CN101186948B - Multiple PCR detection method for sugar-cane mosaic disease and ratoon stunting disease pathogeny - Google Patents
Multiple PCR detection method for sugar-cane mosaic disease and ratoon stunting disease pathogeny Download PDFInfo
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Abstract
The invention discloses a multiple PCR detecting method for pathogen of sugarcane mosaic disease and ratoon stunting disease, which is characterized in that (1), a sheathing leaf at the stem base portion of a sugarcane plant is employed as detecting sample. (2), reverse transcription primer and primer pair of PCR detection cpc1 and cpc2 and RSD primer pair F1 and F2 are designed, sugarcane mosaic virus and genetic fragment of pathogen of sugarcane ratoon stunting disease are amplified. (3), total nucleic acid is extracted, and the RNA thereof is turned into reverse transcription via cDNA. (4),reaction system and amplification program of the one-step method with multiple RT-PCR are designed, and the nucleic acid is amplified. (5), gel electrophoresis analysis and amplification results and specificity are employed, and simultaneously decuple progressive dilution is used to detect the sensibility of the one-step method with multiple RT-PCR. The detecting method provided by the invention accomplishes reverse transcription and PCR reaction and simultaneously detects pathogen of two diseases by extracting the total nucleic acid of sugarcane, which has the advantages of simple operation,economy of time, low cost, high specificity and sensibility and the like.
Description
Technical field
The present invention relates to a kind of detection method of sugarcane disease, mainly be at mosaic of sugarcane virus (Sugarcane mosaic virus, SCMV) and sugarcane ratoon stunting disease (Ratoon Stunting Disease, RSD) pathogenetic bacteria xylem Lai Shi bacillus xylem subspecies (Leifsonia xyli subsp.Xyli, detection method in the time of Lxx).
Background technology
Mosaic of sugarcane (Sugarcane mosaic virus, SCMV) and sugarcane ratoon stunting disease (Ratoon Stunting Disease, RSD) be the main disease that extensively takes place in two kinds of world wides, they are also quite general and serious in each sugarcane district of China, and all cultivation of sugar cane kinds all are subjected to harm in various degree.
Mosaic of sugarcane is caused that by corn mosaic virus corn mosaic virus is positive single strand RNA virus, is one of important member of marmor upsilon section Potyvirus, and virus strain is various, and it is bigger to make a variation.Its main harm sugarcane blade, the intracellular chloroplast(id) particulate of infected plant is grown and suppressed by virus, on number and volume, all be inferior to normal cell, make leaf texture's phenomenon that reveals the green, especially the most obvious with new leaf base symptom, symptom generally is yellowish green alternate irregular embedding line, streak or mottled.Scab length is not of uniform size, is covered with blade, the yellow of disease plant blade, and plant is downgraded, the minimizing of tillering.Part kind symptom when summer high temperature can disappear, promptly so-called " latent disease " phenomenon.The sick sugarcane of perennial root germinates slowly, poor growth, also can show stem canker: promptly water stain shape striped or patch take place in the stem crust, the first purpling redness of outer subcutis, cause necrosis eventually and shrinkage, because necrotic tissue and healthy tissue growth imbalance, cause the stem crust and pushed and break, form so-called stem canker, susceptible strain internode shortens.According to investigations, various places, the south China especially sickness rate of Guangxi upland sugarcane mosaic disease reach more than 30%, production loss 3%-50%.
The sugarcane ratoon stunting disease symptom is more hidden, easily mixes mutually with the infringement symptom that arid causes, and the cripetura of disease plant internode, quality deterioration, reducing sugar increases in the sugarcane juice, and the percent crystallization in massecuite of sucrose reduces.The average infection rate about 50% of sugarcane ratoon stunting disease sugarcane strain, the perennial root time limit longer sickness rate are higher.This disease causes the sugarcane underproduction 12%～37% usually, the underproduction is up to 60% under the situation of arid, sucrose divides reduction by 0.5% (absolute value), and perennial root weakens, and also is 100% infection ratoon stunting disease even anosis nontoxic health seedling used on producing after 5 years.
Prevent the generation of these two kinds of diseases and spread the sane development of sugarcane industry very important.SCMV initial stage outward appearance symptom is not obvious, and RSD does not then have special outward appearance symptom, and this has brought difficulty for these two kinds of diseases of diagnosis.Along with the utilization of sugarcane health seedling on sugarcane production, press for foundation can stablize, accurately, sensitive and can detect the detection method of these two kinds of cause of diseases simultaneously, it can be the production of sugarcane health seedling and application, the exchange of health cane germ plasm resource, kind allocation and transportation etc. effective monitoring means is provided.
Detection method to SCMV has at present: the sugarcane strain is mensuration, biological assay, Electronic Speculum diagnosis, determination of serology, making nucleic acid molecular hybridization, polymerase chain reaction detection methods such as (PCR) intuitively, and wherein PCR is a kind of comparatively sensitive and accurate detection method.Sugarcane RSD diagnostic techniques has experienced 4 stages such as observation of symptoms (cuing open stem inspects), phase-contrast microscopy, immunological technique and PCR rapid detection internally.Because specificity is not obvious, cuts open stem and inspect the Subsidiary Signs that can only infect RSD as detection; If bacteria containing amount hangs down then can not detect with phase microscope; Immunological technique detects that then existing has relatively high expectations to the Lxx number density (requires bacteria containing amount to reach 10
3～10
7More than the cells/ml) and false negative or the too high problem of false positive; Round pcr can detect the RSD that contains 1-10 thalline, is more sensitive and accurate detection method.The detection that sugarcane is carried out these two kinds of diseases at present needs in two steps, and PCR carries out: extract the total RNA of sugarcane and detect SCMV by the RT-PCR method again; Extract total DNA of sugarcane, detect RSD by PCR method again.Detection is comparatively loaded down with trivial details like this, and it is also very high to detect cost.
Summary of the invention
The multi-PCR detection method that the purpose of this invention is to provide a kind of mosaic of sugarcane and ratoon stunting disease cause of disease, it is that foundation can detect the single stage method multi-PCR detection method of these two kinds of cause of diseases simultaneously on the basis of SCMV RT-PCR and the single PCR detection architecture of RSD.
The present invention is an advantage of concentrating single stage method RT-PCR and multiplex PCR, on the basis of the existing single PCR detection architecture of RSD, it is right to be according to the China's Mainland dominant strain of SCMV that SCMV-A and other four strains conserved sequence district design reverse transcription primer that is SCMV-B, D, E, SC and PCR detect primer, foundation has also been optimized single stage method multiple RT-PCR reaction system and response procedures, has accelerated diagnosis speed and has saved testing cost again.
For achieving the above object, the technical solution adopted in the present invention: the characteristic distributions in the sugarcane plant according to corn mosaic virus and sugarcane ratoon stunting disease pathogenetic bacteria, determining to get sugarcane plants stems base portion band sheath blade is test sample, and this has guaranteed exhaustively to detect two kinds of cause of diseases that may exist simultaneously in material to be checked; It is right to be according to known mosaic of sugarcane virus China's Mainland dominant strain that SCMV-A and other four strains gene conserved sequence district design reverse transcription primer that is SCMV-B, D, E, SC and PCR detect primer, guarantees to detect the mosaic of sugarcane virus that China's Mainland and Taiwan Province cause mosaic of sugarcane; Right in conjunction with existing detection sugarcane ratoon stunting disease pathogenetic bacteria primer, the gene fragment of corn mosaic virus and sugarcane ratoon stunting disease cause of disease is increased; Utilize the total nucleic acid extracting method to extract total nucleic acid, and RNA reverse transcription is wherein become cDNA with RT-PCR reverse transcription test kit; Design the reaction system and the amplification program of this single stage method multiple RT-PCR, nucleic acid is increased, utilize gel electrophoresis analysis amplification and specificity, utilize ten times of dilution methods of going forward one by one to detect the susceptibility of this single stage method multiple RT-PCR simultaneously.
Advantage that the present invention is compared with prior art had and positively effect:
At present sugarcane being carried out these two kinds of diseases detects and need in two steps that PCR carries out: (1) is extracted the total RNA of sugarcane and is detected SCMV by the RT-PCR method again; (2) total DNA of extraction sugarcane detects RSD by PCR method again.Detection is comparatively loaded down with trivial details like this, and it is also very high to detect cost.And designed mosaic of sugarcane and the concentrated single stage method RT-PCR of multi-PCR detection method of ratoon stunting disease cause of disease and the advantage of multiplex PCR of the present invention, only need to extract the total nucleic acid of sugarcane, just can detect the cause of disease of these two kinds of diseases after in same PCR pipe, finishing reverse transcription simultaneously by a PCR reaction, have simple to operate, save time, cost is low, also has advantages such as higher specificity and susceptibility simultaneously.Gene fragment and the low energy of utilizing this detection method can specific amplified to go out corn mosaic virus and ratoon stunting disease cause of disease detect 10
-5The total nucleic acid template of ug, early diagnosis and field cause of disease investigation when can be used for SCMV and RSD, can be the production of sugarcane health seedling and application, the exchange of health cane germ plasm resource, kind allocation and transportation etc. effective monitoring means are provided, to the generation that prevents these two kinds of diseases with spread and the sane development of sugarcane industry all has crucial meaning, also the detection to other cause of disease of sugarcane also has certain reference value.
Embodiment
Below the present invention is described in further detail.
The multi-PCR detection method of mosaic of sugarcane that the present invention is designed and ratoon stunting disease cause of disease comprises the following steps:
1, on the sugarcane plant, determine the optimum detection sampling point:
Since SCMV and Lxx separately characteristic distributions in the sugarcane plant, in order to guarantee in material to be checked, can exhaustively detect whether have two kinds of cause of diseases, should be with sugarcane plants stems base portion band sheath blade to be detected as the material that extracts total nucleic acid.
2, be that SCMV-A and other four strains are that nucleic acid conserved sequence district design reverse transcription primer (5 '-CCTTCATCTGCATG-3 ') and the PCR detection primer of SCMV-B, D, E, SC (sequence number in the GenBank database is respectively U7354, U57355, U57356, U57357, D00948) is right: cpc1, cpc2 according to known corn mosaic virus China's Mainland dominant strain; The RSD primer is to being F1, F2; The gene fragment size that is used for augmentation detection mosaic of sugarcane virus is 400bp, and upstream primer cpc1 is 5 '-GAGTTTGATAGGTGGTATGAAGCC-3 ', and downstream primer cpc2 is 5 '-CCTTCATCTGCATGTGGGC-3 '; The primer of gene fragment of Lxx of being used to increase is existing primer: upstream primer F1 is 5 '-CTGGCACCCTGTGTTGTTTTC-3 ', and downstream primer F2 is 5 '-TTCGGTTCTCATCTCAGCGTC-3 ', and the Lxx gene fragment size of amplification is 265bp;
3, the extraction of total nucleic acid and cDNA's is synthetic:
Utilize the RNAplant test kit to extract the sugarcane total nucleic acid: to get the susceptible sample of about 0.1g, liquid nitrogen grinding adds the extraction reagent of 4 ℃ of 0.5ml to the powder, vibration is to thorough mixing, room temperature is placed behind the 5min in 4 ℃ of centrifugal 10min of 12000rpm, add 0.1ml 5MNaCl after supernatant being changed over to the centrifuge tube of new no Rnase, gentleness is mixed and adds the 0.1ml chloroform again, and mixing, 4 ℃ of centrifugal 10min of 12000rpm then for several times turn upside down, get and add isopyknic Virahol again after the upper strata water changes new no Rnase centrifuge tube over to, mixing, 10min, 4 ℃ of centrifugal 10min of 12000rpm are placed in the greenhouse, abandon supernatant, add 1ml 75% ethanol, 4 ℃ of centrifugal 3min of 5000rpm abandon supernatant, the of short duration centrifugal rifle head sucking-off liquid of using then, room temperature is dried 2-3min, and adding 50ul does not have Rnase water dissolution (adding 2ul RNasin Inhibitor), in-70 ℃ of preservations.
Utilize RNA reverse transcription test kit to carry out the synthetic of cDNA: in the PCR pipe, add following sample: cool off 2min on total nucleic acid sample 4ul, the Nuclease-free Water10ul of extraction, SCMV reverse transcription primer 1ul, the rearmounted ice baths of 70 ℃ of heat shock 5min, in pipe, continue to add Nuclease-free Water 1.5ul, 5 * RT Reaction Buffer 5ul, dNTP Mixtuer 1.25ul, RNasin 1.25ul, M-MLV RT1ul, with 42 ℃ of insulations of reaction tubes 60min, termination reaction, reaction solution is exactly the cDNA template in the PCR reaction.
4, the amplification of single stage method multiple RT-PCR:
Set up the single stage method multiple RT-PCR: by the comparison of differential responses condition pcr amplification effect, this single stage method multiple RT-PCR reaction system is designed to the reaction pattern with " 5+20 ": i.e. and reverse transcription thing 5ul+PCR reaction reagent 20ul, carry out reverse transcription and multiplex PCR in same PCR pipe.The reverse transcription reaction system is: 5 * RT Reaction Buffer 1ul, reverse transcription primer (RT) 0.5ul, dNTPs 0.5ul, total nucleic acid extract 1ul, RNAsafe0.25ul, M-MLV Reverse Transcriptase 0.25ul, Nuclease-free Water5ul.After finishing, reaction in same PCR pipe, adds 20ul PCR reaction reagent immediately: Nuclease-free Water 12.35ul, 10 * PCR Buffer 2.5ul, dNTP Mixter2ul, each 1ul of RSD upstream and downstream primer, each 0.5ul of SCMV upstream and downstream primer, Taq archaeal dna polymerase 0.15ul.The pcr amplification program is 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 20s, 53 ℃ of annealing 30s, 72 ℃ of 45s circulate 30 times; Last 72 ℃ are extended 5min.
5, agarose gel electrophoresis is observed amplification:
Prepare 1% sepharose with the TAE electrophoretic buffer, in the TAE electrophoresis chamber, carry out horizontal strip electrophoresis, add the 10ul pcr amplification product, reverse transcription thing with the health cane total nucleic acid is the negative contrast of PCR product of template, with DL2000Marker is standard molecular weight, 100v/85mA electrophoresis 40 minutes, take electrophoresis result with gel imaging system, if amplified band explanation RSD occurs at the 265bp place positive, if amplified band explanation SCMV occurs at the 400bp place positive, if 265bp and 400bp amplified band explanation RSD all appears and SCMV all positive, no amplified band illustrates that RSD and SCMV are all negative.
6, single stage method multiple RT-PCR specificity and sensitivity Detection:
Single stage method multiple RT-PCR sensitivity Detection: the single infection sample of RSD, SCMV that is positive with spectrophotometric determination and the nucleic acid of polyinfection, dilute by ten times of dilution methods of going forward one by one, respectively get 1ul then and carry out the single stage method multiplex PCR, by the gel electrophoresis observations, find out the lowest critical value of the used nucleic acid of the visible special electrophoretic band of naked eyes, draw the susceptibility of single stage method multiple RT-PCR according to this.
Claims (1)
1. the multi-PCR detection method of mosaic of sugarcane and ratoon stunting disease cause of disease is characterized in that: comprise the following steps:
1), on the sugarcane plant, determines the optimum detection sampling point: with the material of sugarcane plants stems base portion band sheath blade to be detected as the extraction total nucleic acid;
2) be that the nucleic acid conserved sequence district design reverse transcription primer that SCMV-A and other four strains are SCMV-B, D, E, SC is that 5 '-CCTTCATCTGCATG-3 ' and PCR detection primer are right: cpc1, cpc2 according to known corn mosaic virus China's Mainland dominant strain; The RSD primer is to being F1, F2; The gene fragment size that is used for augmentation detection mosaic of sugarcane virus is 400bp, and upstream primer cpc1 is 5 '-GAGTTTGATAGGTGGTATGAAGCC-3 ', and downstream primer cpc2 is 5 '-CCTTCATCTGCATGTGGGC-3 '; The primer of gene fragment of Lxx of being used to increase is existing primer: upstream primer F1 is 5 '-CTGGCACCCTGTGTTGTTTTC-3 ', and downstream primer F2 is 5 '-TTCGGTTCTCATCTC AGCGTC-3 ', and the Lxx gene fragment size of amplification is 265bp;
3), the extraction of total nucleic acid and cDNA's is synthetic:
Utilize the RNAplant test kit to extract the sugarcane total nucleic acid: to get the susceptible sample of about 0.1g, liquid nitrogen grinding adds the extraction reagent of 4 ℃ of 0.5ml to the powder, vibration is to thorough mixing, room temperature is placed behind the 5min in 4 ℃ of centrifugal 10min of 12000rpm, add 0.1ml 5MNaCl after supernatant being changed over to the centrifuge tube of new no Rnase, gentleness is mixed and adds the 0.1ml chloroform again, and mixing, 4 ℃ of centrifugal 10min of 12000rpm then for several times turn upside down, get and add isopyknic Virahol again after the upper strata water changes new no Rnase centrifuge tube over to, mixing, 10min, 4 ℃ of centrifugal 10min of 12000rpm are placed in the greenhouse, abandon supernatant, add 1ml 75% ethanol, 4 ℃ of centrifugal 3min of 5000rpm abandon supernatant, the of short duration centrifugal rifle head sucking-off liquid of using then, room temperature is dried 2-3min, and adding 50ul does not have the Rnase water dissolution, add 2ul RNasin Inhibitor, in-70 ℃ of preservations;
Utilize RNA reverse transcription test kit to carry out the synthetic of cDNA: in the PCR pipe, add following sample: cool off 2min on the total nucleic acid sample 4ul of extraction, Nuclease-free Water 10ul, SCMV reverse transcription primer 1ul, 70 ℃ of rearmounted ice baths of heat shock 5min, in pipe, continue to add Nuclease-free Water 1.5ul, 5 * RT Reaction Buffer 5ul, dNTP Mixtuer 1.25ul, RNasin 1.25ul, M-MLV RT1ul, with 42 ℃ of insulations of reaction tubes 60min, termination reaction, reaction solution is exactly the cDNA template in the PCR reaction;
4), the amplification of single stage method multiple RT-PCR:
Set up the single stage method multiple RT-PCR: by the comparison of differential responses condition pcr amplification effect, this single stage method multiple RT-PCR reaction system is designed to the reaction pattern with " 5+20 ": i.e. and reverse transcription thing 5ul+PCR reaction reagent 20ul, carry out reverse transcription and multiplex PCR in same PCR pipe; The reverse transcription reaction system is: 5 * RT Reaction Buffer 1ul, reverse transcription primer RT 0.5ul, dNTPs 0.5ul, total nucleic acid extract 1ul, RNAsafe 0.25ul, M-MLV Reverse Transcriptase 0.25ul, Nuclease-free Water 5ul; After finishing, reaction in same PCR pipe, adds 20ul PCR reaction reagent immediately: Nuclease-free Water 12.35ul, 10 * PCR Buffer 2.5ul, dNTP Mixter2ul, each 1ul of RSD upstream and downstream primer, each 0.5ul of SCMV upstream and downstream primer, Taq archaeal dna polymerase 0.15ul; The pcr amplification program is 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 20s, 53 ℃ of annealing 30s, 72 ℃ of 45s circulate 30 times; Last 72 ℃ are extended 5min;
5), agarose gel electrophoresis is observed amplification:
Prepare 1% sepharose with the TAE electrophoretic buffer, in the TAE electrophoresis chamber, carry out horizontal strip electrophoresis, add the 10ul pcr amplification product, reverse transcription thing with the health cane total nucleic acid is the negative contrast of PCR product of template, with DL2000Marker is standard molecular weight, 100v/85mA electrophoresis 40 minutes, take electrophoresis result with gel imaging system, if amplified band explanation RSD occurs at the 265bp place positive, if amplified band explanation SCMV occurs at the 400bp place positive, if 265bp and 400bp amplified band explanation RSD all appears and SCMV all positive, no amplified band illustrates that RSD and SCMV are all negative;
6), single stage method multiple RT-PCR specificity and sensitivity Detection:
Single stage method multiple RT-PCR sensitivity Detection: the single infection sample of RSD, SCMV that is positive with spectrophotometric determination and the nucleic acid of polyinfection, dilute by ten times of dilution methods of going forward one by one, respectively get 1ul then and carry out the single stage method multiplex PCR, by the gel electrophoresis observations, find out the lowest critical value of the used nucleic acid of the visible special electrophoretic band of naked eyes, draw the susceptibility of single stage method multiple RT-PCR according to this.
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| CN102363819A (en) * | 2011-11-08 | 2012-02-29 | 云南省农业科学院甘蔗研究所 | Primer for detecting sugarcane streak mosaic virus (SCSMV) and detection method thereof |
| CN107805675B (en) * | 2017-11-17 | 2021-05-11 | 黑龙江省农业科学院克山分院 | Potato virus detection kit |
| CN108728581B (en) * | 2018-06-26 | 2021-02-05 | 广西大学 | Multiple RT-PCR method for simultaneously detecting 5 sugarcane viruses, primers and kit thereof |
| CN110951923A (en) * | 2020-01-02 | 2020-04-03 | 云南省农业科学院甘蔗研究所 | Multiplex PCR method for simultaneously detecting four sugarcane seed-borne viruses, primers and kit thereof |
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| CN1281958C (en) * | 2004-04-15 | 2006-10-25 | 云南省农业科学院生物技术研究所 | Cane mosaic virus Yunnan isolate TAS-ELISA test kit and its preparing method |
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| CN1281958C (en) * | 2004-04-15 | 2006-10-25 | 云南省农业科学院生物技术研究所 | Cane mosaic virus Yunnan isolate TAS-ELISA test kit and its preparing method |
Non-Patent Citations (4)
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| 李利君 等.,.利用斑点杂交法和RT-PCR技术检测甘蔗花叶病毒.福建农业大学学报29 3.2000,29(3),342-345. |
| 李利君等.,.利用斑点杂交法和RT-PCR技术检测甘蔗花叶病毒.福建农业大学学报29 3.2000,29(3),342-345. * |
| 邓展云 等.,.广西甘蔗宿根矮化病菌的PCR检测.西南农业学报17 3.2004,17(3),324-327. |
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