CN103045747B - Molecular detection primer for sweet potato black rot germs and application of molecular detection primer - Google Patents
Molecular detection primer for sweet potato black rot germs and application of molecular detection primer Download PDFInfo
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Abstract
The invention provides a molecular detection primer for sweet potato black rot germs and an application of the molecular detection primer. The specific primer comprises an upstream primer DyB1F: 5'-TTCATCGTCAGGAATACCG-3' and a downstream primer DyB2R: 5'-GACCTTGGCTTTCTTGCTGTA-3';; and a specific amplification product with 509 bp of segment length can be amplified from pure DNA (deoxyribonucleic acid) of the sweet potato black rot germs, a tissue of a germ-carrying disease plant and soil by PCR (Polymerase Chain Reaction) amplification and agarose gel electrophoresis so as to detect the sweet potato black rot germs quickly. The specific molecular detection primer and the application thereof can be used for the quick, sensitive and specific detection of the sweet potato black rot germs in the germ-carrying soil, sick bodies, the disease plants and seedlings in the production practice, can also be used for the early diagnosis of field diseases and the monitoring and identification of the germs, and provides the control of the diseases caused by the sweet potato black rot germs with reliable technical and theoretical bases.
Description
Technical field
The invention belongs to that corps diseases detects, qualification and the field of Prevention Technique, more specifically relate to a kind of sweet potato black rot molecular detection primer and application thereof.
Background technology
Sweet potato
(Ipomoea batatas(L.) Lam.) be world food, one of the energy and insutrial crop, China is global maximum sweet potato plantation state.Sweet potato Black Rotten be by
dickeya dadantiiinfect a kind of disease caused, the stem of sweet potato of can seriously causing harm and root, cause a large amount of dead seedling, have a strong impact on the production of sweet potato.Fang Shumin reported the areas such as Putian, Fujian Province, Huian and Jinjiang and the bacillary Black Rotten of sweet potato occurs in 1991, Pathogen identification is
erwiniasp., what damage loss was serious reaches 80 more than %.The prosperous harm to this disease of Roc in 2003 and popularly to investigate, serious diseased plant rate of falling ill reaches 50%, and dead plant rate can reach 35%.Within 2011, yellow vertical flying waits the cities and counties such as Guangzhou, Huizhou, Zhanjiang, Zengcheng, Haifeng county, Boluo, Huidong in report Guangdong that black stem occurs, and Pathogen identification is
erwinia chrysanthemi.The Disease symptoms of two province's reports is similar, for clear and definite this disease pathogen, over the past two years we in Guangdong, Guangxi, Hainan, Fujian etc. economizes the isolation identification that a large amount of disease sample gathering similar symptom carries out pathogenic bacteria, through comparing the pathogenic bacteria of strain more than 100, the strong malignant bacteria that the Sweet Potato that various places black rots is separated is similar on Physiology and biochemistry, the homology of 16S rDNA and 16S-23S rDNA spacer sequence (ITS) all reaches more than 99%, should be same pathogenic bacteria.Blast compares the sequence that shows its 16S rDNA and Dickey, and to belong to the homology of bacterial strain the highest, and in belonging to Dickey further, the 16S rDNA of the type strain of 5 kinds compares discovery, the 16S rDNA sequence of these bacterial strains with
dickeya dadantiithe homology of type strain is the highest, reaches more than 99.5%.In the process of various places disease sample separation, find not to be separated to malignant bacteria in the plant that a lot (about having 30%) black rots, but the non-pathogenic bacteria that separable acquisition is a large amount of, may be physical abuse or after causing plant to be injured by the other reasons such as physiological reason or insect pest saprophytic bacteria enter plant cause blackout rot.Separating resulting shows that the saprophytic tissue that causes having many types bacterium turns black, and causes difficulty to the qualification accurately carrying out disease.Still do not develop for the detection technique of this bacterium at present, isolation identification can only be carried out by conventional art.In order to positive support disease can be held a situation arises, corresponding measure is taked to carry out prevention and control in time, whether need to determine that field has symptom sample is fast that epiphytotic pathogen causes, and traditional pathogenic bacteria certification method needs the longer time, in order to provide to production application more fast, detection method reliably, the present invention is according to bacterium
gyrBgene order design primer establishes sweet potato black rot PCR detection method.
Summary of the invention
The object of this invention is to provide a kind of sweet potato black rot molecular detection primer and application thereof, long to the cycle needed for the biological detection method of sweet potato black rot for prior art, at present again also not about sweet potato black rot molecule and other method for quick, provide a kind of specific molecular of sweet potato black rot to detect primer.
Realize object of the present invention to comprise the following steps:
1. according to sweet potato Black Rotten
gyrBgene order specificity, devises one couple of PCR primers sweet potato black rot to specific amplified effect, and namely the sequence of special molecular detection primer is:
Upstream primer DyB1F:5 '-TTCATCGTCAGGAATACCG-3 '
Downstream primer DyB2R:5 '-GACCTTGGCTTTCTTGCTGTA-3 '
2. the foundation of sweet potato black rot special molecular detection system
(1) from the plant infected by sweet potato black rot or the soil that there is sweet potato black rot, extract the DNA of black rot;
(2) pcr amplification: PCR reaction system 25 μ L, described PCR reaction system is 2.5 μ L 10 × PCR reaction buffers, 2.5 mmol/L Mg
2+, 100 ~ 400 μm of ol/L dNTPs, 1.25U Taq archaeal dna polymerase, each 0.2 μm of ol/L of primer DyB1F and DyB2R and 10 ~ 100 ng template DNAs, all the other are ddH
2o; PCR reaction conditions is: 94 DEG C of denaturation 4 min, 94 DEG C of sex change 35 s, 52 ~ 64 DEG C of annealing 35 s, and 72 DEG C extend 40 s, circulate 30 times, and 72 DEG C extend 5 min;
(3) then get 3 ~ 8 μ L PCR primer weight concentration 1.0% agarose electrophoresiss to be separated, the size result of determination according to amplified production under ultraviolet lamp after ethidium bromide staining.
(4) if when can amplify 509 bp product specifically, can judge to there is sweet potato black rot in described sample; Otherwise there is not sweet potato black rot in described plant or pedotheque.
From the plant tissue infected by sweet potato black rot, extract DNA in described step (1) adopts DNA rapid fractionation method to obtain the DNA of the sweet potato black rot in incidence tissue, the concrete grammar of described DNA rapid fractionation method is: get 300 mg stem tissues, shred with medical operation scissors, adding 1 mL concentration is 0.1 mol/L, pH is the Tris-HCl damping fluid of 8. 0, leave standstill 15 min, centrifugal 2 min of 3000 rpm, take out supernatant liquid to manage to another 1.5 new mL Eppendorf, centrifugal 2 min of 12000 rpm, abandon supernatant, adding 50 μ L concentration is 0.1 mol/L, pH is the Tris-HCl of 8. 0, damping fluid, put on vortex vibrator and throw out is vibrated into suspension, put in boiling water and boil 8 min, centrifugal 5 min of 12000 rpm, get 5 μ L supernatant liquors and join the Tris-HCl that 495 μ L concentration are 0.1 mol/L, pH is in 8. 0 damping fluids, be mixed and namely can be used for PCR reaction.
The concrete grammar extracting black rot DNA in described step (1) from the soil that there is sweet potato black rot is as follows: get the soil powder of 1 g through grinding, put in 10 mL centrifuge tubes, add 2 mL sodium phosphate buffer (0.12 mol/L, pH 8. 0), mixing, put on 30 DEG C of shaking tables, 150 r/min, shake 15 min.8 000 r/min, centrifugal 10 min, get precipitation.Repeat aforesaid operations, get precipitation.Add 3.7 mL DNA extraction liquid (100 mmol/L Tris-HCl, 100 mmol/LEDTA, 100 mmol/L sodium phosphates, 1.5 mol/L NaCl, 0.01 g/mL CTAB, pH8.0, mixing, add 10 μ L Proteinase Ks (20 mg/mL) again, 37 DEG C of shake 30 min on 225 r/min shaking tables, then adding 0.3 mL weight concentration is 20% SDS, 65 DEG C of water-bath 2 h, every 15 ~ 20 min put upside down gently several under, centrifugal 5 min of room temperature 12 000 r/min, collect supernatant, transfer in new 10 mL centrifuge tubes, soil precipitation adds 0.9 mL extracting solution again and 0.1 mL weight concentration is the SDS of 20%, vortex 10s, 65 DEG C of water-bath 10 min, centrifugal 5 min of room temperature 12 000 r/min, collect supernatant liquor and last time supernatant liquor merge.Repeat aforesaid operations, collection supernatant liquor and front twice supernatant liquor merge.Supernatant liquor and isopyknic chloroform, primary isoamyl alcohol mixed solution mixes, chloroform in described mixing solutions: the volume ratio of primary isoamyl alcohol is 24: 1, centrifugal 5 min of 12000 r/min, drawing aqueous phase is transferred in another 10 new mL centrifuge tubes, add 3 mol/L NaAC solution of 0.1 volume and the Virahol of 0.6 times of volume,-20 DEG C of precipitation 1 h, centrifugal 5 min of room temperature 12000 r/min, collection nucleic acid precipitates,-20 DEG C of ethanol adding 700 μ L volumetric concentrations 70% wash, centrifugal 5 min of 12000 r/min, incline and fall supernatant, dissolve with 1 × TE solution after Bechtop dries alcohol-free taste naturally, obtain DNA solution, by UV spectrophotometer measuring DNA concentration and to be diluted to 50 ng/mL stand-by.
Guardian technique of the present invention is primer sequence and the amplification method thereof of the efficient specific amplified of sweet potato black rot.In order to obtain the specific primer sequences of sweet potato black rot, the present invention be separated with the provinces and cities such as China Fujian, Guangdong, Guangxi, Hainan the multiple bacterial strains be separated in the 45 strain sweet potato black rots and Pseudomonas Solanacearum In Sweet Potatoes, sweet potato healthy plant and rotten plant obtained, multiple other plant pathogenic bacteria, erwinia some plant and common several bacteriums for for examination material, adopt CTAB-SDS method to extract strains tested genomic dna, concrete grammar is as follows:
Getting strains tested inoculum 1.5 mL joins in 1.5 mL Eppendorf pipes, 12000 rpm, centrifugal 2 min; Abandon supernatant liquor, add 500 mL 1 × TE, repeatedly blow and beat mixing with rifle head; Add the Proteinase K that 5 mL concentration are 20 mg/mL, 30mL weight concentration 10% SDS, mixes gently, and 37 DEG C of water-baths place 1 h; After bacterium liquid bleach, add 100 mL 5 mol/L NaCl and mix, add 80 mLCTAB extracting solutions, put upside down centrifuge tube light and slowly and for several times sample is fully mixed, 10 min are placed in 65 DEG C of water-baths; 715 mL phenol, chloroform and primary isoamyl alcohol mixing solutions is added, phenol in described mixing solutions: chloroform: the volume ratio of primary isoamyl alcohol is 25: 24: 1, gentle inversion several, centrifugal 5 min of 12000 rpm after cooling; Careful absorption supernatant is managed to another 1.5 new mL Eppendorf, adds isopyknic above-mentioned phenol, chloroform and primary isoamyl alcohol mixing solutions with Aspirate supernatant, puts upside down mixing; Centrifugal 5 min of 12000 rpm, careful absorption supernatant is managed to another 1.5 new mL Eppendorf, add isopyknic dehydrated alcohol with Aspirate supernatant, gentle inversion is mixing for several times, place 30 min for-20 DEG C, centrifugal 5 min of 12000 rpm, abandon supernatant, add 1 mL 70% washing with alcohol precipitation twice, in Bechtop, dry precipitation, add 100 mL 1 × TE solution after alcohol-free taste and dissolve, obtain DNA solution, be diluted to 50 ng/ μ L by UV spectrophotometer measuring DNA concentration, be stored in-20 DEG C for subsequent use; ClustalX software comparison design special primer is applied on the basis of the DNA sequencing fragment of PCR acquisition sweet potato black rot, obtain sweet potato black rot molecular detection primer specific primer sequences, through carrying out PCR checking to the specificity of strains tested and 45 strain sweet potato black rots, PCR reaction system 25 μ L, described PCR reaction system is 2.5 μ L 10 × PCR reaction buffers, 2.5 mmol/L Mg
2+, 100 ~ 400 μm of ol/L dNTPs, 1.25U Taq archaeal dna polymerase, each 0.2 μm of ol/L of primer DyB1F and DyB2R and 10 ~ 100 ng template DNAs, d.d.H
2o supplies 25 μ L; PCR reaction conditions is: 94 DEG C of denaturation 4 min, 94 DEG C of sex change 35 s, 52 ~ 64 DEG C of annealing 35 s, and 72 DEG C extend 40 s, circulate 30 times, and 72 DEG C extend 5 min; Described special primer amplifies the product of 509 bp specifically on sweet potato black rot.This illustrates that this primer can be used to fall ill in production practice the fast and reliable detection of sweet potato black rot in plant tissue and soil and qualification.
Methods and results of the present invention is reliable, easy handling, highly sensitive, high specificity, can be used for the plant that carries disease germs and soil highly sensitive rapid molecular detects, can be used for the early diagnosis of field sweet potato black rot and the monitoring of germ and qualification simultaneously, this technology for sweet potato black rot cause disease show disease before early monitoring, determine that disease control best period tool is of great significance.
remarkable advantage of the present invention:the present invention is directed to present stage does not still have sweet potato black rot Rapid Identification, by sweet potato black rot and other bacterium
gyrBthe multiple compare of analysis of gene order, for sweet potato black rot
gyrBthe specific designs of gene order has gone out pair of primers, highly sensitive rapid molecular for the soil with sweet potato black rot, invalid body, plant and seedling detects, and sets up that sweet potato black rot is quick, easy, high specificity, highly sensitive Monitoring techniques system.This technology can be applicable to sweet potato black rot cause disease show disease before early monitoring, for determining that disease control best period has important effect, for the formulation of control strategy provides scientific basis.
The inventive method is applicable to detection that in soil, invalid body, plant and seedling, sweet potato black rot is fast and reliable and qualification, has important practical value for the microbial disease control of sweet potato Black Rotten in agriculture production; The present invention compared with prior art, has following technical superiority and positively effect:
1, accurately and reliably: detection method utilizes sweet potato black rot
gyrBthe variability of gene order is consistent with DNA-DAN results of hybridization between bacterium kind, has the feature of conservative region and variable region, according to
gyrBthe distinguished sequence design primer of the variable region of gene order detects.To coming from the sweet potato black rot of different areas and the checkings of other bacterial isolates such as China Fujian, Guangdong, Guangxi, Hainan, result has shown very strong specificity, can supply examination pathogenic bacteria, reliable results by precise Identification;
2, practicality is good: a pair Auele Specific Primer gone out designed by the present invention, can be used for the soil of sweet potato black rot, invalid body, plant and seedling highly sensitive rapid molecular detect, therefore present method is practical, and the sweet potato black rot that can meet existing in morbidity plant tissue, soil carries out fast and reliable detection and the needs of qualification;
3, fast easy and simple to handle: application the inventive method, get final product result of determination, without the need to carrying out digestion with restriction enzyme to amplified production after the rapid extraction of germ DNA and the agarose electrophoresis of routine are carried out to sweet potato black rot morbidity plant tissue.General whole testing process can complete in 4 ~ 6 hours.
accompanying drawing illustrates:
The specific PCR amplification figure of the sweet potato black rot that Fig. 1 will detect for the present invention, wherein: swimming lane 1 is DL 2000 bp DNA molecular weight standard, swimming lane 2-7 is sweet potato black rot, swimming lane 8 is potato black rot, swimming lane 9 is paddy rice Phyllostachys pubescens, swimming lane 10 is banana bacterial soft rot bacterium, swimming lane 11 scholar is radish Bacteria erwinia, swimming lane 12 is pseudomonas syringae cloves pvs oryzae and oryzicola, swimming lane 13 is sweet potato potato seasonal febrile diseases bacterium, swimming lane 14 is bacterial wilt of tomato bacterium, swimming lane 15 is bacterial canker of tomato, swimming lane 16 is intestinal bacteria, swimming lane 17 and 18 is genus bacillus, swimming lane 19 is the thin Population of Xanthomonas Oryzae Pv of paddy bacterial, swimming lane 20-24 is other bacteriums that sweet potato is separated, swimming lane 25 is negative control,
Fig. 2 is that the susceptibility of sweet potato black rot of the present invention detects amplification figure; Wherein: swimming lane 1 is DL 1000 bp DNA molecular weight standard, swimming lane 2 is the sweet potato black rot DNA of 100 ng, swimming lane 3 is the sweet potato black rot DNA of 10 ng, swimming lane 4 is the sweet potato black rot DNA of 1 ng, swimming lane 5 is the sweet potato black rot DNA of 100 pg, and swimming lane 6 is the sweet potato black rot DNA of 10 pg, and swimming lane 7 is the sweet potato black rot DNA of 1 pg, swimming lane 8 is the sweet potato black rot DNA of 100 fg, and swimming lane 9 is the sweet potato black rot DNA of 10 fg;
Fig. 3 is the detected result figure of the present invention to incidence tissue; Wherein: swimming lane 1 is DL 2000 bp DNA molecular weight standard, swimming lane 2,3,4 is field morbidity Sweet Potato tissue, and swimming lane 5 is negative control, and 6 is positive control, and swimming lane 7,8 is healthy Sweet Potato tissue;
Fig. 4 is that the present invention is to the Soil K+adsorption result figure that carries disease germs; Wherein: swimming lane 1 is DL 2000 bp DNA molecular weight standard, and swimming lane 2 is negative control, and swimming lane 3,4,5,6,7,8 is the soil around diseased plant, and swimming lane 9 is positive control.
embodiment:
Technology contents of the present invention comprises the specific detection primer of sweet potato black rot, and designed primer and sequence thereof are:
Upstream primer DyB1F:5 '-TTCATCGTCAGGAATACCG-3 '
Downstream primer DyB2R:5 '-GACCTTGGCTTTCTTGCTGTA-3 '
Utilize this primer can go out the product of 509 bp from specific amplified sweet potato black rot.
The specific primer sequences preparation method of sweet potato black rot molecular detection primer is: try material with some kinds of the multiple bacterial strains be separated in sweet potato black rot, Pseudomonas Solanacearum In Sweet Potatoes, sweet potato healthy plant and rotten plant, multiple other plant pathogenic bacteria, erwinia and common several bacteriums for supplying, CTAB-SDS method is adopted to extract each strains tested genomic dna, concrete grammar is as follows: get strains tested inoculum 1.5 mL and join in 1.5 mL Eppendorf pipes, 12000 rpm, centrifugal 2 min; Abandon supernatant liquor, add 500 mL 1 × TE, repeatedly blow and beat mixing with rifle head; Add the Proteinase K that 5 mL concentration are 20 mg/mL, 30mL weight concentration 10% SDS, mixes gently, and 37 DEG C of water-baths place 1 h; After bacterium liquid bleach, add 100 mL 5 mol/L NaCl and mix, add 80 mLCTAB extracting solutions, put upside down centrifuge tube light and slowly and for several times sample is fully mixed, 10 min are placed in 65 DEG C of water-baths; 715 mL phenol, chloroform and primary isoamyl alcohol mixing solutions is added, phenol in described mixing solutions: chloroform: the volume ratio of primary isoamyl alcohol is 25: 24: 1, gentle inversion several, centrifugal 5 min of 12000 rpm after cooling; Careful absorption supernatant is managed to another 1.5 new mL Eppendorf, adds isopyknic above-mentioned phenol, chloroform and primary isoamyl alcohol mixing solutions with Aspirate supernatant, puts upside down mixing; Centrifugal 5 min of 12000 rpm, careful absorption supernatant is managed to another 1.5 new mL Eppendorf, add isopyknic dehydrated alcohol with Aspirate supernatant, gentle inversion is mixing for several times, place 30 min for-20 DEG C, centrifugal 5 min of 12000 rpm, abandon supernatant, add 1 mL 70% washing with alcohol precipitation twice, in Bechtop, dry precipitation, add 100 mL 1 × TE solution after alcohol-free taste and dissolve, obtain DNA solution, be diluted to 50 ng/ μ L by UV spectrophotometer measuring DNA concentration, be stored in-20 DEG C for subsequent use; ClustalX software comparison design special primer is applied on the basis of PCR acquisition sweet potato black rot specific DNA sequence fragment, obtain sweet potato black rot molecular detection primer specific primer sequences, through carrying out PCR checking to the specificity of strains tested and 45 strain sweet potato black rots, described special primer amplifies the product of 509 bp specifically on sweet potato black rot.
The present invention is set forth further below in conjunction with specific embodiment:
embodiment 1: the specific amplification of primer pair sweet potato black rot
1. the specific detection of sweet potato black rot
The DNA extraction method of each bacterial strain is undertaken by above-mentioned SDS-CTAB method, PCR reaction system 25 μ L, comprises 2.5 μ L 10 × PCR reaction buffers, 2.5 mmol/L Mg
2+, 150 μm of ol/L dNTPs, 1.25U Taq archaeal dna polymerase, each 0.2 μm of ol/L of primer DyB1F and DyB2R and 10 ng template DNAs, all the other are ddH
2o; PCR reaction conditions is: 94 DEG C of denaturation 4 min, 94 DEG C of sex change 35 s, 56 DEG C of annealing 35 s, and 72 DEG C extend 40 s, circulate 30 times, and 72 DEG C extend 5 min.
2. detected result
Specific detection result: except sweet potato black rot can amplify except the product of 509 bp specifically, the potato black rot detected, paddy rice Phyllostachys pubescens, all fail to amplify corresponding product for bacterial strain DNA such as banana bacterial soft rot bacterium, radish Bacteria erwinia, sweet potato potato seasonal febrile diseases bacterium, bacterial wilt of tomato bacterium, genus bacillus, intestinal bacteria, there is very strong specificity (Fig. 1).
embodiment 2: the susceptibility of primer pair sweet potato black rot DNA detects
1. the DNA extraction of bacterial detection and dilution: DNA extraction method is undertaken by above-mentioned SDS-CTAB method, sweet potato black rot DNA solution becomes 100 ng/ μ L, 10 ng/ μ L, 1 ng/ μ L, 100 pg/ μ L, 10 pg/ μ L, 1 pg/ μ L, 100 fg/ μ L, 10 fg/ μ L through gradient dilution, then draws 1 μ L and carries out PCR detection.
2. the susceptibility of sweet potato black rot DNA detects
PCR reaction system 25 μ L, comprises 2.5 μ L 10 × PCR reaction buffers, 2.5 mmol/L Mg
2+, 150 μm of ol/L dNTPs, 1.25U Taq archaeal dna polymerase, each 0.2 μm of ol/L of primer DyB1F and DyB2R and template DNA 1 μ L, all the other are ddH
2o; PCR reaction conditions is: 94 DEG C of denaturation 4 min, 94 DEG C of sex change 35 s, 56 DEG C of annealing 35 s, and 72 DEG C extend 40 s, circulate 30 times, and 72 DEG C extend 5 min.Electrophoresis detection amplified production.
3. detected result: in 25 μ L reaction systems, has the sweet potato black rot genomic dna of 100 more than fg can obtain obvious amplified band (Fig. 2).
embodiment 3: the detection of sweet potato black rot in morbidity plant tissue
1. sample collecting: morbidity sweet potato Plant tissue samples picks up from Xin Dian planting base, Foochow.
2.DNA isolation and determination
Morbidity plant tissue adopts DNA rapid fractionation method to obtain the DNA of the sweet potato black rot in incidence tissue, concrete grammar is for getting 300 mg stem tissues, shred with medical operation scissors, adding 1 mL concentration is 0.1 mol/L, pH is the Tris-HCl damping fluid of 8. 0, leave standstill 15 min, centrifugal 2 min of 3000 rpm, take out supernatant liquid to manage to another 1.5 new mL Eppendorf, centrifugal 2 min of 12000 rpm, abandon supernatant, adding 50 μ L concentration is 0.1 mol/L, pH is the Tris-HCl of 8. 0, damping fluid, put on vortex vibrator and throw out is vibrated into suspension, put in boiling water and boil 8 min, centrifugal 5 min of 12000 rpm, get 5 μ l supernatant liquors and join the Tris-HCl that 495 μ l concentration are 0.1 mol/L, pH is in 8. 0 damping fluids, be mixed and namely can be used for PCR reaction.Carry out pcr amplification as stated above, PCR reaction system 25 μ L, comprise 2.5 μ L 10 × PCR reaction buffers, 2.5 mmol/L Mg
2+, 150 μm of ol/L dNTPs, 1.25U Taq archaeal dna polymerase, each 0.2 μm of ol/L of primer DyB1F and DyB2R and 10 ng template DNAs, ddH
2o supplies 25 μ L; PCR reaction conditions is: 94 DEG C of denaturation 4 min, 94 DEG C of sex change 35 s, 56 DEG C of annealing 35 s, and 72 DEG C extend 40 s, circulate 30 times, and 72 DEG C extend 5 min.Electrophoresis detection amplified production.
3. detected result
The results are shown in Figure 3, various diseased tissues all see one clearly molecular weight be the specific band of 509 bp, thus judge that incidence tissue infects sweet potato black rot.
embodiment 4: the detection of sweet potato black rot in pedotheque
1. sample collecting: band soil bacteria is the soil that Xin Dian planting base, Foochow contacts with typical disease plant.
2.DNA isolation and determination
Get the soil powder of 1 g through grinding, put in 10 mL centrifuge tubes, add 2 mL sodium phosphate buffers (0.12 mol/L, pH 8. 0), mixing, put on 30 DEG C of shaking tables, 150 r/min, shake 15 min.8000 r/min, centrifugal 10 min, get precipitation.Repeat aforesaid operations, get precipitation.Add 3.7 mL DNA extraction liquid (100 mmol/L Tris-HCl, 100 mmol/LEDTA, 100 mmol/L sodium phosphates, 1.5 mol/L NaCl, 0.01 g/mL CTAB, pH8.0) mix, add 10 μ L Proteinase Ks (20 mg/mL) again, 37 DEG C of shake 30 min on 225 r/min shaking tables, then adding 0.3 mL weight concentration is 20% SDS, 65 DEG C of water-bath 2 h, every 15 ~ 20 min put upside down gently several under, centrifugal 5 min of room temperature 12000 r/min, collect supernatant, transfer in new 10 mL centrifuge tubes, soil precipitation adds 0.9 mL extracting solution again and 0.1 mL weight concentration is the SDS of 20%, vortex 10s, 65 DEG C of water-bath 10 min, centrifugal 5 min of room temperature 12000 r/min, collect supernatant liquor and last time supernatant liquor merge.Repeat aforesaid operations, collection supernatant liquor and front twice supernatant liquor merge.Supernatant liquor and isopyknic chloroform, primary isoamyl alcohol mixed solution mixes, chloroform in described mixing solutions: the volume ratio of primary isoamyl alcohol is 24: 1, centrifugal 5 min of 12000 r/min, drawing aqueous phase is transferred in another 10 new mL centrifuge tubes, add 3 mol/L NaAC solution of 0.1 volume and the Virahol of 0.6 times of volume,-20 DEG C of precipitation 1 h, centrifugal 5 min of room temperature 12000 r/min, collection nucleic acid precipitates,-20 DEG C of ethanol adding 700 μ L volumetric concentrations 70% wash, centrifugal 5 min of 12000 r/min, incline and fall supernatant, dissolve with 1 × TE solution after Bechtop dries alcohol-free taste naturally, obtain DNA solution, by UV spectrophotometer measuring DNA concentration and to be diluted to 20 ng/mL stand-by.Carry out pcr amplification as stated above, PCR reaction system 25 μ L, comprise 2.5 μ L 10 × PCR reaction buffers, 2.5 mmol/L Mg
2+, 150 μm of ol/L dNTPs, 1.25U Taq archaeal dna polymerase, each 0.2 μm of ol/L of primer DyB1F and DyB2R and 10 ng template DNAs, ddH
2o supplies 25 μ L; PCR reaction conditions is: 94 DEG C of denaturation 4 min, 94 DEG C of sex change 35 s, 56 DEG C of annealing 35 s, and 72 DEG C extend 40 s, circulate 30 times, and 72 DEG C extend 5 min.Electrophoresis detection amplified production.
3. detected result
The results are shown in Figure 4, have in 4 pedotheques amplify one clearly molecular weight be the specific band of 509 bp, thus judge that these soil samples are with sweet potato black rot.
<110> Fujian Academy of Agricultural Sciences Crop Research Institute
<120> sweet potato black rot molecular detection primer and application thereof
<160> 2
<210> 1
<211>19
<212> DNA
<213> artificial sequence
<400> 1
ttcatcgtca ggaataccg 19
<210> 2
<211> 21
<212> DNA
<213> artificial sequence
<400> 2
gaccttggct ttcttgctgta 21
Claims (4)
1. a sweet potato black rot molecular detection primer, is characterized in that: the sequence of described sweet potato black rot molecular detection primer is:
DyB1F ：5’-TTCATCGTCAGGAATACCG -3’
DyB2R ：5’-GACCTTGGCTTTCTTGCTGTA-3’。
2. an application for sweet potato black rot molecular detection primer as claimed in claim 1, is characterized in that: described sweet potato black rot molecular detection primer is applied to the rapid molecular checkout and diagnosis of sweet potato black rot, detects according to following steps:
(1) from the plant infected by sweet potato black rot or the soil that there is sweet potato black rot, extract the DNA of black rot;
(2) with this DNA for described pair of primers DyB1F and DyB2R of template is increased by polymerase chain reaction PCR; PCR reaction system 25 μ L, described PCR reaction system is 2.5 μ L 10 × PCR reaction buffers, 2.5 mmol/L Mg
2+, 100 ~ 400 μm of ol/L dNTPs, 1.25U Taq DNA polysaccharase, each 0.2 μm of ol/L of primer DyB1F and DyB2R and 10 ~ 100 ng template DNAs, all the other are ddH
2o; PCR reaction conditions is: 94 DEG C of denaturation 4 min, 94 DEG C of sex change 35s, 52 ~ 64 DEG C of annealing 35 s, and 72 DEG C extend 40 s, circulate 30 times, and 72 DEG C extend 5 min;
(3) the PCR amplified production agarose electrophoresis of then getting step (2) is separated, observe under ultraviolet lamp after ethidium bromide staining, according to the size result of determination of amplified production, if the product of 509 bp can be amplified specifically, can judge to there is sweet potato black rot in described sample.
3. according to the application of the sweet potato black rot molecular detection primer described in claim 2, it is characterized in that: from the plant tissue infected by sweet potato black rot, extract DNA in described step (1) adopt DNA rapid fractionation method to obtain the DNA of the sweet potato black rot in incidence tissue, the concrete grammar of described DNA rapid fractionation method is for getting 300 mg Sweet Potato tissues, shred with medical operation scissors, adding 1 mL concentration is 0.1 mol/L, pH is the Tris-HCl damping fluid of 8.0, leave standstill 15 min, centrifugal 2 min of 3000 rpm, take out supernatant liquid to manage to another 1.5 new mL Eppendorf, the centrifugal 2min of 12000 rpm, abandon supernatant, adding 50 μ L concentration is 0.1mol/L, pH is the Tris-HCl damping fluid of 8.0, put on vortex vibrator and throw out is vibrated into suspension, put in boiling water and boil 8 min, centrifugal 5 min of 12000 rpm, getting 5 μ L supernatant liquors, to join 495 μ L concentration be the Tris-HCl of 0.1mol/L, pH is in 8.0 damping fluids, namely mixing can be used for PCR reaction.
4. according to the application of the sweet potato black rot molecular detection primer described in claim 2, it is characterized in that: described step (3) is separated for getting 3 ~ 8 μ L PCR amplified production weight concentration 1.0% agarose electrophoresiss.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310006354.2A CN103045747B (en) | 2012-11-12 | 2013-01-09 | Molecular detection primer for sweet potato black rot germs and application of molecular detection primer |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210448667.9 | 2012-11-12 | ||
| CN201210448667 | 2012-11-12 | ||
| CN2012104486679 | 2012-11-12 | ||
| CN201310006354.2A CN103045747B (en) | 2012-11-12 | 2013-01-09 | Molecular detection primer for sweet potato black rot germs and application of molecular detection primer |
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| CN103045747A CN103045747A (en) | 2013-04-17 |
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| CN107868845A (en) * | 2017-12-18 | 2018-04-03 | 福建省农业科学院植物保护研究所 | A kind of sweet potato blackspot bacterium nest-type PRC detection primer and its molecular detecting method |
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| CN105969917B (en) * | 2016-06-12 | 2019-04-19 | 桐乡市恒球皮草制品有限公司 | A kind of standard mink skin nitrate technique |
| CN109371104A (en) * | 2018-11-21 | 2019-02-22 | 广东海洋大学 | A method of detection is extracted convenient for the rice blast ospc gene to rice |
| CN109402288A (en) * | 2018-11-29 | 2019-03-01 | 安徽省农业科学院烟草研究所 | It is a kind of for detecting the primer and detection method of thielaviopsis sp bacterium |
| CN113139461A (en) * | 2021-04-23 | 2021-07-20 | 塔里木大学 | Wheat leaf disease and insect pest detection system for agricultural planting and management method thereof |
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| CN1824799A (en) * | 2005-12-27 | 2006-08-30 | 云南农业大学 | PCR testing and identification optimizing process for carrot black rot |
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| CN107868845A (en) * | 2017-12-18 | 2018-04-03 | 福建省农业科学院植物保护研究所 | A kind of sweet potato blackspot bacterium nest-type PRC detection primer and its molecular detecting method |
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