CN102154485A - Single tube nested PCR (polymerase chain reaction) detection method for wheat stripe rust bacteria and primer thereof - Google Patents
Single tube nested PCR (polymerase chain reaction) detection method for wheat stripe rust bacteria and primer thereof Download PDFInfo
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Abstract
The invention relates to a single tube nested PCR (polymerase chain reaction) detection method for wheat stripe rust bacteria and a primer thereof. The method comprises the following steps: in a single PCR reaction tube, finishing the nested PCR amplification once by an inner pair and an outer pair of wheat stripe rust bacteria specific primers; and judging that wheats or unknown pathogen spores to be detected are wheats infected by the wheat stripe rust bacteria or the wheat stripe rust bacteria by a target segment, wherein the sequences of the nucleotide residues of the inner pair and the outer pair of wheat stripe rust bacteria specific primers are disclosed in SEQ ID No.1, 2, 3 and 4. The method disclosed by the invention has good specificity, is simple and easy to realize, and can be used for early detection for field wheat stripe rust and used for monitoring diseases, and the detection sensitivity is 20fg.
Description
Technical field
The present invention relates to the popular monitoring of Plant diseases field, relate in particular to a kind of one step of stripe rust of wheat single tube nido PCR detection method and primer.
Background technology
Stripe rust of wheat is a most important disease on China's staple food crop wheat, and it harm takes place directly influences wheat yield and quality, threatens national food safety.Since 1949,4 times of China's stripe rust of wheat are very popular and are caused 6.0,3.0,1.8 and 1.3 hundred million kilograms of wheat yield losses respectively.Stripe rust of wheat is that a kind of gas passes the popular disease of Da Qu, south of Gansu Province, Long Dong are acknowledged as China's stripe rust of wheat and get over the summer base, that a situation arises is most important to other popular district development of disease in spring for this zone autumn seedling stripe rust of wheat, realizes that therefore early monitoring and the prevention and control thereof to south of Gansu Province, east, Gansu Province stripe rust of wheat are key links of China's stripe rust of wheat comprehensive regulation.
For a long time, traditional detection method mainly is to detect according to disease symptom, cause of disease morphology, physiological characteristic, this method exists that diagnostic procedure is loaded down with trivial details, the time cycle is long, and the shortcoming that detection accuracy is low, sensitivity is not high, importantly can't realize as yet the not monitoring of the disease of apparent disease intrusion.Along with the development of Protocols in Molecular Biology, particularly based on nucleic acid amplification (PCR) technology make to set up accurately, sensitive, early disease diagnosis efficiently and pathogenic bacteria detect and become possibility.Zhao etc., Wang etc. utilize the difference of wheat stripe rust and the wheat pathogenic bacteria rrna ITS of other leaf portion sequence, and the design Auele Specific Primer has been realized molecular diagnosis and detection to stripe rust of wheat, and this technology is applied for a patent (the patent No.: CN1888886).Nest-type PRC (nested PCR) is in order to improve detection sensitivity, use two cover primers by two-wheeled PCR specific amplification target dna segment, the function of second pair of primer is the section of DNA segment that specific amplification is positioned at first run PCR product, to improve sensitivity and specificity.Wang in 2009 etc. are on the working foundation of Zhao, and design nest-type PRC primer is right, utilize two pipes, two steps nested PCR method, can detect the intrusion of puccinia striiformis behind puccinia striiformis incobation 24h.
Common nest-type PRC normally needs two-step reaction, finishes in two PCR pipes, because there are shortcomings such as sample contamination, complex steps in the high sensitivity of nest-type PRC.Single tube nest-type PRC (single tube nested PCR, stn PCR) is the difference by inside and outside two pairs of primer annealing temperature, is implemented in the single PCR pipe step to finish the nest-type PRC reaction, thereby overcomes the deficiency of common nest-type PRC.Do not see report and the application that detects wheat stripe rust based on the single tube nested PCR method at present both at home and abroad as yet.
Summary of the invention
It is right to the purpose of this invention is to provide one step of good, the highly sensitive wheat stripe rust single tube of specific specificity nest-type PRC primer.
The bacterium amount that the preclinical seedling of puccinia striiformis is carried is few, extremely low with the DNA ratio of germ among the DNA of seedling co-extracted, specificity, susceptibility to primer when carrying out the PCR detection require very high, detection at the preclinical seedling of puccinia striiformis, it is right to the invention provides the used special primer of one step of single tube nest-type PRC detection, and this special primer is to being made up of forward outer primer, reverse outer primer, forward inner primer and reverse inner primer;
Forward outer primer: 5 '-TATGATGGCCACCTTCTCCGTTGT CC-3 ';
Reverse outer primer: 5 '-AAGTATCGGCCG TGTCTCGGGTCA GA-3 ';
Forward inner primer: 5 '-CCTAAGGTGTCT GATACCGTT-3 ';
Reverse inner primer: 5 '-GGCATCAAACATTTGCGA-3 '.
Another object of the present invention provides one step of good, highly sensitive, the simple to operate wheat stripe rust single tube of specific specificity nested PCR detection method.
One step of wheat stripe rust single tube nested PCR detection method of the present invention, be to be template with wheat leaf blade to be measured or unknown pathogenic bacteria spore DNA, it is right to use above-mentioned special primer, in single PCR reaction tubes, carry out pcr amplification, detect the PCR product, the wheat to be measured or the unknown pathogenic bacteria spore that obtain 372bp PCR product are wheat that infects or the strip rust bacteria spore that is subjected to puccinia striiformis.
The mol ratio of above-mentioned forward outer primer and forward inner primer is 1: 150.
The mol ratio of above-mentioned forward outer primer and reverse outer primer is 1: 1; The mol ratio of above-mentioned forward inner primer and reverse inner primer is 1: 1.
In the above-mentioned PCR detection method, the PCR reaction system is conventional PCR reaction system, as Ex Taq Mix 12.5 μ L, and each 1.0 μ L of the above-mentioned positive and negative outer primer of 0.1 μ M, each 1.5 μ L of the forward and reverse inner primer of 10 μ M, 20ng/ μ L template DNA 1.0 μ L use ddH
2O complements to 25.0 μ L.
In the above-mentioned PCR detection method, the PCR response procedures is for being fit to the conventional PCR response procedures of primer amplification, as 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30sec, 60sec, 20 circulations are extended in 72 ℃ of annealing; 95 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 50sec, 35 circulations.
A further object of the present invention provides one step of good, highly sensitive, the simple to operate wheat stripe rust single tube of specific specificity nest type PCR detection reagent.
One step of wheat stripe rust single tube nest type PCR detection reagent of the present invention, it is right to contain above-mentioned special primer.
External except above-mentioned special primer, this test kit can also contain and carries out the needed reagent of pcr amplification, and this reagent comprises Taq archaeal dna polymerase, dNTP, PCR damping fluid etc.
Special primer of the present invention can make a distinction itself and other disease of wheat being according to the primer of the wheat stripe rust microtubule protein gene sequences Design of having reported.The present invention utilizes the single tube nested PCR method, and the minimum concentration that can detect wheat stripe rust DNA is 20fg/ μ L, has improved the sensitivity that detects greatly, also can in time detect being in preclinical pathogenic bacteria.The present invention adopts the single tube nest-type PRC, does not need twice amplification, reduce to detect cost, and method is simple, save time.Method of the present invention can be used for the early detection and the disease monitoring of field stripe rust of wheat, field stripe rust of wheat prevention and control is had important meaningful.
Description of drawings
Fig. 1 wheat stripe rust inner primer Pst-InF/Pst-InR specific detection figure.
Fig. 2 wheat stripe rust inner primer Pst-InF/Pst-InR susceptibility detects figure.
Fig. 3 single tube nest-type PRC (stn PCR) susceptibility detects figure.
The stn PCR of Fig. 4 incubation period wheat leaf blade detects figure.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Experiment material of the present invention:
Wheat stripe rust (Puccinia striiformis f.sp.tritici, Pst) totally 8 physiological strains or fungus strain come from China Agricultural University and plant the monospore breeding of disease epidemiology laboratory; Puccinia triticinia (P.recondite f.sp.tritici, Prt), wheat-straw is embroidered bacterium (P.graminis f.sp.tritici, Pgt), wheat powdery mildew (Blumeria graminis f.sp.tritici, Bgt) be respectively 2 fungus strains, derive from the Chinese Academy of Agricultural Sciences Institute of Plant Protection, Agricultural University Of Hebei, plant disease epidemiology laboratory by Chinese agriculture agriculture university and preserve harvesting; Each 1 fungus strain of rhizoctonia cerealis (Rhizoctoniacereals), gibberellic hypha (Fusarium graminearum), leaf spoting bacteria (Alternariatriticina) and root rotof flax bacterium (Cochliobolus sativus) sees table 1 for details.
The employed fungus strain material of this experiment of table 1.
Main agents:
2 * PCR TaqMix is available from Beijing Mei Laibo medical science and technology company limited, and the sepharose test kit is purchased the company in Sigma, and DNA standard marker is available from Takara company, and primer is synthetic by Shanghai bio-engineering corporation.
Embodiment 1: the checking of puccinia striiformis PCR detection specificity
1 is used to detect the Auele Specific Primer of wheat stripe rust
Pst-stnInF：5’-CCTAAG?GTGTCT?GATACC?GTT-3’
Pst-stnInR：5’-GGCATC?AAACAT?TTGCGA-3’
2 regular-PCRs amplification puccinia striiformis DNA reaction system and response procedures
The 25 μ L reaction systems of regular-PCR amplification puccinia striiformis DNA comprise TaqpreMix 12.5 μ L, each 1.0 μ TL of 10 μ M primer Pst-stnInF, Pst-stnInR, dna profiling 1.0 μ L, ddH
2O 9.5 μ L.On the PCR instrument, carry out pcr amplification: 95 ℃ of pre-sex change 5min according to following program; 95 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 60sec, 35 circulations.
3 required DNA preparations
Adopt the CTAB method to extract wheat stripe rust, leaf rust, stalk embroidery bacterium, white powder germ, pine root fungus, gibberellic hypha, leaf spoting bacteria genomic dna, the trace dna detector detects the DNA quality and also measures DNA concentration, uses ddH
2O transfers to 20ng/ μ L with DNA concentration, and is standby.
4 agarose gel electrophoresis and imaging are observed
Respectively get the pcr amplification product of 5 μ L various pathogenic bacteria DNA, on 1.5% sepharose, carry out electrophoresis, utilize ultraviolet gel imaging observation of use instrument,, judge that then this pathogenic bacteria is a wheat stripe rust if amplified band occurs at the 372bp place.
Fig. 1 is seen in agarose gel electrophoresis result demonstration, and wherein M is standard DNA molecule marker (DNA marker DL 1,000); 1-8 is respectively the different physiological strains of puccinia striiformis (Pst) or fungus strain CYR29, CYR29, CYR 31, CYR 31, CYR32, CYR32, CYR 33, CYR 33,9-15 is respectively puccinia triticinia PHT, THT, wheat-straw is embroidered bacterium 34C2,21C3CTH, wheat powdery mildew E01, E02, fusarium graminearum, wheat leaf spoting bacteria, rhizoctonia cerealis, root rotof flax bacterium, healthy wheat, ddH
2O.Gone out the target stripe of 372bp in several fungus strain DNA cloning of wheat stripe rust, other pathogenic bacteria DNA and contrast all do not amplify target stripe, this result verification the specificity of wheat stripe rust primer Pst-stnInF/Pst-stnInR.
Embodiment 2: the detection sensitivity of uredinial regular-PCR of wheat stripe rust and single tube nest-type PRC relatively
1 is used to detect the Auele Specific Primer of wheat stripe rust
Pst-stnOutF：5’-TATGAT?GGCCAC?CTTCTC?CGTTGT?CC-3’；
Pst-stnOutR：5’-AAGTAT?CGGCCG?TGTCTC?GGGTCA?GA-3’；
Pst-stnInF：5’-CCTAAG?GTGTCT?GATACC?GTT-3’；
Pst-stnInR：5’-GGCATC?AAACAT?TTGCGA-3’；
2 are used for the gradient concentration DNA preparation that susceptibility detects
DNA ddH with concentration known
2O carries out 10 times of gradient dilutions to 10
-8, with this dna profiling as regular-PCR and single tube nest-type PRC.
3 regular-PCR susceptibilitys detect
The 25 μ L reaction systems of regular-PCR amplification puccinia striiformis DNA comprise TaqpreMix 12.5 μ L, each 1.0 μ L of 10 μ M primer Pst-stnInF, Pst-stnInR, dna profiling 1.0 μ L, ddH
2O 9.5 μ L.
On the PCR instrument, carry out pcr amplification: 95 ℃ of pre-sex change 5min according to following program; 95 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 60sec, 35 circulations.
4 single tube nest-type PRC susceptibilitys detect
System to puccinia striiformis single tube PCR reaction is: Ex Taq Mix 12.5 μ L, 0.1 each 1 μ L of the positive and negative outer primer of μ M (Pst-stnOutF/Pst-stnIOutR), each 1.5 μ L of the forward and reverse inner primer (Pst-stnInF/PS-stnInR) of 10 μ M, template DNA 1.5 μ L use ddH
2O complements to 25.0 μ L.
The stn-PCR response procedures is: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30sec, 60sec, 20 circulations are extended in 72 ℃ of annealing; 95 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 50sec, 35 circulations.
5 agarose gel electrophoresis and result are relatively
Respectively get the pcr amplification product of the different DNA concentration gradients of 5 μ L, on 1.5% sepharose, carry out electrophoresis, utilize ultraviolet gel imaging observation of use instrument, observe the detection sensitivity that low DNA concentration that amplified band appears in the 372bp place is PCR.
Fig. 2 and 3 are seen in agarose gel electrophoresis result demonstration, and M is standard DNA molecule marker (DNA marker DL 1,000) among Fig. 2; 1-7 is respectively the gradient DNA of puccinia striiformis (Pst) different concns, is respectively 20ng/ μ L, 2ng/ μ L, 200pg/ μ L, 20pg/ μ L, 2pg/ μ L, 200fg/ μ L, 20fg/ μ L; M is standard DNA molecule marker (DNAmarker DL 2,000) among Fig. 3; 1-7 is respectively the gradient DNA of puccinia striiformis (Pst) different concns, is respectively 2ng/ μ L, 200pg/ μ L, 20pg/ μ L, 2pg/ μ L, 200fg/ μ L, 20fg/ μ L, 2fg/ μ L.The detection sensitivity of regular-PCR and single tube nest-type PRC is respectively 2pg and 20fg, and the latter is the former 100 times.
Embodiment 3: the wheat leaf blade single tube nest-type PRC that infected by wheat stripe rust still to be in the incubation period detects
The preparation of 1 wheat inoculation in seedling stage and testing sample DNA
Adopt spray method to inoculate a leaf phase wheat leaf blade, wheat stripe rust uredospore concentration 0.15mg/mL will preserve moisture in the postvaccinal wheat leaf blade dark, and 5 of 12h, 24h, 2d, 4d, each clip wheat leaf blades of 6d extract DNA respectively.
2 single tube nest-type PRCs detect as yet the not wheat leaf blade of morbidity
DNA with 1 preparation is a template, adopt 25 μ L stn pcr amplification systems: Ex TaqMix 12.5 μ L, 0.1 each 1 μ L of the positive and negative outer primer of μ M (Pst-stnOutF/PS-stnOutR), each 1.5 μ L of the forward and reverse inner primer (Pst-stnInF/Pst-stnInR) of 10 μ M, template DNA 1.5 μ L use ddH
2O complements to 25.0 μ L)
On the PCR instrument, carry out pcr amplification: 95 ℃ of pre-sex change 5min according to following program; 95 ℃ of sex change 30sec, 60sec, 20 circulations are extended in 72 ℃ of annealing; 95 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 50sec, 35 circulations.
3 agarose gel electrophoresis and result observe
Respectively get the pcr amplification product of the DNA of 5 μ L different times sampling, on 1.5% sepharose, carry out electrophoresis, utilize ultraviolet gel imaging observation of use instrument, amplified band occurs, then judge in this time to detect wheat stripe rust at the 372bp place.
Fig. 4 is seen in agarose gel electrophoresis result demonstration, and wherein M is standard DNA molecule marker (DNA marker DL 1,000); 1-5 is respectively wheat leaf blade inoculation 6d, 4d, 2d, 24h, 12h, ddH
2O just can detect the intrusion of strip rust bacteria in inoculation 24h does not show the wheat leaf blade of disease as yet.
Claims (9)
1. the special primer of one step of single tube nest-type PRC detection is right, and this special primer is to being made up of forward outer primer, reverse outer primer, forward inner primer and reverse inner primer;
Forward outer primer: 5 '-TATGATGGCCACCTTCTCCGTTGTCC-3 ';
Reverse outer primer: 5 '-AAGTATCGGCCGTGTCTCGGGTCAGA-3 ';
Forward inner primer: 5 '-CCTAAGGTGTCTGATACCGTT-3 ';
Reverse inner primer: 5 '-GGCATCAAACAT TTGCGA-3 '.
2. the described special primer of claim 1 is to the application in detecting wheat stripe rust.
3. wheat stripe rust single tube one goes on foot nested PCR detection method, be to be template with wheat cdna group DNA to be measured or unknown pathogenic bacteria spore DNA, application rights requires 1 described special primer right, in single PCR reactant pipe, carry out pcr amplification, detect the PCR product, the wheat to be measured or the unknown pathogenic bacteria spore that obtain the PCR product of 372bp are wheat that infects or the strip rust bacteria spore that is subjected to puccinia striiformis.
4. special primer according to claim 3 is right, and the concentration ratio that it is characterized in that described outer primer and described inner primer is 1: 150.
5. right according to claim 3 or 4 described special primers, the mol ratio that it is characterized in that described forward outer primer and described reverse outer primer is 1: 1; The mol ratio of described forward inner primer and described reverse inner primer is 1: 1.
6. special primer according to claim 5 is right, it is characterized in that described PCR reaction system is Ex Taq Mix 12.5 μ L, 0.1 the described forward outer primer of μ M, reverse each 1.0 μ L of outer primer, the described forward inner primer of 10 μ M, reverse each 1.5 μ L of inner primer, 20ng/ μ L template DNA 1.0 μ L use ddH
2O complements to 25.0 μ L.
7. special primer according to claim 5 is right, it is characterized in that described PCR response procedures is 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30sec, 60sec, 20 circulations are extended in 72 ℃ of annealing; 95 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 50sec, 35 circulations.
8. contain one step of the right wheat stripe rust single tube of the described special primer of claim 1 nest type PCR detection reagent.
9. one step of wheat stripe rust single tube according to claim 8 nest type PCR detection reagent is characterized in that also containing and carries out the needed Taq archaeal dna polymerase of pcr amplification, dNTP and/or PCR damping fluid reagent.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586427A (en) * | 2012-01-13 | 2012-07-18 | 河南科技大学 | Method for identifying wheat leaf rust single sorus by utilizing nested PCR (polymerase chain reaction) |
| CN103981265A (en) * | 2014-05-15 | 2014-08-13 | 董志平 | Nested PCR (Polymerase Chain Reaction) high-efficiency detection method for millet rust fungus |
| CN107227364A (en) * | 2017-07-05 | 2017-10-03 | 上海赛安生物医药科技股份有限公司 | DHFR genetic polymorphism detections system and its kit |
| CN108070657A (en) * | 2017-11-21 | 2018-05-25 | 苏州大学附属第医院 | A kind of DNA point mutation immue quantitative detection reagent box |
| CN108165629A (en) * | 2017-11-21 | 2018-06-15 | 苏州大学附属第医院 | A kind of DNA point mutation quantitative detecting method |
| CN108611437A (en) * | 2017-12-25 | 2018-10-02 | 深圳市第三人民医院 | A kind of primer sets and kit detecting zika virus based on one-step method fluorescent quantitation RT-Nested PCR |
| CN109609677A (en) * | 2018-12-26 | 2019-04-12 | 阿勒泰戈宝茶股份有限公司 | The quickly primer and its reagent and kit of detection bluish dogbane grid rest fungus |
| CN111100943A (en) * | 2020-01-16 | 2020-05-05 | 中国热带农业科学院环境与植物保护研究所 | Single-tube nested PCR (polymerase chain reaction) primer pair for detecting sugarcane puccinia hancei and detection method |
| CN113881804A (en) * | 2021-11-11 | 2022-01-04 | 青海省农林科学院 | Primer pair for wheat stripe rust detection, probe and application |
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| CN102586427A (en) * | 2012-01-13 | 2012-07-18 | 河南科技大学 | Method for identifying wheat leaf rust single sorus by utilizing nested PCR (polymerase chain reaction) |
| CN103981265A (en) * | 2014-05-15 | 2014-08-13 | 董志平 | Nested PCR (Polymerase Chain Reaction) high-efficiency detection method for millet rust fungus |
| CN103981265B (en) * | 2014-05-15 | 2016-11-16 | 董志平 | Millet rest fungus nest-type PRC efficient detection method |
| CN107227364A (en) * | 2017-07-05 | 2017-10-03 | 上海赛安生物医药科技股份有限公司 | DHFR genetic polymorphism detections system and its kit |
| CN108165629B (en) * | 2017-11-21 | 2021-05-11 | 苏州大学附属第一医院 | DNA point mutation quantitative detection method |
| CN108165629A (en) * | 2017-11-21 | 2018-06-15 | 苏州大学附属第医院 | A kind of DNA point mutation quantitative detecting method |
| CN108070657A (en) * | 2017-11-21 | 2018-05-25 | 苏州大学附属第医院 | A kind of DNA point mutation immue quantitative detection reagent box |
| CN108070657B (en) * | 2017-11-21 | 2021-05-11 | 苏州大学附属第一医院 | DNA point mutation quantitative determination kit |
| CN108611437A (en) * | 2017-12-25 | 2018-10-02 | 深圳市第三人民医院 | A kind of primer sets and kit detecting zika virus based on one-step method fluorescent quantitation RT-Nested PCR |
| CN109609677A (en) * | 2018-12-26 | 2019-04-12 | 阿勒泰戈宝茶股份有限公司 | The quickly primer and its reagent and kit of detection bluish dogbane grid rest fungus |
| CN111100943A (en) * | 2020-01-16 | 2020-05-05 | 中国热带农业科学院环境与植物保护研究所 | Single-tube nested PCR (polymerase chain reaction) primer pair for detecting sugarcane puccinia hancei and detection method |
| CN111100943B (en) * | 2020-01-16 | 2023-04-14 | 中国热带农业科学院环境与植物保护研究所 | Single-tube nested PCR (polymerase chain reaction) primer pair for detecting sugarcane ophiocordyceps dorsalis and detection method |
| CN113881804A (en) * | 2021-11-11 | 2022-01-04 | 青海省农林科学院 | Primer pair for wheat stripe rust detection, probe and application |
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