CN103361423B - A kind of detection kit of cabbage oxysporum and detection method thereof - Google Patents

Abstract

The invention discloses a kind of detection kit of cabbage oxysporum, the reagent of this test kit comprises detection primer, detect primer and comprise upstream primer Foc-R, downstream primer Foc-S each 1 μ l, the 10 × PCR that concentration is 10pmol/ μ l? Easy? the dNTPs0.5 μ l of Taq damping fluid 2.0 μ l, 10mM, active enzyme concentration are Taq polysaccharase 0.4 μ l, the cabbage oxysporum positive control dna of 1 μ l, the ultrapure water 20ul of weight concentration >=99% of 5U/ml; The sequence 5 '-TCAATGATAGTGACAAGGGTTT-3 ' of Foc-R, the sequence 5 '-AATTTGCTGTGATAGGTGGAT-3 ' of Foc-S; Also disclose detection method, comprise the steps, (1) extracts plant tissue or soil DNA; (2) pcr amplification is carried out to DNA; (3) electrophoretic separation is carried out to amplified production, again through ethidium bromide staining size result of determination according to amplified production under ultraviolet lamp after separation, advantage of the present invention, provides that a kind of accuracy is high, easy and simple to handle, test kit that high specificity and highly sensitive cabbage oxysporum rapid molecular detect and method thereof.

Description

A kind of detection kit of cabbage oxysporum and detection method thereof

Technical field

The invention belongs to the field of corps diseases Testing and appraisal and Plant Quarantine, specifically a kind of detection kit of cabbage oxysporum and detection method thereof.

Background technology

Present known cabbage oxysporum is exactly Fusarium oxysporum sticky group specialized form, the Cabbage Wilt Disease that Fusarium oxysporum sticky group specialized form (Fusariumoxysporumf.sp.Conglutinans) causes is a kind of destructive soil-borne disease, in the whole world, all there is generation in most of wild cabbage producing region, this disease from calendar year 2001 since China's reported first, successively all there is generation in Hebei province's Zhangjiakou City, Jinzhong City, Shanxi Province and Datong City, Shanxi Province, be spreading trend in the generation of China, and increase the weight of year by year, become the important disease affecting China's Cabbage Quality and output.Cabbage Wilt Disease is a kind of soil-borne disease, and pathogenic bacteria can survive the winter formation Infection cycie in soil and invalid body, and its infection ability is strong, plant can be caused within a short period of time dead, and this disease may be propagated from region of disease to non-region of disease.Also seldom there are the means of cost-effective control Cabbage Wilt Disease at present.Therefore, carry out cabbage oxysporum and detect, prevent it from propagating from region of disease to non-region of disease, and carry out diagnostic detection at its their early stage, significant with control to the propagation of Cabbage Wilt Disease.

The detection technique of Fusarium oxysporum is the study hotspot of various countries researchist always, traditional detection method adopts selective medium isolated strains from morbidity soil or plant tissue, again the form etc. of these bacterial strains is identified, thus determine whether there is cabbage oxysporum, or with the naked eye and by microscopy, disease symptom is determined whether to the disease that caused by cabbage oxysporum.Traditional detection method not only time and effort consuming, and require that testing staff has extremely abundant morphology and taxonomy knowledge, the most important is that traditional detection method can not meet in disease control and specifies control approach and the requirement to pathogenic bacteria rapid detection and qualification, and therefore traditional detection method can not meet the needs of Cabbage production and modern plants pathological research.

In general, Molecular Detection means have more specificity sooner than traditional detection method speed, and sensitiveer and accurate and less demanding to the morphological knowledge of operator.At present, comprise RFLP, RAPD, RFLP and SCAR etc. based on DNA or RNA molecule marking method the mirror of Fusarium oxysporum specialized form detect in obtain application.But can be located arbitrarily on genome due to these molecule markers, can be used in public database and the data of other gene comparision fewer, reliable and stable mark also needs to go further screening by the bacterial strain of greater amt.Therefore, the basis of pathogenic bacteria sequencing data of whole genome is designed high special primer to carry out detection to it and be very important.

Summary of the invention

For solving the defect existed in technology of above-mentioned detection cabbage oxysporum, the invention discloses a kind of cabbage oxysporum Molecular Detection reagent and detection kit thereof, its objective is that the cycle needed for the biological detection method for cabbage oxysporum in prior art is long, and not yet have the problem of cabbage oxysporum molecular detecting method at present, by carrying out genome sequencing to cabbage oxysporum, and find out by the method for comparative genomics the genomic fragment that cabbage oxysporum is specific to other wilts, then this specific fragment base sequence for cabbage oxysporum have devised high a pair special primer, highly sensitive rapid molecular for the plant tissue with cabbage oxysporum and soil detects, and provide a kind of accuracy high, easy and simple to handle, the test kit that high specificity and highly sensitive cabbage oxysporum rapid molecular detect, the highly sensitive rapid molecular that this test kit can be used for plant tissue and the soil carried disease germs detects, for cabbage oxysporum cause disease show disease before early detection and diagnosis tool be of great significance.

For achieving the above object, the technical solution used in the present invention is, the invention discloses a kind of detection kit of cabbage oxysporum, this test kit comprises, concentration is each 1 μ l of detection upstream primer Foc-R, downstream primer Foc-S of 10pmol/ μ l, and the dNTPs0.5 μ l of 10 × PCREasyTaq damping fluid 2.0 μ l, 10mM, active enzyme concentration are Taq polysaccharase 0.4 μ l, the cabbage oxysporum positive control dna of 1 μ l, the ultrapure water 20ul of weight concentration >=99% of 5U/ml.Detect the sequence of primer:

The sequence 5 '-TCAATGATAGTGACAAGGGTTT-3 ' of Foc-R,

The sequence 5 '-AATTTGCTGTGATAGGTGGAT-3 ' of Foc-S

The invention also discloses the method adopting described test kit to detect cabbage oxysporum, the method comprises the steps, the plant tissue DNA that (1) utilizes CTAB method to extract to be infected by cabbage oxysporum or utilize PowerSoilDNAIsolationKit kit method to extract the soil DNA infected by cabbage oxysporum;

(2) detection kit is adopted to carry out pcr amplification to the isolated DNA of (1) step;

(3) be that 1.5% agarose gel electrophoresis is separated by the pcr amplification product mass concentration of 6 μ l steps (2), again through ethidium bromide staining size result of determination according to amplified production under ultraviolet lamp after separation, when amplifying 436bp product specifically, can judge to there is cabbage oxysporum in plant tissue or soil.

During the plant tissue DNA that described step (1) utilizes the extraction of CTAB method to be infected by cabbage oxysporum, its concrete operation method is, 1. get the cabbage oxysporum hypha powder after 50mg lyophilize in 1.5ml centrifuge tube, add vibration mixing after the CTAB extracting solution of 900 μ L and the basic sodium sulfonate of 90 μ l mass concentration 10% dodecane;

2. after 1. walking process by said mixture in 55 ~ 60 DEG C of water-bath 1.5h, every 10min puts upside down mixing once, and water-bath is centrifugal 15min after 1.5 hours, and centrifugal speed is 12,000rpm;

3. get supernatant liquor after centrifugal and add isopyknic phenol, chloroform and primary isoamyl alcohol mixing solutions, phenol in described mixing solutions: chloroform: the volume ratio of primary isoamyl alcohol is: 25: 24: 1, centrifugal 5min, and centrifugal speed is 12,000rpm;

4. through 3. walk centrifugal after get supernatant liquor, i.e. aqueous phase, add equal-volume chloroform once, centrifugal 5min, centrifugal speed is 12,000rpm, collect supernatant liquor;

5. be-20 DEG C of dehydrated alcohols of 3mol/LNaAC solution and 2ml to the mass concentration 4. walking supernatant liquor and add 0.1ml, precipitate 12,000rpm centrifugal 5min after 30min at-20 DEG C, remove supernatant liquor lightly;

6.-20 DEG C of ethanol adding 700 μ l volumetric concentrations 70% to the sediment fraction 5. walking abandoning supernatant wash, dissolve with 1 × TE solution after Bechtop dries alcohol-free taste naturally, obtain the DNA solution of bacterial strain, by UV spectrophotometer measuring DNA concentration and to be diluted to 50ng/ μ l stand-by.

Described step 1. in CTAB extracting solution comprise the CTAB that mass concentration is 2%, the Tris-HCl of 100mmol/L, pH8.0, the NaAC of the EDTA of 20mmol/L, pH8.0,1.4mol/L.

The concrete operation method of described step (2) is, 1. the DNA1 μ l of (1) step is got, by the reagent mix of itself and test kit, this reagent comprises each 1 μ l of detection upstream primer Foc-R, downstream primer Foc-S that concentration is 10pmol/ μ l, and the dNTPs0.5 μ l of 10 × PCREasyTaq damping fluid 2.0 μ l, 10mM, active enzyme concentration are the Taq polysaccharase 0.4 μ l of 5U/ml, the ultrapure water 20ul of weight concentration >=99%;

2. the mixture 1. walked is put into PCR instrument device to increase, pcr amplification program is: 94 DEG C of sex change 5min; 94 DEG C of sex change 40sec; 63 DEG C of annealing 30sec; 72 DEG C extend 1min; 35 circulations, last 72 DEG C extend 10min.

The inventive method is applicable to fast and reliable detection and the qualification of cabbage oxysporum in plant tissue and pedotheque, and the disease control caused for cabbage oxysporum in agriculture production has important practical value.The present invention compared with prior art, has following technical superiority and positively effect:

1, high specificity: detection method is the peculiar fragment utilizing whole genome sequence analyze of comparative genomics method to cabbage oxysporum and other wilts to obtain cabbage oxysporum, detects according to specific fragment design primer.To the checking come from from China Beijing, Shanxi Province and external cabbage oxysporum and Fusarium oxysporum sibling species and other common disease bacterium, result has had very strong specificity;

2, practicality is good: a pair Auele Specific Primer gone out designed by the present invention, can be used for being with the plant tissue of cabbage oxysporum and the highly sensitive rapid molecular of soil to detect, therefore present method is practical, and the cabbage oxysporum that can meet existing in band soil bacteria and morbidity plant tissue carries out fast and reliable detection and the needs of qualification;

3, fast easy and simple to handle: application the inventive method, result of determination is got final product, without the need to carrying out digestion with restriction enzyme to amplified production after the agarose gel electrophoresis of pathogenic bacteria DNA extraction, pcr amplification and routine is carried out to the pedotheque and plant tissue of being with cabbage oxysporum.General whole testing process completes within a few hours.

Accompanying drawing explanation

Fig. 1 is cabbage oxysporum Auele Specific Primer PCR primer electrophorogram;

In figure, M:Trans2KMarker; 1-5: cabbage oxysporum; 6-33: other specialized forms of Fusarium oxysporum; 34-39: encountered pathogenic bacteria 40: clear water contrasts.

Fig. 2 is that in disease plant sample, cabbage oxysporum detects electrophorogram;

In figure, M:Trans2KMarker; 1: positive control dna; 2: clear water contrasts; 3-10: disease plant STb gene.

Fig. 3 is that in pedotheque, cabbage oxysporum detects electrophorogram;

In figure, M:Trans2KMarker; 1: positive control dna; 2: clear water contrasts; 3-5: band soil bacteria STb gene.

Embodiment

Content of the present invention is further illustrated below in conjunction with embodiment.

Embodiment one: the detection kit of cabbage oxysporum of the present invention, this test kit comprises, concentration is each 1 μ l of detection upstream primer Foc-R, downstream primer Foc-S of 10pmol/ μ l, and the dNTPs0.5 μ l of 10 × PCREasyTaq damping fluid 2.0 μ l, 10mM, active enzyme concentration are Taq polysaccharase 0.4 μ l, the cabbage oxysporum positive control dna of 1 μ l, the ultrapure water 20ul of weight concentration >=99% of 5U/ml.Detect the sequence of primer:

The sequence 5 '-TCAATGATAGTGACAAGGGTTT-3 ' of Foc-R,

The sequence 5 '-AATTTGCTGTGATAGGTGGAT-3 ' of Foc-S

Test kit of the present invention is adopted to detect the method for cabbage oxysporum, comprise the steps, the plant tissue DNA that (1) utilizes CTAB method to extract to be infected by cabbage oxysporum or utilize PowerSoilDNAIsolationKit kit method to extract the soil DNA infected by cabbage oxysporum;

(2) detection kit is adopted to carry out pcr amplification to the isolated DNA of (1) step;

(3) be that 1.5% agarose gel electrophoresis is separated by the pcr amplification product mass concentration of 6 μ l steps (2), again through ethidium bromide staining size result of determination according to amplified production under ultraviolet lamp after separation, when amplifying 436bp product specifically, can judge to there is cabbage oxysporum in plant tissue or soil.

During the plant tissue DNA that above-mentioned steps (1) utilizes the extraction of CTAB method to be infected by cabbage oxysporum, its concrete operation method is, 1. get the cabbage oxysporum hypha powder after 50mg lyophilize in 1.5ml centrifuge tube, add vibration mixing after the CTAB extracting solution of 900 μ L and the basic sodium sulfonate of 90 μ l mass concentration 10% dodecane;

2. after 1. walking process by said mixture in 55 ~ 60 DEG C of water-bath 1.5h, every 10min puts upside down mixing once, and water-bath is centrifugal 15min after 1.5 hours, and centrifugal speed is 12,000rpm;

3. get supernatant liquor after centrifugal and add isopyknic phenol, chloroform and primary isoamyl alcohol mixing solutions, phenol in described mixing solutions: chloroform: the volume ratio of primary isoamyl alcohol is: 25: 24: 1, centrifugal 5min, and centrifugal speed is 12,000rpm;

4. through 3. walk centrifugal after get supernatant liquor, i.e. aqueous phase, add equal-volume chloroform once, centrifugal 5min, centrifugal speed is 12,000rpm, collect supernatant liquor;

5. be-20 DEG C of dehydrated alcohols of 3mol/LNaAC solution and 2ml to the mass concentration 4. walking supernatant liquor and add 0.1ml, precipitate 12,000rpm centrifugal 5min after 30min at-20 DEG C, remove supernatant liquor lightly;

6.-20 DEG C of ethanol adding 700 μ l volumetric concentrations 70% to the sediment fraction 5. walking abandoning supernatant wash, dissolve with 1 × TE solution after Bechtop dries alcohol-free taste naturally, obtain the DNA solution of bacterial strain, by UV spectrophotometer measuring DNA concentration and to be diluted to 50ng/ μ l stand-by.

Described step 1. in CTAB extracting solution comprise the CTAB that mass concentration is 2%, the Tris-HCl of 100mmol/L, pH8.0, the NaAC of the EDTA of 20mmol/L, pH8.0,1.4mol/L.

The concrete operation method of described step (2) is, 1. the DNA1 μ l of (1) step is got, by the reagent mix of itself and test kit, this reagent comprises each 1 μ l of detection upstream primer Foc-R, downstream primer Foc-S that concentration is 10pmol/ μ l, and the dNTPs0.5 μ l of 10 × PCREasyTaq damping fluid 2.0 μ l, 10mM, active enzyme concentration are the Taq polysaccharase 0.4 μ l of 5U/ml, the ultrapure water 20ul of weight concentration >=99%;

2. the mixture 1. walked is put into PCR instrument device to increase, pcr amplification program is: 94 DEG C of sex change 5min; 94 DEG C of sex change 40sec; 63 DEG C of annealing 30sec; 72 DEG C extend 1min; 35 circulation cycles, last 72 DEG C extend 10min.

When determining detection primer of the present invention, by each repetition the inventive method of sequence of design, finally can make cabbage oxysporum special amplify 436bp product be detection primer, and need lot of experiments that the specificity of this detection primer is described, the present embodiment method is utilized to make the specific test detected: to utilize primer of the present invention except from China Beijing, Shanxi etc. are economized and external cabbage oxysporum DNA can amplify outside the product of 436bp specifically, have detected wilt other 21 specialized forms 28 bacterial strains and 4 kinds of other bacterial strain DNA such as common causative fungi and a kind of pathogenic oomycetes and all fail to amplify spawn, there is very strong specificity, see Fig. 1.

Embodiment two: utilize the method for embodiment one to detect the wilt of falling ill in cabbage plant tissue.

1. sample collecting: Plant tissue samples picks up from little barracks village of Yanqing district of Beijing wild cabbage Vegetable Base

2.DNA isolation and determination: with embodiment one.

3. detected result: the visible Fig. 2 of result, see one clearly molecular weight be the specific band of 436bp, thus judge incidence tissue infect cabbage oxysporum.

Embodiment three: utilize the cabbage oxysporum in the method detection pedotheque of embodiment one.

1. sample collecting: pedotheque picks up from Vegetable & Flower Inst., Chinese Academy of Agriculture Science's experiment greenhouse.

2.DNA isolation and determination:

Pedotheque adopts method described in MOBIO company PowerSoilDNAIsolationKit test kit to extract DNA, the method implemented by mentioned reagent box carries out pcr amplification, PCR reaction system 20 μ l, comprise 2.0 μ l10 × PCR reaction buffers, 10mMdNTPs0.5 μ l, 5U/ μ lEasyTaq polysaccharase 0.4 μ l, each 1 μ l of 10pmol/ μ l primers F oc-R/Foc-S, 5ng/ μ lDNA template 1 μ l, ddH2O supplies 20 μ l.Pcr amplification program is: 94 DEG C of 5min; 94 DEG C of 40sec; 63 DEG C of 30sec; 72 DEG C of 1min; 35cycles, 72 DEG C of 10min.Electrophoresis detection amplified production.

3. detected result; The results are shown in Figure 3, see one clearly molecular weight be the specific band of 436bp, thus judge that pedotheque exists cabbage oxysporum.

The above, not impose any restrictions content of the present invention, every according to the substantial amendment of content technologies of the present invention, all still belongs in the protection domain of content technologies scheme of the present invention.

Claims (4)

1. the detection kit of a cabbage oxysporum, it is characterized in that, the reagent of this test kit comprise detect primer, Taq polysaccharase 0.4 μ l, the cabbage oxysporum positive control dna of 1 μ l, the ultrapure water 20ul of weight concentration >=99% that the dNTPs0.5 μ l of 10 × PCREasyTaq damping fluid 2.0 μ l, 10mM, active enzyme concentration are 5U/ml; Detect primer and comprise each sequence 5 '-TCAATGATAGTGACAAGGGTTT-3 ' of 1 μ l, Foc-R of upstream primer Foc-R, downstream primer Foc-S, the sequence 5 '-AATTTGCTGTGATAGGTGGAT-3 ' of Foc-S that concentration is 10pmol/ μ l.

2. the method adopting test kit described in claim 1 to detect cabbage oxysporum, it is characterized in that, the method comprises the steps, the plant tissue DNA that (1) utilizes CTAB method to extract to be infected by cabbage oxysporum or utilize PowerSoilDNAIsolationKit kit method to extract the soil DNA infected by cabbage oxysporum;

(2) detection kit is adopted to carry out pcr amplification to the isolated DNA of (1) step: the DNA1 μ l 1. getting (1) step, by the reagent mix of itself and test kit, this reagent comprises each 1 μ l of detection upstream primer Foc-R, downstream primer Foc-S that concentration is 10pmol/ μ l, and the dNTPs0.5 μ l of 10 × PCREasyTaq damping fluid 2.0 μ l, 10mM, active enzyme concentration are the Taq polysaccharase 0.4 μ l of 5U/ml, the ultrapure water 20ul of weight concentration >=99%;

2. the mixture 1. walked is put into PCR instrument device to increase, pcr amplification program is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 40sec; 63 DEG C of annealing 30sec; 72 DEG C extend 1min; 35 circulations, last 72 DEG C extend 10min;

(3) be that 1.5% agarose gel electrophoresis is separated by the pcr amplification product mass concentration of 6 μ l steps (2), again through ethidium bromide staining size result of determination according to amplified production under ultraviolet lamp after separation, when amplifying 436bp product specifically, can judge to there is cabbage oxysporum in plant tissue or soil.

3. detect the method for cabbage oxysporum according to claim 2, it is characterized in that, during the plant tissue DNA that described step (1) utilizes the extraction of CTAB method to be infected by cabbage oxysporum, its concrete operation method is, 1. get the cabbage oxysporum hypha powder after 50mg lyophilize in 1.5ml centrifuge tube, add vibration mixing after the CTAB extracting solution of 900 μ L and 90 μ l mass concentration 10% Sodium dodecylbenzene sulfonatees;

2. after 1. walking process by said mixture in 55 ~ 60 DEG C of water-bath 1.5h, every 10min puts upside down mixing once, and water-bath is centrifugal 15min after 1.5 hours, and centrifugal speed is 12,000rpm;

3. get supernatant liquor after centrifugal and add isopyknic phenol, chloroform and primary isoamyl alcohol mixing solutions, phenol in described mixing solutions: chloroform: the volume ratio of primary isoamyl alcohol is 25: 24: 1, centrifugal 5min, and centrifugal speed is 12,000rpm;

4. through 3. walk centrifugal after get supernatant liquor, i.e. aqueous phase, add equal-volume chloroform once, centrifugal 5min, centrifugal speed is 12,000rpm, collect supernatant liquor;

5. be-20 DEG C of dehydrated alcohols of 3mol/LNaAC solution and 2ml to the mass concentration 4. walking supernatant liquor and add 0.1ml, precipitate 12,000rpm centrifugal 5min after 30min at-20 DEG C, remove supernatant liquor lightly;

6.-20 DEG C of ethanol adding 700 μ l volumetric concentrations 70% to the sediment fraction 5. walking abandoning supernatant wash, dissolve with 1 × TE solution after Bechtop dries alcohol-free taste naturally, obtain the DNA solution of bacterial strain, by UV spectrophotometer measuring DNA concentration and to be diluted to 50ng/ μ l stand-by.

4. the method for detection cabbage oxysporum according to claim 3, it is characterized in that, described step 1. in CTAB extracting solution comprise the CTAB that mass concentration is 2%, the Tris-HCl of 100mmol/L, pH8.0, the NaAC of the EDTA of 20mmol/L, pH8.0,1.4mol/L.