CN109811040A - A kind of germ quantitative detecting method based on PMA dyeing - Google Patents
A kind of germ quantitative detecting method based on PMA dyeing Download PDFInfo
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Abstract
A kind of germ quantitative detecting method based on PMA dyeing of the present invention.The described method includes: non-solubility solid plant sample is cut into fragment by (1), phosphate buffer is added after grind away processing, obtains tissue homogenate；(2) homogenate is divided into A, B group, PMA is added to A group, while the water of same volume being added into the homogenate of B group；(3) processing of PMA photo-crosslinking is successively carried out respectively for two groups of A, B, DNA is extracted, DNA profiling is quantitative, quantitative fluorescent PCR processing；(4) according to the Ct value of A, B group, proportion of the live virus in total germ is calculated.This method can be used for judging the plant state of an illness, be classified to the state of an illness；The present invention is able to detect bacterium total amount and viable bacteria amount in plant tissue simultaneously, calculate viable bacteria survival ratio in plant, it provides a kind of for judging the method for evaluating drug effect of pest control method and antimicrobial therapy method, provides a kind of drug effect and the quick evaluation method of prevention and treatment for yellow twig disease control.
Description
Technical field
The present invention relates to a kind of germ quantitative detecting methods based on PMA dyeing, belong to technical field of analysis and detection.
Background technique
Yellow twig is to endanger most serious and most destructive destruction in citrus (including tangerine, mandarin orange, orange, shaddock, trifoliate orange etc.) production
Venereal disease evil.Yellow twig is considered as caused by being specially born in the bacterium of citrus phloem tissue as one kind, and route of transmission includes transferring
It connects and is propagated with diaphorina citri.
The known dip dyeings of Citrus Cultivars and citrus sibling species all by yellow twig germ all at present.The plant of infection after being ill
Strain cannot not only generate economic value, and high the kind such as navel orange, grape fruit of susceptible degree can taper off in several years, and
It there is no the relevant report of yellow twig therapy rehabilitation at present.Therefore the quick, accurate, qualitative of a kind of yellow twig germ and quantitative is provided
Detection, has great importance for the prevention and control and research of yellow twig.
It mainly include at present the hands such as Electronic Speculum detection, immunology detection and PCR detection for the detection method of yellow twig germ
Section.By yellow twig cause of disease bacteria concentration in citrus host plant, the factors such as low, uneven distribution and sample making technology are limited, Electronic Speculum observation
The recall rate of detection is generally between 60%~70%, and there are a large amount of missing inspections；Too strong (the application number of immunology detection specificity
201210093995.1), it can not use between different plants；Although application of the PCR diagnostic method in Citrus Huanglongbing pathogen detection is
Quick, accurate, qualitative and quantitative requirement is realized, the laboratory diagnosis of yellow twig is widely used in；But current PCR diagnosis
Technology is detected to the amount of DNA of yellow twig germ, since bacterium after death still has more DNA residual, PCR means in a short time
The intracorporal dead bacterium of plant, viable bacteria cannot be distinguished.
In addition, the prior art, which is also disclosed, carries out dead, living stems technology contents using PMA, but test object must be
Bacterium solution or other liquid forms, can not be to the bacterial content progress quantitative detection of viable bacteria in the complicated solid tissue of non-solubility.
Summary of the invention
In order to overcome the above technical defects, the present invention provides a kind of for germ content in non-solubility solid plant tissue
Quantitative detecting method.This method provides accurately quantitative analysis for the early detection of the plant diseases such as yellow twig, while being also
It, which is prevented and treated, provides accurate evaluation means.
Technical scheme is as follows:
A kind of germ quantitative detecting method based on PMA dyeing, comprising:
(1) non-solubility solid plant sample is cut into fragment, phosphate buffer (pH=6.5) is added after grind away processing,
Obtain tissue homogenate；
(2) homogenate is divided into A, B group, PMA is added to A group, while the water of same volume being added into the homogenate of B group；
(3) processing of PMA photo-crosslinking is successively carried out respectively for two groups of A, B, DNA is extracted, DNA profiling is quantitative, quantitative fluorescent PCR
Processing；
(4) according to the Ct value of A, B group, proportion of the live virus in total germ is calculated.
Each step is illustrated one by one below.
In step (1), the non-solubility solid plant sample is plant tissue, such as blade, stalk, root system, preferably
Blade is chosen, when actual samples, in the middle part of clip vein, is cut into the fragment of 1mm or so, it is spare.
In step (1), the grind away processing includes:
S1: first grind away, oscillation are carried out to sample, obtain first sample；Wherein, the frequency of the oscillation is 20-30HZ, preferably
Grind away, frequency 30HZ are vibrated using steel ball；
S2: adding phosphate buffer (pH=6.5) into first sample, and grind away, oscillation, form suspension again；
S3: suspension is filtered, and obtains partial size in 0.25mm tissue homogenate below；Wherein, the filtering of suspension is usual
Using the gauze of 60 mesh, bulky grain therein is removed.
In step (1), the pH value of the phosphate buffer is 6-7, preferably 6.5.
In step (2), into the homogenate of A group, addition PMA is to its concentration between 5mg/l-30mg/l；Preferred concentration is 10-
15mg/l。
In step (3), PMA photo-crosslinking processing include: by two groups of shaken wells of A, B, be placed under 0-10 DEG C of environment according to
Secondary progress dark reaction, light reaction；After the reaction was completed, it is centrifuged, abandons supernatant；Supernatant is abandoned in washing, centrifugation, retains precipitating；It is preferred that
Ground, dark reaction handle 30min, and light reaction is to be placed in light reaction 15min under 650W tungsten halogen lamp, and centrifugal speed is 12000 turns.
In step (3), the DNA extraction process includes: that CTAB is added into the PMA photo-crosslinking treated sample to split
Liquid is solved, oscillation is placed in water-bath and cracks；After, the chloroform and isoamyl alcohol mixed liquor that volume ratio is 24:1 is added, shakes up,
Centrifugation, takes supernatant, and isometric isopropanol is added into supernatant, is shaken up, and after cooling, centrifugation abandons supernatant, continuously adds 75% second
Alcohol, centrifugation, takes lower layer to dry, to be programmed colorless and transparent, and the ddH of sterilizing is added2O, dissolution；Wherein, the CTAB lysate
Additional amount be 800ul；
In step (3), the DNA profiling quantitative Treatment includes: the nucleic acid quantification equipment using this field routine to extraction
DNA quantified, two groups of DNA are diluted to two groups of DNA concentration minimum common divisors, be more conducive to improve testing result it is accurate
Property.
In step (3), in the quantitative fluorescent PCR processing, select corresponding specific primer to the disease in plant tissue
Bacterium is cooked PCR quantitative Treatment, carries out viable bacteria quantitative detecting analysis；The germ is preferably Citrus Huanglongbing pathogen.
In step (4), the Ct of A group is the Ct of viable bacteria, and the Ct of B group is the Ct of germ total amount in tissue；Pass through A group and B group
Ct value difference value △ Ct, calculate proportion F, F=2 of the viable bacteria in total flora-△Ct。
The present invention also provides application of the above-mentioned detection method in the Pathogen detection of non-solubility solid plant；It is described it is non-can
Soluble solids plant is not limited to Citrus Cultivars, citrus sibling species sample, also includes other non-solubility solid plants；The disease
Bacterium is not limited to yellow twig, further includes other bacterial plant diseases.
The present invention by the way that grind away processing technique, PMA staining technique and PCR quantitative technique are combined, realize to it is non-can
The detection of Huanglong bacterium viable bacteria and total bacterium in soluble solids living plant tissue.The detection method can be used for judging phytopathy
Feelings are classified the state of an illness；It is able to detect in plant tissue total bacterium amount and viable bacteria amount simultaneously, determines that viable bacteria is deposited plant is intracorporal
Ratio living provides effective evaluating drug effect basis for the prevention and treatment of yellow twig disease.
Detailed description of the invention
Fig. 1 is the flow chart of detection method described in embodiment 1.
Fig. 2 is the Ct value figure of the yellow twig germ in embodiment 1 under different disposal.
In figure: A1 represents the sample for not heating+PMA processing, B1 represents the sample for not heating+not adding PMA processing
This, A2 represent heat treatment+PMA processing sample, B2 represent heat treatment+plus PMA processing sample.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
Viable bacteria and total bacteria count of the embodiment 1 using yellow twig germ in PMA staining technique measurement navel orange blade
As shown in Figure 1, specific assay method is as follows:
(1) material: three Nian Sheng of selection, the Niu Heer navel orange seedling for contaminating yellow twig, as sample；
(2) it pre-processes: taking totally 24 pieces of blade of navel orange seedling, divide two parts equally at random, be placed into 95 DEG C for 12 pieces of a copy of it
Baking oven heats 5min sterilization treatment, as sterilization treatment group；Another 12 pieces do not deal with, as non-sterilization treatment group.
(3) grind away is handled: taking identical mode to distinguish grind away in two parts of (sterilization treatment group, non-sterilization treatment group) blades
Processing；
It is specific as follows by taking non-sterilization treatment group as an example:
I, in the middle part of the vein of clip blade, it is cut into 1mm length vein fragment；
Ii, fragment is mixed, weighs the weight of vein fragment, vein fragment is divided equally with 1.5g portion, be put into 2ml centrifugation
Steel ball grind away is added in Guan Zhong, and the oscillator frequency that grind away uses is 30hz, and the grind away time is 5min, obtains sterilization treatment group
First sample；
Iii, 1ml phosphate buffer (pH=6.5) is added to every pipe first sample, vibrates grind away again, form tissue homogenate；
Iv, tissue homogenate in each centrifuge tube is taken out with liquid-transfering gun from centrifuge tube, is put into test tube and mixes；It will mix again
Tissue homogenate divide equally in 6 1.5ml centrifuge tubes, every triplets are pressed in each centrifuge tube 1ml homogenate, and tissue homogenate is divided
At A group and B group；
Grind away processing is carried out to sterilization treatment group blade using above-mentioned identical step；
So far, it is as follows that tissue homogenate is obtained: not heating group (non-sterilization treatment group) A1, B1, heat treatment group is (at sterilizing
Reason group) A2, B2；
(4) PMA photo-crosslinking is handled: A1 group and A2 group is added in PMA, is homogenized to the concentration of 10mg/l, remaining B1 and B2 add
Enter equivalent sterile water, A1, A2, B1, B2 group are placed in the processing of dark reaction on ice 30min, light reaction 15min by shaken well；Reaction
After the completion, centrifuge tube is taken out, is centrifuged with 12000 turns, abandon supernatant；It is suspended that 600ul sterile water is added, then is left with 12000
The heart abandons supernatant；
(5) DNA of remaining precipitating is extracted: be added in each centrifuge tube 65 DEG C of 800 μ l it is preheated CTAB lysate, vibration
It swings, is placed in 65 DEG C of water-baths and cracks 50 minutes, every 10min shakes once；After 50min, volume ratio is added into centrifuge tube
For 600 μ l of 24:1 chloroform isoamyl alcohol, shake up；12000 turns of centrifugation 10min are put into a centrifuge, until there is liquid level layering up and down；Make
With 1ml liquid-transfering gun, 500 μ l of supernatant solution is drawn, 1.5ml centrifuge tube is moved into, adds isometric 5000 μ l of isopropanol, gently shake up,
5min is placed on ice, puts into a centrifuge 12000 turns of centrifugation 10min, pours out liquid in centrifuge tube, and 75% ethyl alcohol, 600 μ l is added,
10000 turns of centrifugation 5min；Ethyl alcohol in centrifuge tube is poured out, is put into 65 DEG C of baking ovens and dries 10min, becomes colorless and transparent to DNA,
The ddH of sterilizing is added250 μ l of O dissolves 20min；
(6) DNA profiling is quantitative: being quantified using DNA of the nucleic acid quantification equipment to extraction, by the DNA in each centrifuge tube
It is diluted to unified concentration；
(7) quantitative fluorescent PCR is handled: carrying out real-time fluorescence according to DNA of the method in GB/T28062-2011 to extraction
PCR analysis, determines the content of yellow twig specific DNA in A1, B1, A2, B2, obtains each group Ct value, as shown in Figure 2.
As shown in Figure 2, the B2 group Ct value of heat treatment is 22, it is seen that a large amount of DNA target fragments still have；PMA processing
Heat-treated A2 group Ct value is that 32.3, Ct value reaches all yellow twig target fragment quilts in 30 this group of samples described above
PMA is combined, and PMA processing can be in conjunction with the DNA fragmentation of dead bacterium；
And not heat-treated A1 and the Ct value difference value △ Ct of heat-treated A2 group reach 10, illustrate that this programme can
So that the dead bacterium of Huanglong germ and viable bacteria are distinguished；
The Ct value difference value △ Ct of the A1 group of PMA processing and the B1 group without PMA processing is 0.2, and viable bacteria is calculated total
Accounting is 87% (F=2 in flora-△Ct)。
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of germ quantitative detecting method based on PMA dyeing characterized by comprising
(1) non-solubility solid plant sample is cut into fragment, phosphate buffer is added after grind away processing, obtain tissue homogenate；
(2) homogenate is divided into A, B group, PMA is added to A group, while the water of same volume being added into the homogenate of B group；
(3) processing of PMA photo-crosslinking is successively carried out respectively for two groups of A, B, DNA is extracted, DNA profiling is quantitative, at quantitative fluorescent PCR
Reason；
(4) according to the Ct value of A, B group, proportion of the live virus in total germ is calculated.
2. the germ quantitative detecting method according to claim 1 based on PMA dyeing, which is characterized in that in step (1),
The grind away is handled
S1: first grind away, oscillation are carried out to sample, obtain first sample；
S2: adding phosphate buffer into first sample, and grind away, oscillation, form suspension again；
S3: suspension is filtered, and obtains tissue homogenate.
3. the germ quantitative detecting method according to claim 1 or 2 based on PMA dyeing, which is characterized in that the phosphoric acid
The pH value of buffer be 6-7, preferably 6.5.
4. the germ quantitative detecting method according to claim 2 based on PMA dyeing, which is characterized in that the oscillation
Frequency is 20-30HZ.
5. the germ quantitative detecting method according to claim 2 based on PMA dyeing, which is characterized in that gained tissue is even
The partial size of slurry is in 0.25mm or less.
6. -5 any germ quantitative detecting method based on PMA dyeing according to claim 1, which is characterized in that step
(2) in, into the homogenate of A group, addition PMA is to its concentration between 5mg/l-30mg/l；Preferred concentration is 10-15mg/l.
7. the germ quantitative detecting method according to claim 1 based on PMA dyeing, which is characterized in that in step (1),
The non-solubility solid plant sample is plant leaf blade, stalk or root system sample.
8. the germ quantitative detecting method according to claim 1 based on PMA dyeing, which is characterized in that in step (3),
PMA photo-crosslinking processing includes: that two groups of shaken wells of A, B are placed under 0-10 DEG C of environment that successively to carry out dark reaction, light anti-
It answers；After the reaction was completed, it is centrifuged, abandons supernatant；Supernatant is abandoned in washing, centrifugation, retains precipitating.
9. any germ quantitative detecting method based on PMA dyeing of claim 1-8 is in non-solubility solid plant
Application in Pathogen detection.
10. application according to claim 9, which is characterized in that the non-solubility solid plant is Citrus Cultivars, citrus
Sibling species sample；
And/or the germ is bacterial plant disease, preferably yellow twig.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110835655A (en) * | 2019-12-10 | 2020-02-25 | 广东省农业科学院植物保护研究所 | Method for preventing and treating virus disease of trilobate cotton leaf curl |
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Application publication date: 20190528 |