Read the untranslated law here: https://www.vestnesis.lv/ta/id/113980
Cabinet of Ministers Regulations No. 569, Riga, July 26, 2005 (pr. No 43 40) of potato ring rot and control procedure Issued under the plant protection act article 5 paragraph 13 i. General questions 1. determines the order in which combats the potato brown rot disease and light limited its spread.
2. potato light ring suggests the plant quarantine organism Clavibacter michiganensis subsp. sepedonicus (hereinafter body).
3. to establish the potato ring rot, light State plant protection service (hereinafter service): 3.1 regularly checks the potato Solanum tuberosum l. tubers and plants;
3.2. in seed potatoes and potatoes for other uses of Solanum tuberosum l. tuber plants and storage sites and send them to the laboratory for analysis;
3.3. test of potato Solanum tuberosum l. samples visually cut the tubers of potato, Solanum tuberosum l. samples and send them for laboratory analysis, if found in the Visual of potato ring rot symptoms;
3.4. in accordance with the rules referred to in annex laboratory methods laboratory diagnosed;
3.5. apply control and forced out the spread of potato ring rot restriction and control measures (hereinafter referred to as the phytosanitary measures).
4. a Person must not store or replicate the body pure, except as provided in law for trial or scientific purposes and for work on varietal selections of harmful organisms, plants, plant products and contact with them came the arrival of the items and the order.
II. Phytosanitary measures 5. determination if the potato Solanum tuberosum l. Visual inspection service finds of potato ring rot symptoms or in accordance with the provisions referred to in the annex of the immunofluorescence test laboratory analysis of a positive test result is a service shall decide on such a phytosanitary measures: UR5.1.lai prevent the risk of the organism spreading, the person of the load or the potato lot from which the sample was taken from a holding may be moved only under the supervision of the authorities;
5.2. prohibit a person to move from holding other solanaceous plants (Solanaceae) (hereinafter referred to as the host).
6. If the provisions of this annex in future in laboratory tests are not found in samples of the organism, the service specified phytosanitary measures shall be repealed.
7. to determine the provenance of the infection, the service checks the other batches of potatoes have been in connection with the provisions referred to in paragraph 3 of the potatoes.
8. confirms the bright ring of potatoes, if two different provisions referred to in the annex to these laboratory tests are found in the body.
9. If a body is found, the service shall decide on the application of phytosanitary measures. The decision includes this provision 10, 11, 12 and 13 of the type referred to in paragraph 1.
10. in order to determine the prevalence of the infection and the site plan in the infected area (fields, the place of production and the holdings that are in any way associated with the infected potatoes), infected States: 10.1 potato Solanum tuberosum l. tuber or plant or lot from which the sample was taken (hereinafter referred to as the infected potatoes);
10.2. the inventory or parts, agricultural machinery and vehicle, which had been in contact with infected potatoes;
10.3. other items, including packaging, which had been in contact with infected potatoes;
10.4. the field and the place from which you obtained the infected potato yield and the land plan highlights the field and place of production;
10.5. the rural and growing place where potato vegetation period takes samples of potatoes, and the land plan highlights the field and growing location.
11. in order to determine the prevalence of the infection and the site plan in the infected area, the suspect shall be determined: 11.1 all Potato tubers or plants, cultivated or located at the place of production, which sets the rules referred to in paragraph 10;
11.2. the place of production or land and premises related to the tubers or plants determined as contaminated, including growing site that agricultural machinery, vehicles and premises used in common with places of production which has been found in infected potatoes;
11.3. Potato tubers and plants which have been grown this rule 11.2. places or referred to in these places of production found infection detection;
11.4. the place of production or warehouse that stored Potato tubers that are moved from this rule 11.1 and 11.2. bottom of the places referred to in paragraph 1;
UR11.5.iek ārt, agricultural machinery, vehicles, warehouses and other items, including the packaging, which in the previous 12 months were able to come into contact with infected potatoes;
11.6. Potato tubers or plants that came into contact with warehouses or other objects before cleaning and disinfection;
7.3. Potato tubers or plants, which are associated with the infected lots of seed potatoes originating in or coming from the same place with infected potatoes because of the possible spread of the infection, plants reproduction.
12. the service determines the following: 12.1. phytosanitary measures prohibit planting infected and potentially infected potatoes;
12.2. instructs you to do one of the following: 12.2.1. service of destroying infected potatoes;
12.2.2. industrial processing infected potatoes, immediately bringing them directly to the processing plant, which has a suitable waste treatment facilities, warehouse and vehicle disinfection system. These requirements apply if the processing undertaking agrees to rework the infected potatoes;
12.2.3. to execute other phytosanitary measures if the organism have been eliminated the risk;
12.3. ask to meet one of the following: 12.3.1. service of the use of the potentially infected potatoes for their own consumption or to send directly to sales outlets. Potato tubers are delivered packed at the point of sale, and may not be repackaged;
12.3.2. industrial processing potentially infected potatoes, immediately bringing them directly to the processing plant, which has a suitable waste treatment facilities, warehouse and vehicle disinfection system. These requirements apply if the processing undertaking agrees to recycle potentially infected potatoes;
12.3.3. perform other phytosanitary measures where there is a risk of spreading the organism is eliminated.
13. the potato grower asked places of production designated as contaminated, choose and perform one of the following: 13.1. phytosanitary measures in fields defined as contaminated, depending on the intensity of use of the field: 13.1.1. where a phytosanitary measures apply three years after the infection detection: UR188.8.131.52.izn īcin to volunteer for the potatoes and other host plants of the organism;
184.108.40.206. prohibit planting Potato tubers, plants, sow their seeds, beet and other host plants of the organism, until the field is not pārziemojuš at least two of potatoes the next vegetation period;
220.127.116.11. the third growing year if at least two previous periods of vegetation not found pārziemojuš potatoes may be planted to certified seed potatoes for potatoes for food, feed or for processing;
18.104.22.168. current annual production of potatoes under the plant, certified seed may be planted in potatoes, seed production and other types of potatoes (for food, feed, processing);
13.1.2. where a phytosanitary measures applied four years: 22.214.171.124. during this period, destroy the mojušo potato Board and other host plants of the organism;
126.96.36.199. the first three periods of vegetation in the install and maintain black fallow land, pastures with frequent and low intensity nopļaušan or over-grazing;
188.8.131.52. in the fourth growing season of potatoes planting certified seed potatoes or seed other types of potatoes (for food, feed, processing);
13.2. other fields, subject to the change of the plant: 13.2.1. the first vegetation period after infection detection prohibits planted Potato tubers, plants, sow their seeds or other host plants of the organism and asking to destroy the potatoes or permitted volunteer planting only certified seed potatoes potatoes intended for other purposes (such as food processing, feed), if you have destroyed all pārziemojuš potatoes and other host plants of the organism, and the past year has not grown beets;
13.2.2. second period of vegetation after the infection discovery permission to plant certified seed potatoes seed extraction and other types of potatoes (such as food, feed, processing);
13.2.3. each vegetation period defined rules 13.2.1 13.2.2 this. and the bottom point., asking to destroy volunteer potatoes and other host plants of the organism;
13.3. cover areas prohibit the person planting the tubers and plants, as well as other host plants of the organism, except for the production of potatoes used certified seed potatoes, sīkbumbuļ, or meristēmaug, which are tested in service, a complete replacement of the growing medium and cover the area and the cleaning and disinfection of equipment.
14. the service instructs a person to service of: UR14.1.att īrī and disinfect infected or suspect warehouses, agricultural machinery, vehicles and other objects, destroy or disinfected with infected or potentially infected potatoes on in-contact packaging. After disinfection, these objects are no longer considered infected or potentially infected;
14.2. immediately after infection and in each of the next vegetation period up to the first allowed in the year of production of potatoes in fields that were determined to be infected, take all potato production-related machinery and storage area, and cover the areas of cleaning, as well as, if possible, the disinfection;
14.3. the infected area in place for at least three years after the discovery of the infection: 14.3.1. seed potatoes to harvest, store and move separately from other purposes (food, feed, processing) for potatoes;
14.3.2. do all with potato production-related machinery and storage area, and cover the areas of cleaning, as well as, if possible, the disinfection;
14.3.3. the cultivation of potato to use only certified seed potatoes.
15. in order to determine the possible spread of the organism and the original provenance of the infection, the service check: 15.1. Potato tubers and plants associated with contaminated batches of potatoes;
15.2. the farm, located in the vicinity of the holding, which has been found in infected potatoes.
16. If the service finds the original organism material, service to its original material, random seed, basic or higher generation inspection of seed potatoes originating in associated with infected seed potato lot and sent samples for laboratory analysis.
17. Service: 17.1. the decision shall specify the deadline of phytosanitary measures;
17.2. monitor and control the implementation of phytosanitary measures;
17.3. the decision within the time specified in the locations specified for infected or potentially infected, after harvest, take the potato;
17.4. monitor the farms, storage and movement of potato tuber with affiliates, as well as farms that use agricultural machinery on the basis of the Treaty, together with the holding on which it was found infected potatoes;
17.5. the decision shall be accompanied by a map of the infected area.
18. the infected area at least three the next vegetation period after infection detection service Inspector that provision referred to in paragraph 3.
19. when potato breeder has not fulfilled the service specified phytosanitary measures and infected or potentially infected areas are planted infected or potentially infected or non-certified potatoes, service enforced then chemically or mechanically destroyed potato plantations. With the destruction of potato related costs shall be borne by the producers of potatoes.
III. The European Commission and the Member States of the European Union of 20 service information shall immediately inform the European Commission and the Member States of the European Union for each case that found the body, and the notice indicates the following: 20.1. registration number of the holding for plant-health control exposed the plants and plant products circulating in the registry of parties, in which the place of production, set to be infected;
20.2. the phytosanitary certificate or plant passport number attached to the infected host plants to the shipment or lot.
20.3. the infected seeds or other purposes (for example, food processing, animal feed) potatoes varieties;
20.4. Description of the defined infected area and phytosanitary measures laid down therein;
20.5. these rules referred to in point 8 the test result (extract, prepared the slide immunofluorescence analysis, infected Eggplant (Solanum melongen) material and pure cultures of the organism).
21. If the Department intends to apply other phytosanitary measures of infected and potentially infected potatoes, before these measures shall inform the authorities of these measures, the European Commission and the Member States of the European Union.
Informative reference to European Union directive rules included provisions deriving from Council of 4 October 1993 of Directive 93/85/EEC on the control of potato ring rot.
Prime Minister a. Halloween farming Minister – the Minister of the environment r. vējonis Editorial Note: the entry into force of the provisions to 10 august 2005.
Annex a Cabinet of 26 July 2005, regulations No 569 Clavibacter michiganensis subsp. detection and identification of sepedonicus in potato tuber samples i. test sample test 1 sample size is 200 tubers of the standard. You can also test the smaller samples.
2. Preparation of samples for testing: 2.1. Potato tubers, even wash and allow them to apžū;
2.2.ar disinfected scalpel removes potato heel end core piestipr in the definition bark and cut 3-5 mm large pieces of potato;
2.3. place the pieces of potato single-use container and pour with 40-50 ml volume of maceration buffer. It is recommended to cut tissue processed immediately. If immediate treatment is not possible, store the samples up to 72 hours at + 4 ° C to + 10 ° C or up to 24 hours at room temperature;
2.4. sample incubate for four hours with 50 to 100 Shaker revolutions per minute;
2.5. decant the macerate and concentrate bacteria by centrifugation of samples (10000 g) 10 minutes + 4 ° C to + 10 ° C;
2.6. the supernatant liquid, without sediment. The resulting precipitate is dissolved 1.5 ml pellet buffer and further divide the sample analyses: 184.108.40.2060 μl used bacteria Clavibacter michiganensis subsp. sepedonicus and Pseudomonas solanacearum;
220.127.116.110 μl stored reference purposes. Sample reference part 200 μl of sterile glycerol added, mix and store – 75 ° C and-85 ° C;
2.7. during testing, the sample cut vascular and sediment suspension is stored at + 4 ° C to + 10 ° c.
II. test 3 immunofluorescence immunofluorescence (IF) test used for Multiwell microscope microscope slide. On slides with pencil inscription CMS and sample identification number.
4. Prepare decimal dilutions Lees 1/10, 1/100 and 1/1000 the tube with sediment mix. vortex. Pipette Lees and each dilution standard quantity-20 ml-box line (table). The remainder of the line is used for duplicate or second sample.
Table Not diluted 1/10 1/100 1/1000 model 2 1 2 2 2 3 2 4 2 5 1 1 or 2 a duplicate sample sample 2 6 2 7 2 8 2 9 2 10 5. Prepare separate positive control slides (as in). Negative control used in pellet buffer. 5. run it and slide the eye 10 undiluted. Each test kit uses a single slide.
6. the samples shall be dried until the slides are dry. The slides are incubated for 10 minutes at 96% ethanol and firing.
7. Prepare the antibody in IF buffer the working dilution, as indicated in the instructions of the manufacturer.
8. Cover the test boxes with the dilution of antibodies (antibodies used box quantity must be equal to the quantity of sediment used) and incubate for 30 minutes in humid Chamber at room temperature, if antibodies in the instructions of the manufacturer unless otherwise indicated.
9. of the antimatter priekšmetstikl Harvest drops, and the slides carefully with IF buffer flush. Wash twice in five minutes in IF buffer. Carefully dry the excess moisture.
10. preparing IF buffer fluorescīnizotiocianāt (hereinafter referred to as FITC) conjugate dilution work, as indicated in the instructions of the manufacturer.
11. Cover the test boxes with FITC conjugate dilution (dilution of the conjugate box quantity must be equal to the quantity of dilution of the antibodies used) and incubate for 30 minutes in humid Chamber at room temperature if the manufacturer's instructions in the FITC conjugate unless otherwise indicated.
12. Harvest of the slide, drops of conjugate. Rinse and wash as above. Carefully dry the excess moisture.
13.Ar micropipette for each drops 5 to 10 ml of phosphate and glycerol buffer, or similar means of montējoš and cover with a coverslip.
14. The overview slide test boxes with filters for fluorescent microscope suitable for FITC, using oil immersion with a magnification of 1000reiz. Viewing boxes across two diameters at right angles and around the perimeter.
15. First examine positive and negative control slides. Positive control cells must be bright fluorescent and completely painted. If they are not painted, repeat the test. Negative control boxes may not be the typical Fluorescing cells, otherwise the test repeated.
16. Looking at the slide test boxes typical Fluorescing cells with characteristic Clavibacter michiganensis subsp. sepedonicus morphology. The fluorescence intensity must be the same as the dilution of positive control. Cells with incomplete staining or with weak fluorescence ignores. If such a cell is much based on the interpretation of the IF test result.
17. IF test result interpretation: 17.1. If bright Fluorescing cells with characteristic morphology are not found, the IF test is negative;
17.2. If bright Fluorescing cells with characteristic morphology are found, determine the average number of lens cell vision field and calculate the number of cells (N) per ml of resuspended Pellet. On the border IF test is considered to be approximately 103 cell population of re-suspended pellet per: 17.2.1. samples with N greater than 103 cells per ml of resuspended Pellet, the IF test is considered positive;
17.2.2. samples with N less than 103 cells per ml of resuspended Pellet, the IF test is considered positive as possible;
17.3. If you see a large number (> 105 cells per ml) of incomplete or weakly Fluorescing cells, the IF test is considered positive as possible.
III. PCR test 18. Bacteria DNA extraction: 18.1. place 220 µl lizēšan buffer into an Eppendorf tube;
18.2. Add 100 µl sample extract;
18.3. the sample is incubated at 95 ° C for 10 minutes;
18.4. further incubation of sample on ice for 5 minutes;
18.5. add 80 µl of lizozīm concentrate;
18.6. incubate at 37 ° C for a sample of 30 minutes;
11.6. Add 220 µl of solution A, EasyDN inverts the sample;
12.8. incubate at 65 ° C for 30 minutes;
11.7. the 100 μl EasyDN solution B, inverts the sample until the formation of viscous solution;
18.10. Add 500 μl chloroform and shake well;
18.11. centrifuge the sample (15000 g) 20 minutes at + 4 ° C;
18.12. the upper phase into a new microvial;
18.13. Add 1 ml of chilled (-20 ° C) 96% ethanol;
18.14. centrifuge the sample (15000 g) 20 minutes at + 4 ° C;
18.15. decant the liquid phase;
18.16. Add 500 µl of chilled (-20 ° C) 80% ethanol;
18.17. centrifuge the sample (15000 g) 20 minutes at + 4 ° C;
18.18. decant the liquid phase;
18.19. dry the precipitate at room temperature;
18.20. precipitate dissolves 100 μl of sterile distilled water;
18.21. incubate the samples at room temperature for 20 minutes;
18.22. moved sample-20 ° C for further storage.
19. The PCR test progress: 19.1. prepare the PCR reaction mixture;
19.2. PCR reaction tube place 20 ml of reaction mixture;
19.3. place each tube 5 ml of the test sample. Each sample shall be analysed;
19.4. the samples are placed in termoprocesor.
20. The PCR product into agarose gel: 20.1. prepare mixture and pour the agarose gel;
20.2. the gel under 12 μl PCR products;
20.3. the gel side holes insert a 3 µl 100 base pairs (bp) of DNA markers;
20.4. the distribution of the electric field of the PCR product with the power strength of 65 mA and voltage 130 V 60-80 minutes;
20.5. transfer the gel ethidium bromide solution and colour for 30 minutes;
20.6. placed on the gel and transiluminator seen in the PCR products by UV light.
21. the data obtained in the PCR test interpretation: 21.1. If the negative control is not detected by the appropriate hole in the bp length of PCR Amplicon, the PCR test is considered negative;
21.2. If the hole identifies the fragment length under positive control signal, the sample is considered positive.
IV. Biological pathogenic (Eggplant) test and identification tests 22. Eggplant (Solanum melongen) grown in the glasshouse + 21 ° C to + 24 ° C in the day and at night, temperature is not lower than + 15 ° c. Eggplants provide sufficient lighting (approximately 14 hours). Temperature must not be higher than + 30 ° c. Grafting Eggplant third page view development stage. Important to infect as newer au gus, which is more susceptible to infection than small parents, indicator.
23. One sample having 35 plants (25 plants, five plants and five positive control negative control plants). On the potty with Eggplant seedlings of write sample identification number and indicate whether it is a plant of positive control or negative control plants.
24. start Grafting with negative control plants, then takes the sample and in the end – positive control plants.
25. the Pot on the stem between the cotyledons and the first leaf. The day before the inoculation of the plants do not want watering. Stem ieskrāp-0,5 1,0 cm long and three quarters of depth. With a syringe or a pipette pipette sample stems and aizlipin with paraffin to dries. The same Act also with control plants.
26. Positive and negative control plants places separately from the sample.
27. After a week started regular observation of the symptoms. If there is a page on wilting beginning withered tissue may appear dark green mottled spots, tiles, which slowly becomes softer, to reach the stage of necrotic. Necrotic tissue forms a strong yellow pages edge expressed that gradually spreads all over the eggplant. Infected, indicator, which shows the Clavibacter michiganensis subsp. sepedonicus characteristics may not completely die. The plant can regenerate after infections, creating a side shoots and new shoots.
28. the identification of test progress: 28.1. further testing having leaves with symptoms or the stem above the infected sites, disinfected with 70% ethanol, cut into small pieces, cover with sterile water, leave for 15 minutes, then extract the IF and PCR tests carried out;
28.2. If after four weeks of symptoms is observed plant by IF and PCR tests. 1 cm of the stem take part (above the infected sites) of all plants, disinfected with 70% ethanol, cut into small pieces, cover with sterile water and leave to stand for 15 minutes, then extract the IF and PCR tests carried out;
17.6. If you need additional pathogen recontamination from eggplant, testing having leaves with symptoms or the stem above the infected sites, disinfected with 70% ethanol, cut into small pieces, cover with sterile water and leave to stand for 15 minutes, strapped on a yeast peptone glucose agar, incubated at YPG + 21 ° C to + 25 ° c. Colonies are observed each day, they grow very slowly (up to 10 days), is cream in color, details, cupola. Grown colonies and with pure cultures of dressing (diluting the colony with 1 ml pellet buffer colony taking with 1 ml loop) in the IF test. (IF instead of having the test sediment extracts from the colonies.)
29. Also performs the following tests: UR29.1.kr āsošan by gramme (Clavibacter michiganensis subsp. sepedonicus +);
29.2. oxidase test (Clavibacter michiganensis subsp. sepedonicus).
30. biological pathogenic (Eggplant) identification of the test and the test results interpretation: 30.1. If the Eggplant test specimen is negative (no symptoms), but positive control is positive, the negative control is negative and IFA and PCR tests are negative, the sample is considered negative;
30.2. Eggplant test sample, if is negative (no symptoms), but positive control is positive, the negative control is negative and IFA and PCR tests are positive, the sample is considered positive;
30.3. If the Eggplant test sample is positive (symptoms), positive control is positive, but negative control is negative and IFA and PCR tests are positive, the sample is considered positive;
18.9. If confirmatory tests (IF, PCR) results do not match, perform re withdrawal of pathogen of Eggplant;
5. If you are typical of Clavibacter michiganensis subsp. sepedonicus colonies and IF the test is positive, the sample is considered positive;
If there is no typical 30.6. Clavibacter michiganensis subsp. sepedonicus colony and IF test is negative, the sample is considered negative.
31. After the screening test – IF the test and PCR test-indicates whether the found Clavibacter michiganensis subsp. sepedonicus. If the Clavibacter michiganensis subsp. sepedonicus are found, the article added, "Testing continues using biological pathogenic (Eggplant) test and identification tests" and the final opinion on the sample provided by the test results.
V. Use solutions maceration buffer: 18.104.22.168.05 M PBS (PBS) buffer, pH 7.0;
Na2HPO4 4.26 g 32.2. –;
KH2PO4 2.72 g 32.3. –;
32.4. distilled water – 1000 ml dissolve ingredients and 32.5. check pH;
32.6. buffer solution sterilize autoclave + 121 ° C for 15 minutes.
33. Pellet buffer: 33.1.0.01 M phosphate buffer (PBS), pH 7.2;
33.2. Na2HPO4 · 12H2-2.7 g;
33.3. Na2HPO4 · 2H2O – 0.4 g;
20.8. distilled water – 1000 ml dissolve ingredients and 20.8. check pH;
20.9. buffer solution sterilize autoclave + 121 ° C for 15 minutes.
34. IF-buffer: 34.1.0.01 M phosphate buffer (PBS), pH 7.2;
21.3. Na2HPO4 · 12H2-2.7 g;
21.3. Na2HPO4 · 2H2O – 0.4 g;
NaCl 8.0 g 21.4. –;
34.5. distilled water – 1000 ml dissolve ingredients and 21.5. examine the pH;
21.6. buffer solution sterilize autoclave + 121 ° C for 15 minutes.
35. glycerol phosphate buffer: 35.1.0, 1 M phosphate glycerol (PGB) buffer, pH 7.6;
35.2. Na2HPO4 · 12H2-3.2 g;
35.3. NaH2PO4 · 2H2O – 0.15 g;
35.4. glycerol 50 ml; 35.5. distilled water, dissolve 100 ml; 35.6. ingredients and check pH;
22.2. buffer solution sterilize autoclave + 121 ° C for 15 minutes.
36. Lizēšan buffer: 36.1.100 mM NaCl;
36.2.10 mM TRIS-HCl (TRIS (hydroxymethyl) aminoetān) (pH 8.0);
36.3.1 mM EDTA (dihydrated disodium salt) (pH 8.0).
37. Lizozīm 37.1.50 mg of concentrate: lizozīm;
37.2.1 ml in 10 mM TRIS-HCl. 38. Electrophoresis buffer: 38.1.10 times Tae (TrisAcetātaEDT);
38.2. TRIS base – 48.4 g;
23.8.-11.42 ml glacial acetic acid; 23.9. Edta – 3.72 g;
38.5. H2o-up to 1000 ml. 39. Ethidium bromide solution. Once a Tae buffer to add ethidium bromide concentration (10 mg/ml) with a final concentration of 0.5 mg/ml. 40. PCR reaction mixture: no PO box
The quantity of reagent one response (ml) 24.9.
Sterile distilled water 25.0 9.5.
10xPCR buffer 2.5 25.0.
25 mm MgCl2 40.4 3.
dntp's 0.5 25.2.
PS-1 1.0 40.6.
PS-R 1.0 40.7.
NS-7-F 1.0 25.4.
NS-8R 1.0 25.4.
Taq polymerase 0.5 40.10.
NS-8R 1.0 40.11.
Taq polymerase 0.5 34.64.
Sample 5.0 VIII. PCR Primer sequences and used the software table 1 no PO box
The oligonucleotide primer sequence Amplicon length (bp) 1.
PS-1 5 '-gtg ggg tgg ctc CTT ga aa-3 ' 502 2.
PS-R 5 '-tac TGA gat GTT TCA CTT ccc c-3 ' 3.
NS-7-F 5 '-gag GCA ATA ACA ctg TGA tgc-the gg 3 ' 377 4.
NS-8-R 5 '-tcc GCA TCA CCT acg to gg ga-3 ' 2. table no PO box
The number of cycle length temperature (° C) objective 1.
1 3 min Genomic DNA denaturation at 95 2.
10 1 min Genomic DNA denaturation at 95 1 min 1 min 64 72 of Primer hybridization Fragment synthesis 3.
25 30 s Genomic DNA denaturation at 95 30 s 30 s 62-72 Primer hybridization Fragment synthesis 4.
1 5 min 72 Fragments after filling stores while the sample is located in the oven 4-IX. preservation of test material and storage 49. If there is a suspicion of the presence of the organism, because it is of a positive immunofluorescence test result by the method laid down in this annex, and the need to continue the investigation, laboratory tests to confirm or not confirm the presence of the organism in a sample, up to completion of the Save and test the stored until laboratory tests completed : 30.5. If possible, all tubers or plants, of which remove samples;
30.6. the remains of any pellet and in addition prepare for immunofluorescence slides.
50. If in the laboratory analysis in accordance with the methods referred to in this annex, the presence of the organism in a sample is approved, at least one month must be maintained and must be stored: 50.1. point 1 of this annex the specified material;
50.2. a sample of the infected Eggplant material inoculated with that tuber or plant extract;
50.3. The tirage liqueur for this distinction in the organism.
Minister of agriculture, Minister for the environment r. vējonis
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