CN103882125B - A kind of real time fluorescence quantifying PCR method detecting Chinese cabbage verticillium pathogenic bacteria - Google Patents

A kind of real time fluorescence quantifying PCR method detecting Chinese cabbage verticillium pathogenic bacteria Download PDF

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CN103882125B
CN103882125B CN201410089841.4A CN201410089841A CN103882125B CN 103882125 B CN103882125 B CN 103882125B CN 201410089841 A CN201410089841 A CN 201410089841A CN 103882125 B CN103882125 B CN 103882125B
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于拴仓
苏同兵
陈娟
张凤兰
汪维红
余阳俊
张德双
赵岫云
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Beijing Academy of Agriculture and Forestry Sciences
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Abstract

The invention discloses a kind of real time fluorescence quantifying PCR method detecting Chinese cabbage verticillium pathogenic bacteria.Pair of primers disclosed by the invention, by SEQ? ID? DNA molecular shown in No.1 and SEQ? ID? DNA molecular composition shown in No.2.Method disclosed by the invention be one fast, resistance to verticillium wilt molecular diagnosis system accurately and efficiently, for good basis has been established in Chinese cabbage resisting verticillium Screening germplasm and resistance transformation; In addition, the method also can be used for Chinese cabbage verticillium prediction, and the safety in production for Chinese cabbage is significant.

Description

A kind of real time fluorescence quantifying PCR method detecting Chinese cabbage verticillium pathogenic bacteria
Technical field
The present invention relates to a kind of real time fluorescence quantifying PCR method detecting Chinese cabbage verticillium pathogenic bacteria.
Background technology
Mostly verticillium is to infect by Huang verticillium dahliae, the black and white Verticillium that Verticillium belongs to that wither the soil-borne vascular bundle disease caused from root, the host range of this disease is wide, there is different physiological strains, its Microsclerotia survival time in soil is long, there is the ability of highly resisting poor environment, difficulty of prevention and cure is large, has caused various crop such as cotton, tomato, capsicum, eggplants at present and has had a strong impact on, be called as " cancer " of plant.
Chinese cabbage belongs to Cruciferae Brassica genus, and be the maximum vegetable crop of China's cultivated area, become international vegetable crop at present gradually.According to Ministry of Agriculture's statistics, nearly 4,000 ten thousand mu of China's Chinese cabbage Annual planting area, accounts for about 15% of the total sown area of vegetables, and visible Chinese cabbage is very important in China's vegetable basket project.At present the research of Chinese cabbage disease is focused mostly in virus disease, soft rot, the large disease of oidium three, and there occurs very serious verticillium in Beijing and some extensive Chinese cabbage production bases in recent years, and in ascendant trend year by year.Research shows that this disease is the Chinese cabbage verticillium caused by verticillium dahliae (Verticilliumdahliae), this disease starts morbidity in bag heart stage, and its Symptoms is that blade is withered and yellow, and plant is downgraded, the blackening of vascular bundle browning, then can cause Chinese cabbage to be had no harvest time serious.
Traditional host resistance qualification is generally adopt greenhouse artificial infection idenfication and field Disease garden identification, but is subject to the impact of envrionment conditions, human factor and non-targeted pathogenic bacteria, brings certain error to qualification result.When verticillium occurs, the symptom occurred the earliest is that radical leaves turns yellow, and further developing is that growth retardation appears withered spot, in blade, wilts, leaf abscission, last fibrovascular system browning look.Verticillium artificial inoculation Disease Resistance Identification, generally in tri-leaf period, adopts and dips in root inoculation method, and because Chinese cabbage seedling lignifying level is low, after inoculation verticillium wilt pathogen, slow seedling is slow, and blade also easily occurs etiolation, brings very large difficulty to the differentiation of verticillium symptom.Therefore, to identify the resistance to verticillium wilt of Chinese cabbage breeding material in early days, a kind of simple, sensitive, detection of pathogens technology accurately must be set up.
In detection of pathogens method, Real-Time Fluorescent Quantitative PCR Technique has unique advantage, has been widely used in the detection of phytopathogen and plant virus.The method utilizes Auele Specific Primer to the DNA of target compound in the vegetable material that increases, and more adequately can detect the relative content of pathogenic bacteria in different tissues, differing materials.Gayoso etc. apply Real-Time Fluorescent Quantitative PCR Technique and have detected pathogenic bacteria content in the capsicum and tomato of being infected by verticillium dahliae (Verticilliumdahliae), find that the relative content of germ in disease plant is higher than disease-resistant plant, and the plant of different genotype show pathogenic bacteria in the severity of illness and plant body relative content have dependency.Dan etc. utilize the number of pathogenic bacteria DNA content in potato plant to evaluate the anti-sense level of plant to verticillium.Tradition pathogenic bacteria content detection adopts colony counting method usually, the dull and stereotyped detection method of such as NP-10, but the method bacterial isolate bacterium from diseased plant probably needs two weeks consuming time, therefore can not be used for large batch of detection practice; Secondly the method effectively can not distinguish the sibling species of verticillium dahliae.Compared with traditional detection of pathogens method, qRT-PCR technology has the advantages such as sensitive, quick, special.Concrete advantage is as follows:
1. the specificity detected.Effectively can distinguish the sibling species of verticillium dahliae.Fungi has the conservative property of height in planting at rDNA-ITS section, there is again polymorphism widely, be widely used in the taxonomic identification of germ, monitoring and disease screening etc. between genus or between planting.
2. the susceptibility detected.The Schwellenwert detected can reach 5 × 10 -3ng μ L -1.
3. the rapidity detected.Again to the total time utilizing real-timePCR technology to obtain pathogenic bacteria content being no more than 8 hours from preparation of samples to DNA extraction.
4. the high flux property detected.Utilize 384 modules of Roche Holding Ag LightCycler480PCR instrument, the accurate quantitative analysis of 384 sample pathogenic bacterias in 50 minutes, can be realized.
5. the popularity of test material growth time.This technology can detect the former bacterium infection conditions of verticillium in any period from Chinese cabbage seedling to strain.
Summary of the invention
The object of this invention is to provide a kind of real time fluorescence quantifying PCR method detecting Chinese cabbage verticillium pathogenic bacteria.
Pair of primers provided by the invention, is made up of the DNA molecular shown in the DNA molecular shown in SEQIDNo.1 and SEQIDNo.2.
The test kit of detection by quantitative Chinese cabbage verticillium pathogenic bacteria also belongs to a protection scope of the present invention, and this test kit comprises above-mentioned primer pair.
In mentioned reagent box, described Chinese cabbage verticillium pathogenic bacteria is verticillium dahliae (Verticilliumdahliae), is specially Chinese cabbage verticillium pathogenic bacteria BCHW10-2.
The real time fluorescence quantifying PCR method of detection by quantitative Chinese cabbage verticillium pathogenic bacteria also belongs to a protection scope of the present invention, and the method comprises the steps:
(1) DNA of the verticillium pathogenic bacteria standard substance of extraction is carried out 10 times of gradient dilutions, obtain the DNA of the verticillium pathogenic bacteria of gradient dilution, with it for template, with the DNA molecular shown in the DNA molecular shown in SEQIDNo.1 and SEQIDNo.2 for primer carries out real-time fluorescence quantitative PCR amplification, with the Log value of template concentrations for X-coordinate, reach the cycle number of set threshold value experience for ordinate zou with the fluorescent value of each reacting hole, production standard curve, obtains typical curve formula 1;
(2) genomic dna of the root not contaminating the Chinese cabbage of any germ extracted, stem or leaf texture is carried out twice gradient dilution, obtain the DNA of the sample of gradient dilution, with it for template, with the primer of the GAPDH that increases for primer carries out real-time fluorescence quantitative PCR amplification, with the Log value of template concentrations for X-coordinate, reach the cycle number of set threshold value experience for ordinate zou with the fluorescent value of each reacting hole, production standard curve, obtains typical curve formula 2;
(3) with the genomic dna of the root of Chinese cabbage to be checked, stem or leaf texture for template, with the DNA molecular shown in the DNA molecular shown in SEQIDNo.1 and SEQIDNo.2 for primer carries out real-time fluorescence quantitative PCR amplification, obtain cycle number CP1, bring CP1 value into typical curve formula 1, obtain the absolute content of Chinese cabbage verticillium pathogenic bacteria in Chinese cabbage to be checked; With the genomic dna of the root of Chinese cabbage to be checked, stem or leaf texture for template, carry out real-time fluorescence quantitative PCR amplification with GAPDH primer for primer, obtain cycle number CP2, bring CP2 value into typical curve formula 2, obtain the GAPDH absolute content of measuring samples; Chinese cabbage Neihuang County to be checked wither disease pathogen bacterium relative content=Chinese cabbage to be checked in the absolute content/GAPDH absolute content of Chinese cabbage verticillium pathogenic bacteria.
In aforesaid method, the PCR reaction system of described real-time fluorescence quantitative PCR is as follows: real-time fluorescence quantitative PCR amplification buffer (2 ×) 5 μ L, and concentration is each 1 μ L of the primer of 10 μMs, template 3 μ L;
The trade name of described real-time fluorescence quantitative PCR amplification buffer (2 ×) is SYBRGreenIMaster (2 ×), from 480cSYBRGreenIMaster test kit, this test kit is purchased from Roche Holding Ag, and catalog number is 04887352001;
PCR program is as follows: denaturation 95 DEG C, 5min; 95 DEG C of sex change 30s, 60 DEG C of renaturation 30s, 72 DEG C extend 50s, 40 circulations;
Melting curve production process: 95 DEG C of 5s, 65 DEG C of 60s, every 10s temperature is risen progressively 0.5 DEG C to 97 DEG C end, continuous detecting fluorescence intensity.
In above-mentioned arbitrary described method, the DNA concentration of the verticillium pathogenic bacteria BCHW10-2 extracted in described step (1) is 50ng μ L -1;
The genomic dna concentration of the root of the Chinese cabbage of extracting in described step (2), stem or leaf texture is 200ng μ L -1;
Described typical curve formula 1 is Y=-3.553X+10.04;
Described typical curve formula 2 is Y=-3.193X+23.22.
In above-mentioned arbitrary described method, described verticillium pathogenic bacteria is Chinese cabbage verticillium pathogenic bacteria BCHW10-2.
Application in the Chinese cabbage verticillium pathogenic bacteria of above-mentioned primer pair in detection Chinese cabbage also belongs to protection scope of the present invention;
Or,
The application whether above-mentioned primer pair infects in Chinese cabbage verticillium pathogenic bacteria in detection Chinese cabbage also belongs to protection scope of the present invention.
The application that mentioned reagent box is detecting the Chinese cabbage verticillium pathogenic bacteria in Chinese cabbage also belongs to protection scope of the present invention;
Or,
The application whether mentioned reagent box infects in Chinese cabbage verticillium pathogenic bacteria in detection Chinese cabbage also belongs to protection scope of the present invention.
In above-mentioned arbitrary described application, described Chinese cabbage verticillium pathogenic bacteria is verticillium dahliae (Verticilliumdahliae), is specially Chinese cabbage verticillium pathogenic bacteria BCHW10-2.
Verticillium has developed into one of soil-borne disease important in Chinese cabbage production, excavates disease-resistant gene resource, and cultivating stable, permanent disease-resistant kind, is the effective way preventing Chinese cabbage verticillium pandemic.Method provided by the invention be one fast, resistance to verticillium wilt molecular diagnosis system accurately and efficiently, for good basis has been established in Chinese cabbage resisting verticillium Screening germplasm and resistance transformation; In addition, the method also can be used for verticillium prediction, and the safety in production for Chinese cabbage is significant.
Accompanying drawing explanation
Fig. 1 is the specific detection result of primer.
Fig. 2 is the sensitivity technique result of primer.
Fig. 3 is the incidence of Chinese cabbage artificial inoculation verticillium pathogenic bacteria.
Fig. 4 utilizes real-time fluorescence quantitative PCR to the qualification of the material of artificial inoculation verticillium pathogenic bacteria.
Fig. 5 is Chinese cabbage verticillium field incidence and the qualification utilizing real-time fluorescence quantitative PCR.
Fig. 6 is the typical curve of amplification verticillium pathogenic bacteria.
Fig. 7 is the typical curve of amplification Chinese cabbage genomic dna.
Fig. 8 is the amplification melting curve of primer HW1 and GAPDH.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Chinese cabbage verticillium pathogenic bacteria BCHW10-2(Verticilliumdahliae) document " Han Ruijuan; Geng Lihua; Wang Weihong, in fastening storehouse, Zhu Yuelin; Zhang Fenglan; Yu Yangjun, Zhao Xiuyun, Zhang Deshuan. the Pathogen identification of Beijing area Chinese cabbage verticillium. " gardening journal " the 03rd phase in 2012 " in be disclosed, the public can obtain from Beijing City Agriculture and Forestry Institute, and this Pseudomonas is in verticillium dahliae (Verticilliumdahliae).
480cSYBRGreenIMaster test kit is purchased from Roche Holding Ag, and catalog number is 04887352001.
Chinese cabbage downy mildew (HyaloperonosporaparasiticaConstant. (Pers.exFr.)) document " Li Hui; in fastening storehouse; Zhang Fenglan; Yu Yangjun; Zhao Xiuyun, Zhang Deshuan, Zhao Xiang. with the molecular markers development .HEREDITAS (2011) of Chinese cabbage downy mildew resistance main effect QTL linkage; 33 (11): 1271-1278 " in be disclosed, the public can obtain from Beijing City Agriculture and Forestry Institute.
GAPDH primer is disclosed in document " QiJN; YuSC; ZhangFL; etal.ReferenceGeneSelectionforReal-TimeQuantitativePolym eraseChainReactionofmRNATranscriptLevelsinChineseCabbage (BrassicarapaL.ssp.pekinensis) [J] .PlantMolBiolRep; 2010,28:597 – 604 ".
Embodiment 1, design of primers
One, design of primers
It is as follows that the rDNA-ITS sequence (NCBI accession number be: JN564038) special according to verticillium pathogenic bacteria designs specific amplification primer:
HW1-F：5’-GTTACAGCTCGCATCGGAGT-3’（SEQIDNo.1）
HW1-R：5’-CAGCGGGTATTCCTACCTGA-3’（SEQIDNo.2）
The object fragment length of amplification is 100bp.
Two, primer specificity and sensitivity technique
(1) primer specificity detects
Extract the leaf tissue not infecting the Chinese cabbage of Chinese cabbage verticillium pathogenic bacteria, infect the leaf tissue of the Chinese cabbage of Chinese cabbage downy mildew, Chinese cabbage verticillium pathogenic bacteria BCHW10-2, infects the DNA of the sick leaf of the Chinese cabbage of Chinese cabbage verticillium pathogenic bacteria, sick stem and old complaint, and with ddH 2o is blank.
Respectively with the DNA of said extracted for template, HW1-F and HW1-R is that primer carries out pcr amplification, obtains pcr amplification product, and the agarose gel electrophoresis analytical results of each product is as shown in Figure 1.
In Fig. 1, M is DNAmarker; 1 template is ddH 2o; 2 templates are not from healthy tree (infecting the Chinese cabbage of Chinese cabbage verticillium pathogenic bacteria); 3 templates carry out the Chinese cabbage of self-infection Chinese cabbage downy mildew; 4 templates are from BCHW10-2; 5,6,9 templates are from sick leaf; 7,10 templates are from sick stem; 8 templates are from old complaint.
Fig. 1 shows, HW1-F and HW1-R is to detecting that Chinese cabbage verticillium pathogenic bacteria has specificity.
(2) primer sensitivity technique
Adopt Roche 480cSYBRGreenIMaster test kit, with the DNA of BCHW10-2 reference culture, for template, (concentration is 50ng μ L -1), carry out ten times of dilutions, obtain 5 gradients, with it for template, with HW1-F and HW1-R for primer, carry out real-time fluorescence quantitative PCR.
PCR reaction system (10 μ L): SYBRGreenIMaster (2 ×) 5 μ L, HW1-F (concentration is 10 μMs) 1 μ L, HW1-R (concentration is 10 μMs) 1 μ L, template DNA 3 μ L.
PCR program: denaturation 95 DEG C, 5min; 95 DEG C of sex change 30s, 60 DEG C of renaturation 30s, 72 DEG C extend 50s, 40 circulations.
Result as shown in Figure 2.
In Fig. 2, the bacterium liquid DNA being from left to right followed successively by 1,10,100,1000,10000 times of dilution is template, carries out the amplification curve of real-time fluorescence quantitative PCR.
Fig. 2 shows, the minimum concentration of 30 circulation internal standard curve detection is 50ng μ L -1× 10 -4, prove that the sensitivity of primer is good.
Embodiment 2, fluorescence quantifying PCR method detect verticillium pathogenic bacteria in sample
One, sample prepares
Greenhouse sample: extracted from nutrition pot (naturally hindering root) by the seedling growing to three leaves each kind Chinese cabbage wholeheartedly, with Chinese cabbage verticillium pathogenic bacteria BCHW10-2 spore suspension, (concentration is 1 × 10 7individual/mL) soak seedling root 15 minutes, then seedling is planted again in the nutrition pot of 6 × 6cm, simultaneously to inoculate sterilized water for contrast; The plant of inoculation pathogenic bacteria is sampled, to connect the plant of sterilized water as a control group after 2,3,4 and 5 weeks inoculation verticillium pathogenic bacteria respectively.Often organize material and get 5 strains, root system, stem and blade are separately preserved, ultra low temperature vacuum freezes in sample instrument for subsequent use after freeze-drying.
Field sample: get its blade as sample the Chinese cabbage of field field planting each kind of 10 weeks.
Fig. 3 is the incidence of Chinese cabbage artificial inoculation verticillium pathogenic bacteria BCHW10-2, wherein A 1for the plant of V2-1 kind inoculation sterilized water after 2 weeks; A 2-A 5be respectively the V2-1 kind inoculation pathogenic bacteria plant of 2,3,4,5 weeks; B 1for the plant of V2-2 kind inoculation sterilized water after 2 weeks; B 2-B 5for the V2-2 kind inoculation pathogenic bacteria plant of 2,3,4,5 weeks; C 1for the plant of V2-8 kind inoculation sterilized water after 2 weeks; C 2-C 5for the V2-8 kind inoculation pathogenic bacteria plant of 2,3,4,5 weeks; D 1for the plant of V2-9 kind inoculation sterilized water after 2 weeks; D 2-D 5for the V2-9 kind inoculation pathogenic bacteria plant of 2,3,4,5 weeks.
Two, the DNA of each sample of extraction step one gained, and concentration is adjusted to 200ng/ μ L.
Three, real-time fluorescence quantitative PCR
Adopt Roche 480cSYBRGreenIMaster test kit, the DNA that respectively organizes of greenhouse sample extracted with step 2 for template, with HW1-F and HW1-R for primer carries out real-time fluorescence quantitative PCR amplification.
PCR reaction system (10 μ L): SYBRGreenIMaster (2 ×) 5 μ L, HW1-F (concentration is 10 μMs) 1 μ L, HW1-R (concentration is 10 μMs) 1 μ L, template DNA (concentration is 200ng/ μ L) 3 μ L.
PCR program: denaturation 95 DEG C, 5min; 95 DEG C of sex change 30s, 60 DEG C of renaturation 30s, 72 DEG C extend 50s, 40 circulations;
Melting curve production process: 95 DEG C of 5s, 65 DEG C of 60s, every 10s temperature is risen progressively 0.5 DEG C to 97 DEG C end, continuous detecting fluorescence intensity.
The real-time fluorescence quantitative PCR detected result of greenhouse sample as shown in Figure 4.
Fig. 4 A-4D is respectively artificial inoculation verticillium pathogenic bacteria BCHW10-22, the plant of 3,4,5 weeks.
Fig. 4 shows, adopt fluorescent quantitative PCR technique, utilize primer HW1-F and HW1-R, have detected the content of the Chinese cabbage verticillium pathogenic bacteria BCHW10-2 in four kinds of Chinese cabbage inbred lines plant (V2-1, V2-2, V2-8 and V2-9) bodies, find that germ content in each plant in root and stem is apparently higher than leaf.
Connect the 3rd week after being ill and the 4th week, in blade, Chinese cabbage verticillium pathogenic bacteria BCHW10-2 relative content shows notable difference in anti-sense material, verticillium pathogenic bacteria content in susceptible self-mating system V2-8 and V2-9 is apparently higher than disease-resistant self-mating system V2-1 and V2-2, germ content order is from high to low V2-8 > V2-9 > V2-1 > V2-2, this is consistent with the disease severity that verticillium symptom observation (Fig. 3) reflects, the relative content of visible leaf portion pathogenic bacteria accurately can reflect the Resistant Difference of differing materials.
Chinese cabbage verticillium field incidence and utilize real-time fluorescence quantitative PCR qualification result respectively as shown in Figure 5.
18 Chinese cabbage inbred lines that in Fig. 5, each digitized representation incidence is different.
Fig. 5 shows, utilize real-time fluorescence quantitative PCR detect the Chinese cabbage verticillium field obtained fall ill the content results of each self-mating system Chinese cabbage verticillium germ BCHW10-2 and each Chinese Cabbage incidence basically identical.
Four, the content of verticillium pathogenic bacteria in detection by quantitative sample
(1) making of amplification verticillium pathogenic bacteria typical curve
With the DNA of verticillium pathogenic bacteria BCHW10-2 for template, mother liquid concentration is 50ng μ L -1, undertaken diluting (1,10 by 10 times of gradients with CTAB/NaCl solution (50g/LCTAB, 0.5mol/LNaCl, surplus is water) -1, 10 -2, 10 -3with 10 -4doubly), obtain the DNA of each dilution verticillium pathogenic bacteria BCHW10-2, with it for template, the typical curve of the fluorescent quantitative PCR of verticillium pathogenic bacteria BCHW10-2 is obtained according to the method for step 3, with the Log value of template concentrations for X-coordinate, reach the cycle number of set threshold value experience for ordinate zou with fluorescent signal in reaction tubes, make verticillium pathogenic bacteria amplification typical curve, as shown in Figure 6, typical curve formula 1:Y=-3.553X+10.04 is obtained.
(2) making of amplification Chinese cabbage genomic dna typical curve
With the genomic dna of cabbage leaves tissue, for template, (concentration is 200ng μ L -1), with CTAB/NaCl solution (50g/LCTAB, 0.5mol/LNaCl, surplus is water) undertaken diluting by twice and (dilute 1 for original concentration respectively, 0.5, 0.25, 0.125, 0.0625, 0.03125), with each dilution DNA sample for template, according to the method for step 3, primer is replaced with GAPDH primer, obtain the typical curve of the fluorescent quantitative PCR of Chinese cabbage genomic dna, with the Log value of template concentrations for X-coordinate, the cycle number of set threshold value experience is reached for ordinate zou with fluorescent signal in reaction tubes, production standard curve, as shown in Figure 7, obtain typical curve formula 2:Y=-3.193X+23.22.
The object making this curve is using glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) as reference gene, carries out homogenization to sample DNA concentration,
(3) with the DNA of Chinese cabbage verticillium pathogenic bacteria BCHW10-2 for template, with HW1(HW1-F and HW1-R) be primer, with the genomic dna of not susceptible cabbage leaves for template, with GAPDH primer for primer carries out real-time fluorescence quantitative PCR, melting curve as shown in Figure 8.
Fig. 8 shows, the DNA of verticillium pathogenic bacteria special primer HW1 to Chinese cabbage verticillium pathogenic bacteria BCHW10-2 is utilized to carry out real-time fluorescence quantitative PCR analysis, the genomic dna of GAPDH primer pair Chinese cabbage is utilized to carry out real-time fluorescence quantitative PCR analysis, find that the amplification melting curve of Chinese cabbage verticillium pathogenic bacteria BCHW10-2 and Chinese cabbage DNA all has sharp keen simple spike, without the appearance of primer dimer, explanation design of primers is reasonable, and specificity is higher; The Tm value of HW1 and GAPDH amplified production is respectively 80 DEG C and 86 DEG C, and the two has notable difference.
(4) detection of verticillium pathogenic bacteria content in sample
With the DNA of measuring samples for template, respectively with HW1 primer pair and GAPDH primer for primer, carry out real-time fluorescence quantitative PCR according to the method for step 3, obtain the absolute content of verticillium pathogenic bacteria in sample and reference gene (GAPDH) according to typical curve formula 1 and typical curve formula 2 respectively.
The absolute quantitation pattern utilizing Lightcyclerversion2.0 (RocheDiagnostics) software is the content of verticillium pathogenic bacteria and reference gene in analytic sample respectively, and wither the relative content=verticillium pathogenic bacteria DNA absolute content/reference gene DNA absolute content of disease pathogen bacterium in sample Neihuang County.Statistical study SPSS18.0, Sigmaplot12.0 and Excel2010 software carries out.

Claims (13)

1. pair of primers, is made up of the DNA molecular shown in the DNA molecular shown in SEQIDNo.1 and SEQIDNo.2.
2. a test kit for detection by quantitative Chinese cabbage verticillium pathogenic bacteria, this test kit comprises primer according to claim 1.
3. test kit according to claim 2, is characterized in that: described Chinese cabbage verticillium pathogenic bacteria is verticillium dahliae (Verticilliumdahliae).
4. test kit according to claim 3, is characterized in that: described verticillium dahliae (Verticilliumdahliae) is Chinese cabbage verticillium pathogenic bacteria BCHW10-2.
5. a real time fluorescence quantifying PCR method for detection by quantitative Chinese cabbage verticillium pathogenic bacteria, the method comprises the steps:
(1) DNA of the verticillium pathogenic bacteria standard substance of extraction is carried out 10 times of gradient dilutions, obtain the DNA of the verticillium pathogenic bacteria of gradient dilution, with it for template, with the DNA molecular shown in the DNA molecular shown in SEQIDNo.1 and SEQIDNo.2 for primer carries out real-time fluorescence quantitative PCR amplification, with the Log value of template concentrations for X-coordinate, reach the cycle number of set threshold value experience for ordinate zou with the fluorescent value of each reacting hole, production standard curve, obtains typical curve formula 1;
(2) genomic dna of the root not contaminating the Chinese cabbage of any germ extracted, stem or leaf texture is carried out twice gradient dilution, obtain the DNA of the sample of gradient dilution, with it for template, with the primer of the GAPDH that increases for primer carries out real-time fluorescence quantitative PCR amplification, with the Log value of template concentrations for X-coordinate, reach the cycle number of set threshold value experience for ordinate zou with the fluorescent value of each reacting hole, production standard curve, obtains typical curve formula 2;
(3) with the genomic dna of the root of Chinese cabbage to be checked, stem or leaf texture for template, with the DNA molecular shown in the DNA molecular shown in SEQIDNo.1 and SEQIDNo.2 for primer carries out real-time fluorescence quantitative PCR amplification, obtain cycle number CP1, bring CP1 value into typical curve formula 1, obtain the absolute content of Chinese cabbage verticillium pathogenic bacteria in Chinese cabbage to be checked; With the genomic dna of the root of Chinese cabbage to be checked, stem or leaf texture for template, carry out real-time fluorescence quantitative PCR amplification with GAPDH primer for primer, obtain cycle number CP2, bring CP2 value into typical curve formula 2, obtain the GAPDH absolute content of measuring samples; Chinese cabbage Neihuang County to be checked wither disease pathogen bacterium relative content=Chinese cabbage to be checked in the absolute content/GAPDH absolute content of Chinese cabbage verticillium pathogenic bacteria.
6. method according to claim 5, is characterized in that: the DNA concentration of the verticillium pathogenic bacteria of extracting in described step (1) is 50ng μ L -1;
The genomic dna concentration of the root of the Chinese cabbage of extracting in described step (2), stem or leaf texture is 200ng μ L -1;
Described typical curve formula 1 is Y=-3.553X+10.04;
Described typical curve formula 2 is Y=-3.193X+23.22.
7. the method according to claim 5 or 6, is characterized in that: described verticillium pathogenic bacteria is Chinese cabbage verticillium pathogenic bacteria BCHW10-2.
8. primer pair according to claim 1 is detecting the application in the Chinese cabbage verticillium pathogenic bacteria in Chinese cabbage.
9. whether primer pair according to claim 1 infects the application in Chinese cabbage verticillium pathogenic bacteria in detection Chinese cabbage.
10. test kit according to claim 2 is detecting the application of the Chinese cabbage verticillium pathogenic bacteria in Chinese cabbage.
Whether 11. test kits according to claim 2 infect the application in Chinese cabbage verticillium pathogenic bacteria in detection Chinese cabbage.
12.-11 arbitrary described application according to Claim 8, is characterized in that: described Chinese cabbage verticillium pathogenic bacteria is verticillium dahliae (Verticilliumdahliae).
13. application according to claim 12, is characterized in that: described verticillium dahliae (Verticilliumdahliae) is Chinese cabbage verticillium pathogenic bacteria BCHW10-2.
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