CN102453755A - Method and application for quantitatively detecting quantity of late blight bacteria in soil - Google Patents
Method and application for quantitatively detecting quantity of late blight bacteria in soil Download PDFInfo
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- CN102453755A CN102453755A CN2010105220832A CN201010522083A CN102453755A CN 102453755 A CN102453755 A CN 102453755A CN 2010105220832 A CN2010105220832 A CN 2010105220832A CN 201010522083 A CN201010522083 A CN 201010522083A CN 102453755 A CN102453755 A CN 102453755A
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Abstract
The invention provides a method and application for quantitatively detecting the quantity of potato late blight bacteria in soil, in particular to a method for quantitatively detecting the quantity of live late blight bacteria in potato growing soil by using the common PCR (Polymerase Chain Reaction) technology and the Real Time PCR technology and through specific primers 5'-CGGCGGCTGCTGGCTTTAT-3' and 5'-GCTCAGACCGAAGTCCAAACG-3'. According to the method provided by the invention, the quantity of potato late blight bacteria in soil can be quantitatively detected.
Description
Technical field
The present invention relates to the method and the application of phytophthora infestans content of molds in a kind of detection by quantitative soil, can quantitative, special detection soil in late blight of potato germ, belong to agricultural biological technical field.
Background technology
The late blight of potato is a kind of destructive disease of yam, and oneself classifies the first disease of world food crop as.Late blight generally takes place in the potato planting district, and the field production loss reaches 20%～50%, stores sth. in a cellar loss and can reach 5%～30%, the time even can cause total crop failure of being very popular.In recent years, the popular frequency and the popularity degree of late blight increase the weight of gradually, become the significant obstacle that yam produces.Yam is the fourth-largest food crop (Reader in the world; 2009); It has been first big country that produces of yam in the world that the loss that world's late blight of potato causes has reached 6,700,000,000 dollars of (Haverkort et al., 2008) China, China annual because of the late blight loss up to 1,000,000,000 dollars.
The late blight of potato, mainly by phytophthora infestans (
Phytophthora infestans) cause.This germ mainly with the in spite of illness stem tuber of form in soil of oospore and zoospore, passes the winter in the undesirable root, gets over the summer.Under the adapt circumstance condition, late disease bacteria produces sporocyst and propagates the plant around infecting with air-flow.As long as thereby soil contains the some amount germ or plants the potato seed of carrying disease germs of only a few down, generation (Andrivon, 1995 that may cause full wafer ground late blight; Zwankhuizen et al., 1998).Therefore plantation yam soil late disease bacteria content of molds is carried out quick specific detection; The generation of late blight and popular is predicted and prevented and treated particularly crucial; It also is the major measure (Zwankhuizen et al., 1998) of following the tracks of late blight generating process in the potato raw growth process and then effectively preventing disease.
The main method and the technology that detect at present soil and potato seed late disease bacteria content of molds have: 1. field detection method; This method through the main tissue conditions observing the late blight of potato and cause as detecting appraisal evidence; Just can detect when having only potato tissue that tangible symptom takes place; Accuracy is relatively poor, time and effort consuming (5.1.1.2 among the GB7331-2003); 2. laboratory detection method, this method is utilized the Physiology and biochemistry characteristics of the main pathogenic bacterium of yam, cultivates and means such as biochemistry detect through microscope, pathogenic bacteria recovery.Accuracy is high than the field detection method, but sense cycle is long, for the prevention detection hysteresis (GB6682) of late blight.3. molecular biology method (Judelson and Tooley, 2000; Niepold and Schober – Butin, 1995; Tooley., 1997; Trout et al., 1997), these methods all are to utilize
P.infestansGenomic dna sequence design primer detects potato leaf, stem tuber etc.
P. infestansContent, detect potato tuber and blade behind the inoculation late disease bacteria like design such as Niepold primer, but this method can only detect the late disease bacteria of inoculation potato tissue, sensitivity lower (Niepold and Schober – Butin., 1995); Trout etc. (Trout et al., 1997) utilize primer I TS5 and PINF to detect the late disease bacteria in the natural potato piece, and sensitivity is higher, but specificity is relatively poor.Judelson and Tooley are according to Tumor-necrosis factor glycoproteins design primer in the full genome of late disease bacteria, and this primer can increase
P. infestansITS (Internal Transcribed Spaces) sequence, specificity high (Judelson and Tooley, 2000).But this research directly is not used for late blight of potato potato seed and the germ-carrying detection of leaf.Xu Anchuan etc. design primer 08-3 and 08-4 detect the seed potato different sites late blight situation of hiding respectively, and susceptibility and specificity be all than higher (Xu Anchuan etc., 2004), but do not have realization that the late disease bacteria in the potato planting district soil is detected.
The test kit that detects at present potato seed and blade late disease bacteria content of molds has: 1. phytophthora infestans molecular detection primer and using method thereof; This detection method has mainly designed the special primer (upstream primer INF1 and downstream primer INF2) of a Phytophthora infestans; Through pcr amplification and agarose gel electrophoresis, can in phytophthora infestans pure dna, germ-carrying plant and potato piece, amplify specific amplified product (application number/patent No.: 200910111735) that fragment length is 324bp specifically.But this method can not provide quantizating index for the detection of late blight in the yam production.The most important thing is still do not have effective detection method as the detection of content of molds in late blight dormancy and the main place-soil propagated.
Comprehensive above analysis can be found out; Prior art can't be fast, accurately; Efficiently, detection by quantitative soil late blight of potato germ number specifically, thereby can't effectively follow the tracks of late blight generating process in the potato raw growth process, and propose effectively prevention disease measure in view of the above.
In view of the prior art defective; The invention provides the method for phytophthora infestans in a kind of detection by quantitative soil; This method can be fast, accurately, efficiently, late blight of potato germ number in the detection by quantitative soil specifically, satisfy the production needs that people prevent and treat the late blight of potato.
Reference:
Andrivon?D.?Biology,?ecology?and?epidemiology?of?the?potato?late?blight?pathogen?
Phytophthora?inf?estans?in?soil.?
Phytopathology,?1995,?85?:?1053-1056.
Haverkort?A.J.?et?al.?Societal?costs?of?late?blight?in?potato?and?prospects?of?durable?resistance?through?cisgenic?modification.?
Potato?Res.?2008,?51,?47-57.
Judelson?H.S.?and?Tooley?P.W.?Enhanced?PCR?methods?for?detection?and?quantification?of?
Phytophthora?infestans?in?plants.?
Phytopathology,?2000,?90:?1112-1115.
Niepold?FB?and?S,?Butin?Application?of?the?PCR?technique?to?detect?
Phytophthora?infestans?in?potato?tubers?and?leaves.?
Microbiol?Res,?1995,?150:?379-385.
Reader?J.?Potato:?A?History?of?the?Propitious?Esculent.?2009?Yale?Univ.?Press.
Tooley?P.W.,?Bunyard?B.A.,?Carras?M.M.?and?Hatziloukas?E.?Development?of?PCR?primers?from?internal?transcribed?spacer?region?2?for?detection?of?
Phytophthora?species?infecting?potatoes.?
Appl.?Environ.?Microbiol,?1997,?63:1467-1475.
Trout?C.L.,?Ristaino?J.B.,?Madritch?M.?and?Wangsomboondee?T.?Rapid?detection?of?
Phytophthora?infestans?in?late?blight?potatoes?and?tomatoes?using?PCR.?
Plant?Dis,?1997,?81:1042-1048.
Wangsomboondee?T.?and?Ristaino?J.B.?Optimization?of?Sample?Size?and?DNA?Extraction?Methods?to?Improve?PCR?Detection?of?Different?Propagules?of?
Phythphtoora?infestans.?
Plant?Dis.2002,?86?(3):?247-253.
Zwankhuizen?M.J.F.?Govers?J.C.,?Zadoks.?Development?of?potato?late?blight?eipidemics:?disease?foci,?disease?gradients?and?infeciton?sources.?
Phytopathology,?1998?,?88?:?754-763.
Xu Anchuan, Luo Wenfu, Yang Yanli, Zhao Haiyan. the germ-carrying Molecular Detection of late blight of potato potato seed. Chinese yam, 2004,18 (2): 72-76.
Summary of the invention
The purpose of this invention is to provide the method and the application of late blight of potato content of molds in a kind of detection by quantitative soil.This method is that regular-PCR technology and Real Time PCR thereof are combined specifically; Utilize special primer; Quantity to late blight live body germ in the potato growing ground soil is carried out detection by quantitative; By method provided by the present invention, phytophthora infestans content of molds in the detection by quantitative soil exactly.The special primer that this method is used is:
Forward primer (PIF): 5 '-CGGCGGCTGCTGGCTTTAT-3 ';
Reverse primer (PIR): 5 '-GCTCAGACCGAAGTCCAAACG-3 '
The concrete steps of this method are following:
1., make up typical curve: with reference culture (
P.infestansATCC) 10 times of dilution DNA to 10 of DNA liquid (concentration is 100ng/ μ l)
2Ng/ μ l, 10ng/ μ l, 1ng/ μ l, 10
-1Ng/ μ l, 10
-2Ng/ μ l, 10
-3Ng/ μ l, 10
-4Ng/ μ l; (concentration is respectively 10 in Real Time pcr amplification pipe, to add dna profiling 1 μ l
-1Ng/ μ l, 10
-2Ng/ μ l, 10
-3Ng/ μ l, 10
-4Ng/ μ l, 10
-5Ng/ μ l), adds SYBR-Green fluorescent labeling reagent mixture (SYBR green PCR Master Mix, Applied Biosystems) 10 μ l, add each 0.5ul of Auele Specific Primer PIF/PIR (10 μ M), aseptic deionized water to TV 20 μ l.3 parallel laboratory tests are done in each reaction.The setting program of real-time fluorescence quantitative PCR appearance is: 50 ℃-5 min, 95 ℃-10 min; With 95 ℃-20s, 60 ℃-1min, circulate 35 times then; At last at 72 ℃ of insulation 5min.After full cycle was accomplished, sample 0.03 ℃/s, was increased to 95 ℃ from 60 ℃ under 35 ℃ of gradients.After reaction finishes, derive Microsoft Excel, judge the amplification efficiency of each sample according to the solubility curve of reaction; Every group reaction is averaged, give up Ct greater than 30 reaction (thresholds cycles, Ct); Log value with dna profiling content is an X-coordinate; Reaction Ct is the ordinate zou mapping, and the dependency of calculated curve, R
2>=0.99 thinks that dependency is good, and amplification is credible.
2., the earth 0.5 that fetches earth restrains adding DNA extraction liquid (100 mmol/L, Tris-HCl; 100 mmol/L EDTA, 100 mmol/L sodium phosphates, 1.5mol/L NaCl; 2% CTAB, pH8.0) 1.2ml extracts DNA according to the CTAB method; Through the quality of determined by ultraviolet spectrophotometry extraction DNA, standard is that OD260/280 is about 1.8, as the dna profiling in following several steps.
3., in the amplification pipe, add dna profiling 2 μ l (50ng), adding 10 * Tris-HCl damping fluid (pH8.3,500mM KCl, 15mM MgCl
2) 3 μ l; DNTP (2.5mM) 2 μ l; Each 1 μ l of special primer (PIF/PIR, 10 μ M); Taq enzyme (5U/ μ l) 0.2 μ l and 2% Blotto (10% skim-milk and 0.2% NaN
3), add aseptic deionized water to TV 25 μ l.
4., above-mentioned reaction system is carried out regular-PCR amplification, specific procedure is: 95 ℃-4min; With 95 ℃-55s, 60 ℃-1min, 72 ℃-45s, circulate 40 times then; At last at 72 ℃ of insulation 5min.
5., the sample after will increasing detects at agarose gel electrophoresis, sees that the 101bp place has or not band, if having, has phytophthora infestans to exist, if do not find obvious amplified band, explains that this bacterium does not exist or content is very low.
6., (concentration is respectively 10 in Real Time pcr amplification pipe, to add dna profiling 1 μ l
-1Ng/ μ l, 10
-2Ng/ μ l, 10
-3Ng/ μ l, 10
-4Ng/ μ l, 10
-5Ng/ μ l), adds SYBR-Green fluorescent labeling reagent mixture (SYBR green PCR Master Mix, Applied Biosystems) 10 μ l, add each 0.5 μ l of Auele Specific Primer PIF/PIR (10 μ M), aseptic deionized water to TV 20 μ l.
7., above-mentioned reaction system is carried out Real Time pcr amplification, specific procedure is: 50 ℃-5min, and 95 ℃-10min; With 95 ℃-20s, 60 ℃-1min, circulate 35 times then; At last at 72 ℃ of insulation 5min.After full cycle was accomplished, sample 0.03 ℃/s, was increased to 95 ℃ from 60 ℃ under 35 ℃ of gradients.
8., the Ct value that obtains of test sample, calculate the dna content of corresponding sample phytophthora infestans through Ct=-3.3282log [DNA]+31.994.Because the late disease bacteria content of molds is directly proportional in the dna content of pedotheque phytophthora infestans and the soil; So can pass through the height of the indirectly quantitative pedotheque phytophthora infestans content of molds of height of pedotheque phytophthora infestans dna content, thereby reach the purpose of late disease bacteria content of molds in the detection by quantitative soil.
According to method provided by the invention; All reagent comprise CTAB method agents useful for same of the present invention: the primer that is screened; The enzyme or the reagent of required usefulness in the various PCR reactions; Realize other enzyme essential to the invention or reagent or material, can liquid form or lyophilisate pack into and be prepared into test kit in one or more acceptable containers, be used for detection by quantitative soil late blight of potato content of molds.
Embodiment:
The building process of embodiment 1 typical curve
With reference culture (
P.infestansATCC) 10 times of dilution DNA to 10 of DNA liquid (concentration is 100ng/ μ l)
2Ng/ μ l, 10ng/ μ l, 1ng/ μ l, 10
-1Ng/ μ l, 10
-2Ng/ μ l, 10
-3Ng/ μ l, 10
-4Ng/ μ l; (concentration is respectively 10 in Real Time pcr amplification pipe, to add dna profiling 1 μ l
-1Ng/ μ l, 10
-2Ng/ μ l, 10
-3Ng/ μ l, 10
-4Ng/ μ l, 10
-5Ng/ μ l), adds SYBR-Green fluorescent labeling reagent mixture (SYBR green PCR Master Mix, Applied Biosystems) 10 μ l, add each 0.5 μ l of Auele Specific Primer PIF/PIR (10 μ M), aseptic deionized water to TV 20 μ l.3 parallel laboratory tests are done in each reaction.The setting program of real-time fluorescence quantitative PCR appearance is: 50 ℃-5 min, 95 ℃-10 min; With 95 ℃-20s, 60 ℃-1min, circulate 35 times then; At last at 72 ℃ of insulation 5min.After full cycle was accomplished, sample 0.03 ℃/s, was increased to 95 ℃ from 60 ℃ under 35 ℃ of gradients.After reaction finishes, derive Microsoft Excel, judge the amplification efficiency of each sample according to the solubility curve of reaction; Every group reaction is averaged, give up Ct greater than 30 reaction (thresholds cycles, Ct); Log value with dna profiling content is an X-coordinate; Reaction Ct is the ordinate zou mapping, and the dependency of calculated curve, obtains the higher typical curve (R of dependency
2=0.9984), amplification is credible.
[0019]
Late disease bacteria content of molds in the embodiment 2 detection by quantitative soil
1, get each 0.5g of three kinds of potato planting soil (sandy soil Sandy, clay Clay and vegetable mould Muck) respectively, each adds DNA extraction liquid (100 mmol/L, Tris-HCl; 100 mmol/L EDTA, 100 mmol/L sodium phosphates, 1.5mol/L NaCl; 2% CTAB, pH8.0) 1.2ml extracts DNA according to the CTAB method; Through the quality of determined by ultraviolet spectrophotometry extraction DNA, standard is that OD260/280 is about 1.8.
2, in the amplification pipe, add dna profiling 2 μ l (50ng), add 10 * Tris-HCl damping fluid (pH8.3,500mM KCl, 15mM MgCl
2) 3 μ l; DNTP (2.5mM) 2 μ l; Each 1 μ l of special primer (PIF/PIR, 10 μ M); Taq enzyme (5U/ μ l) 0.2 μ l and 2% Blotto (10% skim-milk and 0.2% NaN
3), add aseptic deionized water to TV 25 μ l.
3, above-mentioned reaction system is carried out the regular-PCR amplification, specific procedure is: 95 ℃-4min; With 95 ℃-55s, 60 ℃-1min, 72 ℃-45s, circulate 40 times then; At last at 72 ℃ of insulation 5min.
4, the sample after will increasing carries out agarose gel electrophoresis and detects, and the DNA cloning result of three kinds of soil is presented at 101bp place all has obvious amplified band, explains that this bacterium existence and content are higher.
5, (concentration is respectively 10 in Real Time pcr amplification pipe, to add dna profiling 1 μ l
-1Ng/ μ l, 10
-2Ng/ μ l, 10
-3Ng/ μ l, 10
-4Ng/ μ l, 10
-5Ng/ μ l), adds SYBR-Green fluorescent labeling reagent mixture (SYBR green PCR Master Mix, Applied Biosystems) 10ul, add each 0.5 μ l of Auele Specific Primer PIF/PIR (10 μ M), aseptic deionized water to TV 20 μ l.
6, above-mentioned reaction system is carried out Real Time pcr amplification, specific procedure is: 50 ℃-5min, and 95 ℃-10min; With 95 ℃-20s, 60 ℃-1min, circulate 35 times then; At last at 72 ℃ of insulation 5min.After full cycle was accomplished, sample 0.03 ℃/s, was increased to 95 ℃ from 60 ℃ under 35 ℃ of gradients.
7, after amplification finishes,, pass through Ct=-3.3282log [DNA]+31.994 (R again the Ct value that test sample obtains
2=0.9984) calculates the dna content (like following table) of corresponding sample phytophthora infestans.：
Because the late disease bacteria content of molds is directly proportional in the dna content of pedotheque phytophthora infestans and the soil; So can be through the indirect quantitatively height of pedotheque phytophthora infestans content of molds of the height of pedotheque phytophthora infestans dna content; Upward show that three kinds of soil all have a large amount of phytophthora infestans, and the vegetable mould content of molds silty loam content of molds clay content of molds.
Claims (4)
1. the method and the application of phytophthora infestans content of molds in the detection by quantitative soil is characterized in that the special primer that this method contains is that following primer is right, and the protection base of not fixing a number can be added in the two ends of these primers, restriction enzyme site or joint:
Forward primer (PIF): 5 '-CGGCGGCTGCTGGCTTTAT-3 '
Reverse primer (PIR): 5 '-GCTCAGACCGAAGTCCAAACG-3 '.
2. the method and the application of phytophthora infestans content of molds in a kind of detection by quantitative soil according to claim 1 is characterized in that the primer that this method adopts can make fluorescent mark or isotopic labeling on the primer band when synthetic.
3. the test kit of phytophthora infestans content of molds in the detection by quantitative soil is characterized in that the special primer that contains in the test kit is that following primer is right, and the protection base of not fixing a number can be added in the two ends of these primers, restriction enzyme site or joint:
Forward primer (PIF): 5 '-CGGCGGCTGCTGGCTTTAT-3 '
Reverse primer (PIR): 5 '-GCTCAGACCGAAGTCCAAACG-3 '.
4. the test kit of phytophthora infestans content of molds in a kind of detection by quantitative soil according to claim 3 is characterized in that the primer that is contained in this test kit can make fluorescent mark or isotopic labeling on the primer band when synthetic.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102453756A (en) * | 2010-10-28 | 2012-05-16 | 兰州大学 | Reagent kit for quantitatively detecting quantity of late blight bacteria in soil |
| CN106841077A (en) * | 2017-02-21 | 2017-06-13 | 安徽省农业科学院园艺研究所 | A kind of method for quick of the late blight of potato |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101643788A (en) * | 2009-05-12 | 2010-02-10 | 福建省农业科学院植物保护研究所 | Detection primer of potato late blight bacterium molecules and use method thereof |
| CN102453756A (en) * | 2010-10-28 | 2012-05-16 | 兰州大学 | Reagent kit for quantitatively detecting quantity of late blight bacteria in soil |
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| CN101643788A (en) * | 2009-05-12 | 2010-02-10 | 福建省农业科学院植物保护研究所 | Detection primer of potato late blight bacterium molecules and use method thereof |
| CN102453756A (en) * | 2010-10-28 | 2012-05-16 | 兰州大学 | Reagent kit for quantitatively detecting quantity of late blight bacteria in soil |
Non-Patent Citations (2)
| Title |
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| 徐安传等: "马铃薯晚疫病种薯带菌的分子检测", 《中国马铃薯》, vol. 18, no. 02, 25 April 2004 (2004-04-25) * |
| 陈庆河等: "双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用", 《植物病理学报》, vol. 39, no. 06, 31 December 2009 (2009-12-31) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102453756A (en) * | 2010-10-28 | 2012-05-16 | 兰州大学 | Reagent kit for quantitatively detecting quantity of late blight bacteria in soil |
| CN106841077A (en) * | 2017-02-21 | 2017-06-13 | 安徽省农业科学院园艺研究所 | A kind of method for quick of the late blight of potato |
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