, and lay the foundation for developing potato detection kit quick, easy, sensitive, high specificity.
Summary of the invention
Purpose of the present invention is intended to overcome above-mentioned the deficiencies in the prior art, and a kind of two step method multiple RT-PCR solidified detection reagent kit for potato virus disease and detection method thereof are provided.
For achieving the above object, technical scheme of the present invention is:
1. quote the Oligonucleolide primers that potato virus detects in the prior art, comprising:
Oligonucleolide primers is right: 5 '-TAGCACAACACAGGCCACAG-3 '
5 '-GGCAGCATTTCAGCTTC-3 ' (sequence number: NO.1);
Oligonucleolide primers is right: 5 '-ACGTCCAAAATGAGAATGCC-3 '
5 '-TGGTGTTCGGTGATGTGACCT-3 ' (sequence number: NO.2);
Oligonucleolide primers is right: 5 '-CGCGCTAACAGAGTTCAGCC-3 '
5 '-GCAATGGGGGTCCAACTCAT-3 (sequence number: NO.3);
Oligonucleolide primers is right: 5 '-GTTGGAGAATTCAAGATCCTGG 3 '
5 '-TTTCTCTGCCACCTCATCG 3 ' (sequence number: NO.4)
Oligonucleolide primers is right: 5 '-TGGCGAACACCGAGCAAATG-3 '
5 '-ATGATCGAGTCCAAGGGCACTG-3 ' (sequence number: NO.5).
Wherein NO.1 is used to the potato virus X that increases, and the amplified production size is 562bp; NO.2 is used to the marmor upsilon that increases, and the amplified production size is 480bp; NO.3 is used to the corium solani that increases, and the amplified production size is 336bp; NO.4 is used to the marmor solani that increases, and the amplified production size is 255bp, and NO.5 is used to the potato virus S that increases, and the amplified production size is 187bp.
2. preparation is used for the synchronous reverse transcription two-step approach dual RT-PCR immobilization test kit of compound marmor upsilon that infects in field (being called for short PVY) and corium solani (being called for short PLRV), is made up of following various detection reagent:
The sample viral RNA extracts reagent,
Viral RNA reverse transcription immobilization reagent,
CDNA amplification immobilization reagent,
Innoxious PVY and PLRV positive reference substance,
Healthy potato plant negative control product;
Its key is that it is 0.4～0.5% triton x-100 and 0.35～0.45%NaSO that described sample viral RNA extracts reagent
3Mixture at normal temperatures; Described innoxious PVY and PLRV positive reference substance are made by following steps:
(1) plant marmor upsilon, corium solani on healthy potato plant,
(2) extract marmor upsilon and corium solani is compound after infecting viral RNA,
(3) the upstream and downstream primer of 5 kinds of potato viruses of design amplification, the feature of upstream primer are that 5 ' end has the T7 promoter sequence,
(4) go dual RT-PCR increase PVY, PLRV dna fragmentation mixture,
(5) PVY, PLRV dna fragmentation mixture be template transcribe PVY, PLRV RNA fragment mixture;
Described viral RNA reverse transcription immobilization reagent, cDNA amplification immobilization reagent are respectively immobilization mixture freeze-drying glue pearls; Wherein, viral RNA reverse transcription immobilization reagent is made up of synchronous reverse transcription marmor upsilon of dual RT-PCR and corium solani, contains following component in this reagent:
The component final concentration
5x reverse transcription damping fluid 1X,
25mM MgCl
2 3mmol/L，
25mM dNTPs 1.0～1.5mmol/L，
40U/ μ L RNA enzyme inhibitors 5～10Unit/10uL,
200U/ μ L MMLV ThermoScript II 50～100Unit/10uL,
25 μ M PVY: PLRV antisense primer 0.30～0.50: 0.70～0.90,
25ng/ μ L testing sample or RNA template 1.0～2.5 μ L,
The biomacromolecule stablizer adds to 10 μ L,
Employed antisense primer is that Oligonucleolide primers is right:
5’-ACGTCCAAAATGAGAATGCC-3’
5 '-TGGTGTTCGGTGATGTGACCT-3 ' (sequence number No.2),
Right with Oligonucleolide primers: 5 '-CGCGCTAACAGAGTTCAGCC-3 '
5 '-GCAATGGGGGTCCAACTCAT-3; (sequence number No.3);
CDNA amplification immobilization reagent is made up of the double PCR reaction of synchronous amplification marmor upsilon and corium solani cDNA, contains following component in this reagent:
The component final concentration
10xPCR damping fluid 1X,
25mM MgCl
2 2.0～2.5mmol/L，
25mM dNTPs 0.4～0.5mmol/L，
5U/ μ L Taq warm start polysaccharase 1.5～2.5Unit/25uL,
25ng/ μ l PVY: PLRV (upstream and downstream primer to) 0.30～0.40: 0.15～0.30,
The biomacromolecule stablizer adds to 21 μ L,
Employed primer is that Oligonucleolide primers is right:
5’-ACGTCCAAAATGAGAATGCC-3’
5 '-TGGTGTTCGGTGATGTGACCT-3 ' (sequence number: NO.2),
Right with Oligonucleolide primers: 5 '-CGCGCTAACAGAGTTCAGCC-3 '
5 '-GCAATGGGGGTCCAACTCAT-3 (sequence number: NO.3),
Reverse transcription synthetic cDNA 4 μ L are as the template of PCR reaction.
3, preparation is used for the triple immobilization RT-PCR of the synchronous reverse transcription two-step approach test kit of the compound potato virus X virus that infects in field (being called for short PVX), marmor solani (being called for short PVA), potato virus S (being called for short PVS), is made up of following various detection reagent:
The sample viral RNA extracts reagent,
Viral RNA reverse transcription immobilization reagent,
CDNA amplification immobilization reagent,
Innoxious PVX, PVA and PVS positive reference substance,
Healthy potato plant negative control product;
Its key is that it is 0.4～0.5% triton x-100 and 0.35～0.45%NaSO that described sample viral RNA extracts reagent
3Mixture at normal temperatures; Described innoxious PVX, PVA and PVS positive reference substance are made by following steps:
(1) inoculation potato virus X, marmor solani, potato virus S on healthy potato plant,
(2) extract potato virus X virus, marmor solani, potato virus S is compound after infecting viral RNA,
(3) the upstream and downstream primer of 5 kinds of potato viruses of design amplification, the feature of upstream primer are that 5 ' end has the T7 promoter sequence,
(4) carry out triple RT-PCR increase PVX, PVA, PVS dna fragmentation mixture,
(5) with PVX, PVA, PVS dna fragmentation mixture be template transcribe PVX, PVA, PVS RNA
The fragment mixture is as the innoxious positive control of triple RT-PCR;
Described viral RNA reverse transcription immobilization reagent, cDNA amplification immobilization reagent are respectively immobilization mixture freeze-drying glue pearls; Wherein, viral RNA reverse transcription immobilization reagent is made up of triple RT-PCR marmor upsilons of synchronous reverse transcription and corium solani, contains following various component in this reagent:
The component final concentration
5X reverse transcription damping fluid 1X,
25mM MgCl
2 3mmol/L，
25mM dNTPs 1.5～2.0mmol/L，
40U/ μ L RNA enzyme inhibitors 5.0～10.0Unit/10uL,
200U/ μ L MMLV ThermoScript II 50～100Unit/10uL,
25 μ M PVX: PVA: PVS primer 0.30～0.40: 0.80～1.00: 0.80～1.00,
Innoxious positive reference substance 1.0～2.5 μ L of 25ng/ μ L,
25ng/ μ L testing sample or RNA template 1.0～2.5 μ L,
The biomacromolecule stablizer adds to 10 μ L,
Employed primer is that Oligonucleolide primers is right:
5’-TAGCACAACACAGGCCACAG-3’
5 '-GGCAGCATTTCAGCTTC-3 ' (sequence number: NO.1),
Right with Oligonucleolide primers: 5 '-GTTGGAGAATTCAAGATCCTGG 3 '
5 '-TTTCTCTGCCACCTCATCG 3 ' (sequence number: NO.4),
Right with Oligonucleolide primers: 5 '-TGGCGAACACCGAGCAAATG-3 '
5 '-ATGATCGAGTCCAAGGGCACTG-3 ' (sequence number: NO.5);
CDNA amplification immobilization reagent is made up of synchronous amplification potato virus X, marmor solani and the reaction of potato virus S cDNA triple PCR, contains following component in this reagent:
The component final concentration
10x PCR damping fluid 1X,
25mM MgCl
2 2.5～3.0mmol/L，
25mM dNTPs 0.4～0.5mmol/L，
5U/ μ L Taq warm start polysaccharase 1.5～2.5Unit/25 μ L,
25ng/μLPVX∶PVA∶PVS 0.35～045∶0.15～0.25∶0.10～0.20，
The biomacromolecule stablizer adds to 21 μ L,
Employed primer is that Oligonucleolide primers is right:
5’-TAGCACAACACAGGCCACAG-3’
5 '-GGCAGCATTTCAGCTTC-3 ' (sequence number: NO.1),
Right with Oligonucleolide primers: 5 '-GTTGGAGAATTCAAGATCCTGG 3 '
5 '-TTTCTCTGCCACCTCATCG 3 ' (sequence number: NO.4),
Right with Oligonucleolide primers: 5 '-TGGCGAACACCGAGCAAATG-3 '
5 '-ATGATCGAGTCCAAGGGCACTG-3 ' (sequence number: NO.5),
Reverse transcription synthetic cDNA 4 μ L are as the template of PCR reaction.
The immobilization mixture freeze-drying glue pearl of viral RNA reverse transcription immobilization reagent of the present invention, cDNA amplification immobilization reagent be utilize the biomacromolecule stablizer through vacuum lyophilization be prepared from can the normal temperature storing pcr amplification immobilization mixture freeze-drying glue pearl.And the biomacromolecule stablizer is 0.05-0.1% gelatin, 0.1～0.2% polysaccharide, 0.02～0.05%BAS by weightmeasurement ratio, and all the other mix for pure water.
4, be provided for the multiple viral RNA extracting method of potato, carry out according to the following steps:
(1) the special-purpose vat liquor of preparation potato virus RNA, promptly the sample viral RNA in the test kit extracts reagent, and it is by 0.4～0.5% triton x-100 and 0.35～0.45%NaSO
3At normal temperatures with the sterilization purified water mixing solutions of no RNA enzyme;
(2) blade 5～10mg, leaf stalk 20～30mg, stem 30～40mg or the stem tuber 50～60mg that adds the sample potato plant in 100 μ L potato virus RNA vat liquors respectively carries out lixiviate 30min under 37 ℃;
(3) the centrifugal 10min of 10000r/min gets supernatant and is directly used in RT-PCR.
5, possessing under the situation of above-mentioned 1,2,3,4, condition, the multiple RT-PCR detection method of the multiple potato virus of a kind of synchronous detection is provided, its step is as follows:
(1) right according to distinguished sequence design design potato virus X, marmor upsilon, corium solani, marmor solani, the potato virus S Auele Specific Primer of potato virus, this primer is respectively the 1st a described primer;
(2) adopt the multiple viral RNA of simple and easy extraction rapid extraction potato (i.e. the 4th described method), add PCR reaction supressor agent for releasing simultaneously; This agent for releasing is with parts by weight 0.3-0.5%NaSo3, and 0.01-0.1%Triton X-100 with the sterilization pure water of an amount of no RNA enzyme, mixes at normal temperatures and stirs gained solution;
(3), add marmor upsilon, corium solani specificity downstream primer and reverse transcription reaction component and carry out the synthetic cDNA of reverse transcription for dual RT-PCR;
(4), add potato virus X, marmor solani, potato virus S specificity downstream primer and reverse transcription component and carry out the synthetic cDNA of reverse transcription for triple RT-PCR; Carry out positive control and negative control simultaneously; Viral RNA reverse transcription immobilization reagent;
(5) with reverse transcription synthetic cDNA as template, dual RT-PCR add respectively marmor upsilon, corium solani Auele Specific Primer to and the PCR reactive component carry out pcr amplification; Triple RT-PCR add respectively potato virus X, marmor solani, potato virus S Auele Specific Primer to and the PCR reactive component carry out pcr amplification; Carry out quantitatively product of positive control and negative control simultaneously; Be cDNA amplification immobilization reagent in the test kit.
(6) amplified production is carried out gel electrophoresis, after the colour developing of GV fluorescence dye, on the gel imaging instrument, take a picture.The present invention obtains significantly success through experiment, and its part is tested and detected data and asks for an interview Fig. 1, Fig. 2 and table 1.As shown in Figure 1, use the dual RT-PCR detection and have potato virus PVX and PVY or PVA and PLRV; Show that detection architecture is special, sensitive.
As shown in Figure 2: use triple RT-PCR detections and have potato virus PVX, PVS and PVA; Show that triple detection architecture are accurately with special.
Table 1 is potato virus RT-PCR actual detected result.
As seen from Table 1, employing RT-PCR technology is carried out actual detected to the part potato sample of different sources, and the result shows that method proposed by the invention has high specificity, and is highly sensitive, the characteristics that accuracy is good.
Table 1 potato virus RT-PCR actual detected
| The potato sample | Sample source | The potato kind | Sample size | Potato virus | ||||
| PVX | PVA | PVS | PVY | PLRV | ||||
| Virus-free seed potato | The Chongqing WuLong | No. 3, Hubei Province potato | 9 | 1 | 0 | 3 | 0 | 0 |
| Yongchuan, Chongqing | In No. 2, potato in No. 3, the potato | 3 | 0 | 0 | 0 | 0 | 0 | |
| Wushan, Chongqing | No. 3, Hubei Province potato | 1 | 0 | 0 | 0 | 0 | 0 | |
| Jiangjin, Chongqing | No. 2, middle potato | 2 | 0 | 0 | 0 | 0 | 0 | |
| Chongqing Yu Bei | No. 3, middle potato | 9 | 0 | 0 | 0 | 0 | 0 | |
| Tissue cultured seedling | Chongqing Yu Bei | No. 3, Hubei Province potato | 39 | 0 | 0 | 0 | 0 | 0 |
| The Yaan, Sichuan | No. 3, middle potato | 34 | 0 | 2 | 0 | 0 | 0 | |
| Tissue cultural seedlings of free | Chongqing Yu Bei | No. 3, Hubei Province potato | 6 | 0 | 0 | 1 | 0 | 0 |
| Land for growing field crops potato seed plant | Yongchuan, Chongqing | No. 2, middle potato | 25 | 0 | 0 | 0 | 14 | 0 |
| The Yaan, Sichuan | No. 3, middle potato | 14 | 0 | 0 | 0 | 6 | 0 | |
| Chongqing Yu Bei | No. 4, Chongqing of Sichuan | 30 | 10 | 0 | 12 | 0 | 0 | |
| Land for growing field crops potato seed plant | Chongqing Yu Bei | The land for growing field crops conventional variety | 44 | 6 | 12 | 15 | 5 | 10 |
Adopt technique scheme, the technical progress that the present invention gives prominence to is:
1, can the synchronous detection marmor upsilon and the test kit of the immobilization test kit of corium solani and the triple RT-PCR synchronous detection of two-step approach potato virus X, marmor solani, potato virus S;
2, the present invention is based on Molecular Detection, utilize simple and easy quick microwave leaching technology of RNA and multiple RT-PCR technology, only needing micro-material directly to carry out RT-PCR to multiple potato virus detects, outstanding advantage just is fast, highly sensitive, high specificity, required test material is few, overcome existing to the semeiologic routine diagnostic method False Rate height of potato virus disease foundation, traditional biological detection method is wasted time and energy, general immunological method is difficult to the few phloem virus of detection level as the defectives such as virus in PLRV and the dormancy potato seed, and the sensitivity of single stage method multiple RT-PCR is low, unsettled shortcoming.
3, because the present invention can detect 2-3 kind potato virus in speciality aspect sensitivity and the specificity and while, therefore be particularly suitable for the Rapid identification of Virus Type in early stage Rapid identification seed potato original silkworm egg and original seed band poison situation and potato leaf, leaf stalk, stem and the stem tuber sample.
4, two step method multiple RT-PCR detection technique and immobilization test kit especially are fit to the evaluation and the monitoring of field multiple virus of compound potato of infecting and biography virulent aphis worm carrier state, and time saving and energy saving, can reduce the detection cost.
5, because RNA extraction time weak point of the present invention, shortened viral detection time, utilize starting materials to carry out RT-PCR now and detect and only to need 4-6 hour, be particularly suitable for potato primary stock, the original seed seedling carries out the detoxification check, to guarantee to organize the quality of training potato seed.
In sum, adopt the present invention, can carry out potato virus disease molecular biology identification or detection, the production and the quality surveillance of germchit of potato of taking off poison had vital role the various materials that obtained in seed potato seedling and the field investigation process.
Description of drawings
Fig. 1 uses dual RT-PCR amplification potato virus PVX and PVY; PVA and PLRV contrast figure, wherein 1 row: 100bp dna segment size criteria; 2 row: blank; 3-4 row: PVX and PVA; 5 row: blank; 6-7 row: PVY and PLRV; 8 row: negative control.
Fig. 2 is triple RT-PCR amplification potato virus detection figure, wherein 1 row: 100bp dna segment size criteria; The 2-5 row: RT-PCR detects PVX, PVA and PVS; 6 row: negative control.
Embodiment
Embodiment one, two-step approach two-fold RT-PCR synchronous detection marmor upsilon, corium solani solidified detection reagent kit and detection method.
One, the component of solidified detection reagent kit:
The sample viral RNA extracts reagent, PVY, PLRV viral RNA reverse transcription immobilization reagent, cDNA amplification immobilization reagent, innoxious PVY, PLRV positive control, healthy plant negative control.
Two, the preparation of immobilization test kit:
1, detection method comprises the steps:
1) extract viral RNA: the special-purpose vat liquor of preparation potato virus RNA (is 0.5% triton x-100 and contain final concentration 0.4%NaSO
3At normal temperatures with the mixed solution of the pure water of an amount of no RAN enzyme); Take out potato sample to be checked, blade 10mg, leaf stalk 30mg, the stem 40mg or the stem tuber 60mg that add the sample potato plant in 100 μ L potato virus RNA vat liquors respectively carry out lixiviate 30min under 37 ℃; The centrifugal 10min of 10000r/min gets supernatant liquor and is directly used in inverse transcription polymerase chain reaction.Mix 37 ℃ the time heating, stir 5 minutes gained solution;
2) two pairs of primers of design: a pair of Oligonucleolide primers is used to the marmor upsilon that increases: 5 '-ACGTCCAAAATGAGAATGCC-3 ', 5 '-TGGTGTTCGGTGATGTGACCT-3 ' (sequence number: NO.2); Another is used to the corium solani that increases to Oligonucleolide primers: 5 '-CGCGCTAACAGAGTTCAGCC-3 ', 5 '-GCAATGGGGGTCCAACTCAT-3 (sequence number: NO.3);
3) downstream primer among adding NO.2 and the NO.3 is in the reverse transcription reaction system, and the reverse transcription reaction system is: 1 * reverse transcription reverse transcription reaction damping fluid is 3mmol/L MgCl
21.5mmol/L dNTPs, 10 U RNA enzyme inhibitorss, 100 U MMLV ThermoScript II, antisense primer PVY: PLRV is 0.50: 0.90, the viral RNA 2.5 μ L that extract, the ultrapure water of biomacromolecule stablizer 0.4% gelatin, 0.2% polysaccharide, 0.05%BAS, no RNA enzyme complements to 10uL.Reaction system mixed place the pcr amplification instrument after centrifugal again (Bio-Rad USA) carries out the PCR reaction, and reaction uses water as blank, makes positive control with innoxious PVY and PLRV viral RNA, makes negative control with healthy potato plant.Response procedures is a reverse transcription reaction: 25 ℃ of 10min, 42 ℃ of 1h, 95 ℃ of 5min.
4) add NO.2 and NO.3 primer in the PCR reaction system, the pcr amplification reaction system is: the anti-record of 4 μ L synthetic cDNA is as the PCR reaction template, 1 * PCR damping fluid, 2.5mM/L MgCl
2, 0.5mM/L dNTPs, Taq archaeal dna polymerase 2.5U, just antisense primer is 0.40: 0.30 to PVY: PLRV; Biomacromolecule stablizer 0.05% gelatin, 0.1% polysaccharide, 0.03%BAS, deionized water complement to 25uL.With reaction system mix place again after centrifugal the quantitative pcr amplification instrument (Bio-Rad USA) carries out the PCR reaction, and the pcr amplification program is 94 ℃ of sex change 1min, 60 ℃ of renaturation 1min, 72 ℃ are extended 1min; Totally 30 circulations, last 72 ℃ of 10min.
5) amplified production detects: amplified reaction is got 10 μ l amplified productions and is added the sample-loading buffer that 1 μ l contains tetrabromophenol sulfonphthalein after finishing, and the agarose gel electrophoresis with 2% detects, damping fluid is 1 * TAE, 100V electrophoresis 30min, the GV fluorescent dye, the dna fragmentation size criteria adopts 100bp DNA ladder.Gel imaging system observation electrophoresis result and photographic recording.
2, detected result:
If detecting with potato virus disease RNA is that the positive control of template has the target band to produce, negative control and blank do not have the target band to produce, and test result of samples is consistent with the target band size of positive control, the band that promptly produces the 480bp size then is a PVY virus, the band that produces the 336bp size is PVY, if produce the band of 480bp, 336bp simultaneously, then be PVY, PLRV is compound to be infected.As Fig. 1, shown in Figure 2.
Embodiment two, the triple RT-PCR synchronous detection of two-step approach potato virus X, marmor solani, potato virus S solidified detection reagent kit and detection method.
One, the component of solidified detection reagent kit:
The sample viral RNA extracts reagent, PVX, PVA, PVS viral RNA reverse transcription immobilization reagent, cDNA amplification immobilization reagent, innoxious PVX, PVA, PVS positive control, healthy plant negative control.
Two, the preparation of immobilization test kit:
1, detection method comprises the steps:
1) extract viral RNA: the special-purpose vat liquor of preparation potato virus RNA (is 0.5%Triton X-100 and contain final concentration 0.4%NaSO
3At normal temperatures with the mixed solution of the pure water of an amount of no RAN enzyme); Take out potato sample to be checked, blade 5mg, leaf stalk 20mg, the stem 30mg or the stem tuber 50mg that add the sample potato plant in 100 μ L potato virus RNA vat liquors respectively carry out lixiviate 30min under 37 ℃, the centrifugal 10min of 10 000r/min gets supernatant liquor and is directly used in inverse transcription polymerase chain reaction.
2) two pairs of primers of design: a pair of Oligonucleolide primers is used to the potato virus X that increases: 5 '-TAGCACAACACAGGCCACAG-3 ', 5 '-GGCAGCATTTCAGCTTC-3 ' (sequence number: NO.1); A pair of Oligonucleolide primers is used to the marmor solani that increases: 5 '-GTTGGAGAATTCAAGATCCTGG 3 ', 5 '-TTTCTCTGCCACCTCATCG 3 ' (sequence number: NO.4); Another is used to the potato virus S that increases to Oligonucleolide primers: 5 '-TGGCGAACACCGAGCAAATG-3 ', 5 '-ATGATCGAGTCCAAGGGCACTG-3 ' (sequence number: NO.5).
3) downstream primer among adding NO.1, NO.4 and the NO.5 is in the reverse transcription reaction system, and the reverse transcription reaction system is: 1 * reverse transcription reaction damping fluid is 3mmol/L MgCl
21.5mmol/L dNTPs, 5U RNA enzyme inhibitors, 50～100U MMLV ThermoScript II, antisense primer PVX: PVA: PVS is 0.40: 0.80～1.00: 0.80, the viral RNA 1 μ L that extracts, the ultrapure water of biomacromolecule stablizer 0.08% gelatin, 0.2% polysaccharide, 0.02%BAS, no RNA enzyme complements to 10uL.Reaction system mixed place the pcr amplification instrument after centrifugal again (Bio-Rad USA) carries out the PCR reaction, and reaction uses water as blank, makes positive control with innoxious PVX, PVA and PVS viral RNA, makes negative control with healthy potato plant.Response procedures is a reverse transcription reaction: 25 ℃ of 10min, 42 ℃ of 1h, 95 ℃ of 5min.
4) add NO.1, NO.4 and NO.5 primer in the PCR reaction system, the pcr amplification reaction system is: 4 μ L reverse transcription synthetic cDNA are as the PCR reaction template, 1 * PCR damping fluid, 2.5mmol/L MgCl
2, 0.4mmol/L dNTPs, Taq archaeal dna polymerase 1.5 U, just antisense primer is 0.35: 0.15: 0.10 to PVX: PVA: PVS; Biomacromolecule stablizer 0.1% gelatin, O.2% polysaccharide, 0.04%BAS, deionized water complement to 25uL.With reaction system mix place again after centrifugal the pcr amplification instrument (Bio-Rad USA) carries out the PCR reaction, and the pcr amplification program is 94 ℃ of sex change 1min, 60 ℃ of renaturation 1min, 72 ℃ are extended 1min; Totally 30 circulations, last 72 ℃ of 10min.
5) amplified production detects: after amplified reaction finishes, get 10 μ l amplified productions and add the sample-loading buffer that 1 μ l contains tetrabromophenol sulfonphthalein, agarose gel electrophoresis with 2% detects, damping fluid is 1 * TAE, 100V electrophoresis 30min, the GV fluorescent dye adopts 100bp DNA ladder as the dna fragmentation size criteria.Gel imaging system observation electrophoresis result and photographic recording.
Detected result is judged:
If detecting with potato virus disease RNA is that the positive control of template has the target band to produce, negative control and blank do not have the target band to produce, and test result of samples is consistent with the target band size of positive control, the band that promptly produces the 562bp size then is a PVX virus, the band that produces the 255bp size is PVA, the band that produces the 187bp size is PVS, if produce the band of 562bp, 255bp and 187bp size simultaneously, then for PVX, PVA with PVS is compound infects.
Claims (1)
1. synchronous reverse transcription two-step approach dual RT-PCR immobilization test kit that is used for compound marmor upsilon that infects in field (be called for short PVY) and corium solani (abbreviation PLRV), form by following various detection reagent:
The sample viral RNA extracts reagent,
Viral RNA reverse transcription immobilization reagent,
CDNA amplification immobilization reagent,
Innoxious PVY and PLRV positive reference substance,
Healthy potato plant negative control product;
It is 0.4～0.5% triton x-100 and 0.35～0.45%NaSO that described sample viral RNA extracts reagent
3Mixture at normal temperatures; Described innoxious PVY and PLRV positive reference substance are made by following steps:
(1) inoculate marmor upsilon, corium solani on healthy potato plant,
(2) extract marmor upsilon and corium solani is compound after infecting viral RNA,
(3) the upstream and downstream primer of 5 kinds of potato viruses of design amplification, the feature of upstream primer are that 5 ' end has the T7 promoter sequence,
(4) carry out dual RT-PCR increase PVY, PLRV dna fragmentation mixture,
(5) with PVY, PLRV dna fragmentation mixture be template transcribe PVY, PLRV RNA fragment mixture;
Described viral RNA reverse transcription immobilization reagent, cDNA amplification immobilization reagent are respectively immobilization mixture freeze-drying glue pearls; Wherein, viral RNA reverse transcription immobilization reagent is made up of synchronous reverse transcription marmor upsilon of dual RT-PCR and corium solani, contains following component in this reagent:
The component final concentration
5x reverse transcription damping fluid 1X,
25mM MgCl
2 3～5mmol/L，
25mM dNTPs 1.0～1.5mmol/L，
40U/ μ L RNA enzyme inhibitors 5～10Unit/10uL,
200U/ μ L MMLV ThermoScript II 50～100Unit/10uL,
25 μ M PVY: PLRV antisense primer 0.30～0.50: 0.70～0.90,
25ng/ μ L testing sample or RNA template 1.0～2.5ng/ μ L
The biomacromolecule stablizer adds to 10 μ L
Employed antisense primer is that Oligonucleolide primers is right:
5’-ACGTCCAAAATGAGAATGCC-3’
5 '-TGGTGTTCGGTGATGTGACCT-3 ' (sequence number No.2),
Right with Oligonucleolide primers: 5 '-CGCGCTAACAGAGTTCAGCC-3 '
5 '-GCAATGGGGGTCCAACTCAT-3; (sequence number No.3);
CDNA amplification immobilization reagent is made up of the double PCR reaction of synchronous amplification marmor upsilon and corium solani cDNA, contains following component in this reagent:
The component final concentration
10xPCR damping fluid 1X,
25mM MgCl
2 2.0～2.5mmol/L，
25mM dNTPs 0.4～0.5mmol/L，
5U/ μ L Taq warm start polysaccharase 1.5～2.5Unit/25uL,
25n μ L PVY: PLRV upstream and downstream primer is to 0.30～0.40: 0.15～0.30,
The biomacromolecule stablizer adds to 21 μ L,
Employed primer is that Oligonucleolide primers is right:
5’-ACGTCCAAAATGAGAATGCC-3’
5 '-TGGTGTTCGGTGATGTGACCT-3 ' (sequence number: NO.2),
Right with Oligonucleolide primers: 5 '-CGCGCTAACAGAGTTCAGCC-3 '
5 '-GCAATGGGGGTCCAACTCAT-3 (sequence number: NO.3),
Reverse transcription synthetic cDNA4 μ L is as the template of PCR reaction;
It is characterized in that: described biomacromolecule stablizer is 0.05-0.1% gelatin, 0.1～0.2% polysaccharide, 0.02～0.05%BAS by weightmeasurement ratio, and all the other mix for pure water.
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