CN102453755B - 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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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 method and the application of phytophthora infestans content of molds in a kind of detection by quantitative soil, can quantitatively, late blight of potato germ in specific detection soil, belong to agricultural biological technical field.
Background technology
The late blight of potato is a kind of destructive disease of potato, and oneself is classified as the first disease of world food crop.Late blight generally occurs in potato planting district, and field yield loss reaches 20% ~ 50%, and storing sth. in a cellar loss can reach 5% ~ 30%, and the time of being very popular even can cause total crop failure.In recent years, the popular frequency of late blight and popularity degree increase the weight of gradually, become the significant obstacle that potato produces.Potato is the fourth-largest food crop (Reader in the world, 2009), the loss that world's late blight of potato causes has reached 6,700,000,000 dollars of (Haverkort et al., 2008) China has been the first big country of potato production in the world, and China is every year because late blight loss is up to 1,000,000,000 dollars.
The late blight of potato, causes primarily of phytophthora infestans (Phytophthora infestans).This germ mainly with the in spite of illness stem tuber of form in soil of oospore and zoospore, passes the winter in undesirable root, more summer.Under adapt circumstance condition, late disease bacteria produces sporocyst and propagates the plant of infecting surrounding with air-flow.As long as thus soil is containing some amount germ or the potato seed of carrying disease germs of planting lower only a few, generation (Andrivon, 1995 of full wafer ground late blight may be caused; Zwankhuizen et al., 1998).Therefore quick specific detection is carried out to plantation potato soil late disease bacteria content of molds, prediction and prevention carries out to the Occurrence & epidemic of late blight particularly crucial, also be the major measure (Zwankhuizen et al., 1998) of following the tracks of late blight generating process and then effectively pre-disease prevention in potato raw growth process.
Main method and the technology of current detection soil and potato seed late disease bacteria content of molds have: 1. Fields detection method, this method is by observing the Main Tissues symptom that causes of the late blight of potato as Testing and appraisal evidence, just can detect when only having potato tissue that obvious symptom occurs, accuracy is poor, time and effort consuming (in GB7331-2003 5.1.1.2); 2. test in laboratory method, this method utilizes the Physiology and biochemistry feature of potato Main Pathogenic Bacteria, is detected by means such as microscope, pathogenic bacteria recovery cultivation and biochemistry.Comparatively Fields detection method is high for accuracy, but sense cycle is long, and the prevention for late blight detects delayed (GB6682).3. molecular biology method (Judelson and Tooley, 2000; Niepold and Schober – Butin, 1995; Tooley., 1997; Trout et al., 1997), these methods are all utilize P.infestans genomic dna sequence to design primer, detect the content of the P.infestans such as potato leaf, stem tuber, as the design primers such as Niepold detect the potato tuber after inoculation late disease bacteria and blade, 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 natural potato block, and sensitivity is higher, but specificity is poor.Judelson and Tooley is according to tumor-necrosis factor glycoproteins design primer in late disease bacteria full-length genome, this primer can increase ITS (the Internal Transcribed Spaces) sequence of P.infestans, specificity high (Judelson and Tooley, 2000).But this research is not directly used in the detection that late blight of potato potato seed and leaf carry disease germs.Xu Anchuan etc. design primer 08-3 and 08-4, detect seed potato different sites late blight respectively to hide situation, susceptibility and specificity all higher (Xu Anchuan etc., 2004), but do not have to realize detecting the late disease bacteria in potato planting district soil.
The test kit of current detection potato seed and blade late disease bacteria content of molds has: 1. phytophthora infestans molecular detection primer and using method thereof, this detection method major design special primer of one Phytophthora infestans (upstream primer INF1 and downstream primer INF2), through pcr amplification and agarose gel electrophoresis, in phytophthora infestans pure dna, the plant carried disease germs and potato block, specific amplified product (application number/patent No.: 200910111735) that fragment length is 324bp can be amplified specifically.But this method, can not provide quantizating index for the detection of late blight in potato production.The most important thing is still there is no effective detection method as the detection of content of molds in late blight dormancy and main place-soil of propagating.
Comprehensive above analysis can be found out, prior art cannot fast, accurately, efficiently, detection by quantitative soil late blight of potato germ number specifically, thus effectively cannot follow the tracks of late blight generating process in potato raw growth process, and propose effectively pre-disease prevention measure accordingly.
In view of prior art defect, the invention provides the method for phytophthora infestans in a kind of detection by quantitative soil, the method can fast, accurately, efficiently, late blight of potato germ number in detection by quantitative soil specifically, meets the need of production that people prevent and treat the late blight of potato.
Reference:
Andrivon D.Biology,ecology and epidemiology of the potato late blight pathogen Phytophthora infestans in soil.Phytopathology,1995,85:1053-1056.
Haverkort A.J.et al.Societal costs of late blight in potato and prospects of durable resistancethrough cisgenic modification.Potato Res.2008,51,47-57.
Judelson H.S.and Tooley P.W.Enhanced PCR methods for detection and quantification ofPhytophthora infestans in plants.Phytopathology,2000,90:1112-1115.
Niepold FB and S,Butin Application of the PCR technique to detect Phytophthora infestans inpotato tubers and leaves.Microbiol Res,1995,150:379-385.
Reader J.Potato:A History of the Propitious Esculent.2009Yale Univ.Press.
Tooley P.W.,Bunyard B.A.,Carras M.M.and Hatziloukas E.Development of PCR primers frominternal transcribed spacer region 2for 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 Phytophthorainfestans 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 Methodsto 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:diseasefoci,disease gradients and infeciton sources.Phytopathology,1998,88:754-763.
Xu Anchuan, Luo Wenfu, Yang Yanli, Zhao Haiyan. the Molecular Detection that late blight of potato potato seed is carried disease germs. potatoes, 2004,18 (2): 72-76.
Summary of the invention
The object of this invention is to provide method and the application of late blight of potato content of molds in a kind of detection by quantitative soil.The method is combined regular-PCR technology and Real Time PCR thereof specifically, utilize special primer, detection by quantitative is carried out to the quantity of late blight live body germ in potato growing ground soil, by method provided by the present invention, can phytophthora infestans content of molds in detection by quantitative soil exactly.The special primer that the method uses is:
Forward primer (PIF): 5 '-CGGCGGCTGCTGGCTTTAT-3 ';
Reverse primer (PIR): 5 '-GCTCAGACCGAAGTCCAAACG-3 '
The concrete steps of the method are as follows:
1., typical curve is built: by reference culture (P.infestans) DNA liquid (concentration is 100ng/ μ l) 10 times of dilution DNA to 10
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; In RealTime pcr amplification pipe, adding DNA profiling 1 μ l, (concentration is respectively 10
-1ng/ μ l, 10
-2ng/ μ l, 10
-3ng/ μ l, 10
- 4ng/ μ l, 10
-5ng/ μ l), add SYBR-Green fluorescent labeling reagent mixture (SYBR green PCR Master Mix, Applied Biosystems) 10 μ l, add Auele Specific Primer PIF/PIR (10 μMs) each 0.5ul, aseptic deionized water is to cumulative volume 20 μ l.3 parallel laboratory tests are done in each reaction.The setting program of real-time fluorescence quantitative PCR instrument is: 50 DEG C of-5min, 95 DEG C of-10min; Then with 95 DEG C of-20s, 60 DEG C of-1min, circulate 35 times; Finally at 72 DEG C of insulation 5min.After whole circulation completes, sample, under 35 DEG C of gradients, 0.03 DEG C/s, is increased to 95 DEG C from 60 DEG C.After reaction terminates, derive Microsoft Excel, the amplification efficiency of each sample is judged according to the solubility curve of reaction, every group reaction is averaged, and gives up the reaction (thresholds cycles, Ct) that Ct is greater than 30, with the log value of DNA profiling content for X-coordinate, reaction Ct is ordinate zou mapping, and the dependency of calculated curve, R
2>=0.99 thinks that dependency is good, and amplification is credible.
2., fetch earth earth 0.5 gram, add DNA extraction liquid (100mmol/L, Tris-HCl, 100mmol/L EDTA, 100mmol/L sodium phosphate, 1.5mol/L NaCl, 2%CTAB, pH8.0) 1.2ml, DNA is extracted according to CTAB method, extracted the quality of DNA by determined by ultraviolet spectrophotometry, standard is OD260/280 is about 1.8, as the DNA profiling of lower a few step.
3., in 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; Special primer (PIF/PIR, 10 μMs) each 1 μ l; 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 cumulative volume 25 μ l.
4., to above-mentioned reaction system carry out regular-PCR amplification, specific procedure is: 95 DEG C of-4min; Then with 95 DEG C of-55s, 60 DEG C of-1min, 72 DEG C of-45s, circulate 40 times; Finally at 72 DEG C of insulation 5min.
5., by amplification after sample detect at agarose gel electrophoresis, see that 101bp place is with or without band, if having, have phytophthora infestans to exist, if do not find obvious amplified band, illustrate this bacterium do not exist or content 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), add SYBR-Green fluorescent labeling reagent mixture (SYBRgreen PCR Master Mix, Applied Biosystems) 10 μ l, add each 0.5 μ l of Auele Specific Primer PIF/PIR (10 μMs), aseptic deionized water is to cumulative volume 20 μ l.
7., to above-mentioned reaction system carry out Real Time pcr amplification, specific procedure is: 50 DEG C of-5min, 95 DEG C of-10min; Then with 95 DEG C of-20s, 60 DEG C of-1min, circulate 35 times; Finally at 72 DEG C of insulation 5min.After whole circulation completes, sample, under 35 DEG C of gradients, 0.03 DEG C/s, is increased to 95 DEG C from 60 DEG C.
8., detect the Ct value that obtains of sample, calculated the DNA content of corresponding sample phytophthora infestans by Ct=-3.3282log [DNA]+31.994.Because the DNA content of pedotheque phytophthora infestans is directly proportional to late disease bacteria content of molds in soil, so by the height of the height indirect quantification pedotheque phytophthora infestans content of molds of pedotheque phytophthora infestans DNA content, thus the object of late disease bacteria content of molds in detection by quantitative soil can be reached.
According to method provided by the invention, all reagent comprises CTAB method agents useful for same of the present invention: the primer screened, enzyme needed in various PCR reaction or reagent, realize other enzyme essential to the invention or reagent or material, can in liquid form or lyophilisate load in one or more acceptable container and be prepared into test kit, for late blight of potato content of molds in detection by quantitative soil.
Embodiment:
The building process of embodiment 1 typical curve
By reference culture (P.infestans) DNA liquid (concentration is 100ng/ μ l) 10 times of dilution DNA to 10
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; In Real Time pcr amplification pipe, adding DNA profiling 1 μ l, (concentration is respectively 10
-1ng/ μ l, 10
-2ng/ μ l, 10
-3ng/ μ l, 10
-4ng/ μ l, 10
-5ng/ μ l), add SYBR-Green fluorescent labeling reagent mixture (SYBR green PCR Master Mix, AppliedBiosystems) 10 μ l, add each 0.5 μ l of Auele Specific Primer PIF/PIR (10 μMs), aseptic deionized water is to cumulative volume 20 μ l.3 parallel laboratory tests are done in each reaction.The setting program of real-time fluorescence quantitative PCR instrument is: 50 DEG C of-5min, 95 DEG C of-10min; Then with 95 DEG C of-20s, 60 DEG C of-1min, circulate 35 times; Finally at 72 DEG C of insulation 5min.After whole circulation completes, sample, under 35 DEG C of gradients, 0.03 DEG C/s, is increased to 95 DEG C from 60 DEG C.After reaction terminates, derive Microsoft Excel, the amplification efficiency of each sample is judged according to the solubility curve of reaction, every group reaction is averaged, and gives up the reaction (thresholds cycles, Ct) that Ct is greater than 30, with the log value of DNA profiling content for X-coordinate, reaction Ct is ordinate zou mapping, and the dependency of calculated curve, obtain the typical curve (R that dependency is higher
2=0.9984), amplification is credible.
Late disease bacteria content of molds in embodiment 2 detection by quantitative soil
1, three kinds of potato planting soil (sandy soil Sandy, clay Clay and vegetable mould Muck) each 0.5g is got respectively, respectively add DNA extraction liquid (100mmol/L, Tris-HCl, 100mmol/L EDTA, 100mmol/L sodium phosphate, 1.5mol/L NaCl, 2%CTAB, pH8.0) 1.2ml, extracts DNA according to CTAB method, extracted the quality of DNA by determined by ultraviolet spectrophotometry, standard is OD260/280 is about 1.8.
2, in 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; Special primer (PIF/PIR, 10 μMs) each 1 μ l; 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 cumulative volume 25 μ l.
3, carry out regular-PCR amplification to above-mentioned reaction system, specific procedure is: 95 DEG C of-4min; Then with 95 DEG C of-55s, 60 DEG C of-1min, 72 DEG C of-45s, circulate 40 times; Finally at 72 DEG C of insulation 5min.
4, the sample after amplification is carried out agarose gel electrophoresis detection, the DNA cloning result of three kinds of soil is presented at 101bp place all obvious amplified band, illustrates that this bacterium exists and content is 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), add SYBR-Green fluorescent labeling reagent mixture (SYBR greenPCR Master Mix, Applied Biosystems) 10ul, add each 0.5 μ l of Auele Specific Primer PIF/PIR (10 μMs), aseptic deionized water is to cumulative volume 20 μ l.
6, carry out Real Time pcr amplification to above-mentioned reaction system, specific procedure is: 50 DEG C of-5min, 95 DEG C of-10min; Then with 95 DEG C of-20s, 60 DEG C of-1min, circulate 35 times; Finally at 72 DEG C of insulation 5min.After whole circulation completes, sample, under 35 DEG C of gradients, 0.03 DEG C/s, is increased to 95 DEG C from 60 DEG C.
7, after amplification terminates, the Ct value that detection sample obtains, then by Ct=-3.3282log [DNA]+31.994 (R
2=0.9984) DNA content (as following table) of corresponding sample phytophthora infestans is calculated.：
Because the DNA content of pedotheque phytophthora infestans is directly proportional to late disease bacteria content of molds in soil, so can by the height of the height indirect quantification pedotheque phytophthora infestans content of molds of pedotheque phytophthora infestans DNA content, upper table shows that three kinds of soil are all with a large amount of phytophthora infestans, and vegetable mould content of molds > silty loam content of molds > clay content of molds.
Claims (1)
1. a method of phytophthora infestans DNA in detection by quantitative soil, is characterized in that the concrete steps of the method are as follows:
1., typical curve is built: the reference culture P.infestans by concentration being 100ng/ μ l, DNA liquid dilutes 10 times to 10
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; In Real Time PCR increases pipe, add DNA template 1 μ l, concentration is respectively 10
-1ng/ μ l, 10
-2ng/ μ l, 10
-3ng/ μ l, 10
-4ng/ μ l, 10
-5ng/ μ l, add SYBR-Green fluorescent labeling reagent mixture SYBR greenPCR Master Mix 10 μ l, add Auele Specific Primer, forward primer is (PIF): 5 '-CGGCGGCTGCTGGCTTTAT-3 ', reverse primer is (PIR): 5 '-GCTCAGACCGAAGTCCAAACG-3 ', PIF/PIR 10 μMs of each 0.5ul, aseptic deionized water is to cumulative volume 20 μ l; 3 parallel laboratory tests are done in each reaction; The setting program of real-time fluorescence quantitative PCR instrument is: 50 DEG C of-5 min, 95 DEG C of-10 min; Then with 95 DEG C of-20s, 60 DEG C of-1min, circulate 35 times; Finally at 72 DEG C of insulation 5min; After whole circulation completes, sample, under 35 DEG C of gradients, 0.03 DEG C/s, is increased to 95 DEG C from 60 DEG C; After reaction terminates, derive EXCEL form, the amplification efficiency of each sample is judged according to the solubility curve of reaction, every group reaction is averaged, and gives up the reaction (thresholds cycles, Ct) that Ct is greater than 30, with the log value of DNA template content for X-coordinate, reaction Ct is ordinate zou mapping, and the dependency of calculated curve, R
2>=0.99 thinks that dependency is good, and amplification is credible;
2., fetch earth earth 0.5 gram, add DNA extraction liquid 1.2ml:100 mmol/L, Tris-HCl, 100 mmol/L EDTA, 100mmol/L sodium phosphate, 1.5mol/L NaCl, 2% CTAB, pH8.0, DNA is extracted according to CTAB method, extracted the quality of DNA by determined by ultraviolet spectrophotometry, standard is OD260/280 is about 1.8, as the DNA template of lower a few step;
3., in amplification pipe 50ng DNA profiling 2 μ l is added, add 10 × containing 500mM KCl, 15mM MgCl
2, the Tris-HCl damping fluid 3 μ l of pH8.3; 2.5mM dNTP 2 μ l; Special primer PIF/PIR, 10 μMs of each 1 μ l; 5U/ μ l Taq enzyme 0.2 μ l and 2% is containing 10% skim-milk and 0.2% NaN
3blotto, add aseptic deionized water to cumulative volume 25 μ l;
4., to above-mentioned reaction system carry out regular-PCR amplification, specific procedure is: 95 DEG C of-4min; Then with 95 DEG C of-55s, 60 DEG C of-1min, 72 DEG C of-45s, circulate 40 times; Finally at 72 DEG C of insulation 5min;
5., by amplification after sample detect at agarose gel electrophoresis, see that 101bp place is with or without band, if having, have phytophthora infestans to exist, if do not find obvious amplified band, illustrate this bacterium do not exist or content very low;
6., in Real Time PCR increases pipe add DNA template 1 μ l, concentration is respectively 10
-1ng/ μ l,
10
-2ng/ μ l, 10
-3ng/ μ l, 10
-4ng/ μ l, 10
-5ng/ μ l, add SYBR-Green fluorescent labeling reagent mixture SYBR green PCR Master Mix 10 μ l, add each 0.5 μ l of Auele Specific Primer PIF/PIR 10 μMs, aseptic deionized water is to cumulative volume 20 μ l;
7., to above-mentioned reaction system carry out Real Time PCR to increase, specific procedure is: 50 DEG C of-5min, 95 DEG C of-10min; Then with 95 DEG C of-20s, 60 DEG C of-1min, circulate 35 times; Finally at 72 DEG C of insulation 5min; After whole circulation completes, sample, under 35 DEG C of gradients, 0.03 DEG C/s, is increased to 95 DEG C from 60 DEG C;
8., detect the Ct value that obtains of sample, calculated the DNA content of corresponding sample phytophthora infestans by Ct=-3.3282log [DNA]+31.994.
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| 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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| 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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| 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;陈庆河等;《植物病理学报》;20091231;第39卷(第06期);参见材料与方法部分 * |
| 马铃薯晚疫病种薯带菌的分子检测;徐安传等;《中国马铃薯》;20040425;第18卷(第02期);参见材料与方法部分 * |
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