Cabinet of Ministers Regulations No. 364 in Riga on 29 may, 2007 (pr. No 31 61) potato brown rot of combat and control procedure Issued under the plant protection act, article 13 i. General questions 1. determines the order in which combats the potato ring rot in dark and limited its spread. 2. potato dark ring suggests the plant quarantine organism Ralstonia solanacearum (Smith) Yabuuc et al. (hereinafter body). 3. To establish the dark ring of potatoes, State plant protection service (hereinafter service): 3.1 regularly checks the potato Solanum tuberosum l. plants and tubers (hereinafter potatoes) and eat tomato Lycopersicon esculentum Mill. (SYN. Lycopersicon lycopersicum (l.) H. Karsten) plants, other than fruit and seeds (hereinafter referred to as the organism);
3.2. take seed and other potatoes for use of storage of samples and send them for laboratory testing;
3.3 check visually by cutting potatoes for Potato tubers. If it is found in the potato brown rot of potatoes of Visual signs, take samples and send them for laboratory testing;
3.4. when assessing the risk of the organism spreading, check: 3.4.1. knockturn Solanace family au gus, including wild plants of this species (hereinafter referred to as the other organism);
3.4.2. surface water used for irrigation and spraying of host plants;
3.4.3. the company, engaged in the processing and packaging of host plants, liquid waste, used for irrigation and watering the host plant;
3.4.4. the growing medium, soil and solid waste from the production host processing and packaging companies;
3.5. check reservoir and taking water samples for laboratory testing, if the received information from another Member State of the European Union that it infected Syme niekaug watering using surface water that flows into Latvia;
3.6. diagnosed organisms in accordance with the provisions of 1, 2, 3, and 4;
3.7. the isolation and cultivation of the organisms used in these rules referred to in annex 5, medium;
3.8. test procedures used in these rules referred to in annex 6, buffer;
3.9. contamination levels determined in accordance with the provisions of annex 7;
3.10. laboratory analysis carried out in accordance with these rules 8, 9 and 10;
3.11. determines, controls and forcibly take control of the organism and control measures (hereinafter referred to as the health measures). 4. A Person must not be stored or reproduced, agent of brown rot of potatoes in pure cultures, except in the cases laid down in the legislation for trial or scientific purposes and for work on varietal selections of harmful organisms, plants, plant products and contact with them in the introduction into and movement of objects. II. determination of phytosanitary measures 5. If, by laboratory testing in accordance with the provisions referred to in annex 1 of the rapid diagnostic test-immunofluorescence test (IF) of a positive result, and this is confirmed by polymerase chain reaction (PCR) test or fluorescence in situ hybridisation (FISH) testing, service shall decide on the following health measures: 5.1 prohibited move host tubers or plants, or the party from holding a sample was taken except if the host is moved to the service of the organism have been eliminated and the risk;
5.2. at the risk of the organism spreading of prohibited person evaluation move from holding other holding grown host plants;
5.3. take measures to ascertain the presence of the organism may cause. 6. If the subsequent laboratory tests in accordance with the provisions set out in annex 1 of the bioassay tests of the samples and identification of the body is not found, the Department under certain phytosanitary measures shall be repealed. 7. to determine the provenance of the organism, the service checks a lot of potatoes that were associated with the rule mentioned in paragraph 5 potatoes. 8. If the subsequent laboratory tests in accordance with the provisions set out in annex 1 of the bioassay and identification tests are found organisms in the sample, the service validates the dark ring of potatoes. 9. If the found body, service shall decide on the application of phytosanitary measures in accordance with the provisions of 10, 11, 12, 13, 14 and 15. 10. in order to determine the organism and mark the land 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 host plants and their cargo or the lot from which the sample was taken (the-infected host plants);
10.2. the inventory or parts, agricultural machinery and vehicle which had contact with the infected host plants;
10.3. other items, including packaging, which had contact with the infected host plants;
10.4. the field, cover the area and the place of production from which the infected host plants for harvest;
10.5. the field, cover the area and the raising of host plants of vegetation which takes samples of host plants;
10.6. other host plants from which the sample was taken;
10.7. the surface water from which the sample was taken. 11. in order to determine the organism and the site plan in the infected area, the suspect shall be determined: 11.1. all host plants grown at the place of production, which sets the rules referred to in paragraph 10;
11.2. production sites which are linked to the infected host plants, also of the place of production, which is a joint technique of farming vehicles and rooms with growing site that is found in the infected host;
11.3. the host which has grown 11.2. these provisions in the place of production referred to in or after the capture of the organism found in the place of production, set to be infected;
11.4. warehouses or premises which are stored or processed in the host plant of this rule 11.2. referred to in subparagraph places of production;
11.5. equipment, agricultural machinery, vehicles, warehouse or parts and other items, including containers and packaging, which during the previous 12 months were able to come into contact with the infected host plants;
11.6. host plants, which come in contact with this rule 11.5. the items referred to in point before cleaning and disinfection;
7.3. potatoes, which are associated with the infected lots of seed potatoes or tomatoes, origin, coming from the same place of production with infected tomatoes, if in accordance with the provisions referred to in point 6 of the inspection and test results, the body was not found, however, there is a risk of the organism spreading, plants reproduction;
11.8. the place where grown this rule 11.7. saimn referred to in paragraph iekaug;
7.4. host breeding sites that use irrigation or spraying water, defined as contaminated;
11.10. host plants grown on fields flooded with surface water designated as contaminated. 12. the service determines the following: 12.1. phytosanitary measures prohibit planting infected and potentially infected host plants;
12.2. instructs you to do one of the following: 12.2.1. service of the burn the infected host plants;
12.2.2. after heat treatment that destroys the organism infected niekaug for use of the feedingstuff Syme;
12.2.3. infected host plants deeply burying waste dumps, from which the body can not get agricultural land and which are not in contact with the water reservoirs, which could be used for irrigation of agricultural land or watering;
12.2.4. industrial processing infected host plants, immediately bringing them directly to the processing plant, which has a suitable waste treatment facilities, warehouses and vehicles both cleaning and disinfecting equipment;
12.2.5. host plants durum production waste (including rejected scale and bark) and any other solid waste (for example, soil, stones and other debris), which have been in contact with SAIM niekaug: 12.2.5.1. burying landfills, bringing the industry directly to the landfill and the way to avoid their loss and the body would not be able to get agricultural land, as well as this site would not have contact with the water reservoirs, where water can be used for irrigation of agricultural land or watering;
12.2.5.2. destroyed;
12.2.5.3. do other phytosanitary measures where there is a risk of spreading the organism is eliminated;
12.2.6. host liquid production waste: 12.2.6.1. If they contain solid particles, before tipping the filter or induct solids to separate them, and other solid waste burying or destroying it in accordance with this regulation and section 12.2.5.2.12.2.5.1.;
12.2.6.2. before tipping it to be fired at least 30 minutes at the minimum temperature of 60 ° C; 12.2.6.3. do other phytosanitary measures against the organism into the agricultural land and reservoirs, which can be used for irrigation of agricultural land or watering;
12.2.7. do other phytosanitary measures if the organism have been eliminated the risk;
12.2.8. potentially infected Potato tubers: 12.2.8.1. under the supervision of the use of the service for other purposes (for example, or send directly to the sales outlets). Potato tubers agricultural sales site delivered packed. Potatoes intended for planting after cleansing and disinfection may be processed in the same place;
12.2.8.2. industrially processed food potatoes, packing and immediately bringing directly to the processing plant, which are suitable for waste disposal and disinfection equipment and transport means departing cleaning and disinfecting equipment;
12.2.8.3. do other phytosanitary measures in accordance with the laws and regulations in the field of plant quarantine, prevention of the risk of the organism spreading;
12.2.9. other parts of host plants, including trunks and leaves: 12.2.9.1. destroyed;
12.2.9.2. do other phytosanitary measures in accordance with the laws and regulations in the field of plant quarantine, prevention of the risk of the organism spreading. 13. the service instructs a person to cover areas and places of production, defined as contaminated, according to the intensity of use of the field to select and perform one of the following measures: 13.1 if the phytosanitary measures apply for at least five years after the capture of the organism: 13.1.1. four years after the capture of the organism: 13.1.1.1. destroy volunteer potato, tomato plants, solanaceous weeds and other host plants of the organism;
13.1.1.2. must not be planted Potato tubers and plants, tomato plants, Brassica genus of plants and other host plants, as well as sow their seeds, if it can spread the organism;
13.1.2. If at least two previous periods of vegetation not found pārziemojuš potatoes, tomato plants, solanaceous weeds and other host plants of the organism, in the fifth year of the growing potatoes may be planted for potatoes for food, feed or for processing. These potatoes after harvesting service checks in accordance with paragraph 3 of this rule;
13.1.3. the first potato or tomato production under the year crop rotation, service this provision referred to in paragraph 3;
13.2. If the phytosanitary measures shall apply for a period of six years after the capture of the organism: 13.2.1. destroy volunteer potato, tomato plants, solanaceous weeds and other host plants of the organism;
13.2.2. the first three periods of vegetation in the install and maintain black fallow land, pasture land, and often low-mow or intensive grazing, sow the seed for the taking of lawns or crops;
13.2.3. the next two periods of vegetation in the field to grow the plants, not the host;
13.2.4. next potato or tomato-growing season that follows this rule 13.2.2 and 13.2.3.. the period referred to, may be grown potato seed production, or for other purposes (such as food, feed, processing) for potato production. The resulting tubers shall be tested in accordance with the provisions of 1, 2, 3 and 4 of the annex. 14. After the volunteer potato, tomato plant, solanaceous weeds and other host plants of the organism spreading risk prevention service-the organism infected area in fields or on places of production other than those referred to in paragraph 13 of these regulations, subject to the rotation: 14.1 after the first vegetation period prohibit the capture of the organism to plant the tubers, plants, or other host plants of the organism and asking to destroy volunteer potato, tomato plants and other host plants of the organism, or permission to plant only certified seed potatoes for other purposes (consumption of potatoes for , processing, animal feed production, if it is destroyed) all pārziemojuš potatoes, tomato plants and other host plants of the organism;
14.2. in the second period of vegetation after the infection detection may be planted to certified seed potatoes and seed for other purposes (food, feed, processing) for potato production;
14.3. the third growing seasons after the discovery of the infection permitted the seeds or other purposes (food processing, forage) production of potatoes for planting potatoes or certified seed potatoes that are directly derived from certified seed potatoes and have tested the service;
14.4. in each of these rules and 14.3 14.2. bottom vegetation period referred to in paragraph quoted in destroying the potatoes shall, tomato plants and other host plants of the organism. The harvested tubers shall be tested in accordance with the provisions of 1, 2, 3 and 4 of the annex. 15. the service determines the following: 15.1 the phytosanitary measures cover areas prohibit planting Potato tubers and plants or seeds, 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, and is fully nomanīt growth substrate, as well as cover has been cleaned and disinfected areas and equipment. To prevent the spread of the organism, the Department carried out irrigation and watering system monitoring;
15.2. instructs the service of the person: 15.2.1 to clean up or disinfect the infected or suspect warehouses, agricultural machinery, vehicles and other objects, destroy or disinfect the infected and potentially infected host plants on in-contact packaging. These objects after disinfection, are no longer considered infected or potentially infected;
15.2.2. immediately after each of the organism and the next vegetation period allowed to the first year of production of potatoes in fields that were identified as infected, take all potato production-related machinery and inventory, cover the area cleaning and, if possible, also for disinfection;
15.3. cultivation sites on the infected area for at least three years after the capture of the organism a person asked: 15.3.1. seed potatoes to harvest, store and move separately from other objectives (such as food, feed, processing) for potatoes or on seed potatoes and potatoes for processing before consumption to make cleaning and dez infection;
15.3.2. make machinery, storage and production sites for cleaning and, if possible, also for disinfection;
15.3.3. all places of production designated as contaminated, use only certified seed potatoes and the harvested tubers shall be tested in accordance with the provisions of 1, 2, 3 and 4 of the annex. 16. in order to determine the possible spread of the organism and the original provenance of the organism, the service check: 16.1. potatoes, associated with contaminated batches of potatoes;
16.2. the cultivation site, located not far from the place of production which has been found in infected potatoes;
16.3. the cultivation site, located not far from the surface water flooding or otherwise come into contact with the infected field. 17. in order to determine the provenance of the organism, the service check: 17.1. other this provision mentioned in paragraph 10.1 below host plants cultivation related sites: 17.1.1. where host plants are grown or tumor whose origin is associated with the infected host plants;
17.1.2. tumors which are grown or the host that will monitor the service as suspected of being infected with the organism;
17.1.3. in which potatoes are grown or tumor whose origin is associated with the potatoes at the place of production, set to be infected with the organism;
17.1.4. where host plants are grown and which is adjacent to the place of production, defined as infected, including breeding sites that share the use of agricultural machinery, vehicles and rooms with a farm, which is found in the infected host;
17.1.5. which is used for the irrigation and spraying of surface waters, which is identified as contaminated or suspected of being infected;
17.1.6. which is used for the irrigation and spraying of surface waters, which is used in common with places of production, which is defined as contaminated or suspected to be infected;
17.1.7. which submerged or have been flooded with surface water is defined as contaminated or suspected of being infected;
17.2. surface water used for irrigation or spraying, or applūdinājuš in the field or place of production designated as contaminated;
17.3. other solanaceous plants in the wild. 18. If the service finds the original organism material, service checks the original material, random seed, pre-basic or basic seed potatoes originating in associated with contaminated batches of seed potatoes, and the sample sent to the laboratory for testing. 19. Service: 19.1. the decision shall specify the deadline of phytosanitary measures. The decision shall be accompanied by a map of the infected area;
19.2. the monitor and control of phytosanitary measures;
19.3. in places defined as contaminated, after the harvest, within the time limit laid down in the decision to check the potatoes;
19.4. check irrigation and watering systems and, if necessary, shall determine the prohibition of the use of water, to prevent the risk of the organism spreading;
19.5. monitor the farms, warehouses and Potato tubers or tomatoes with the movements of the related undertakings, as well as production sites, which use the agricultural technique, together with the growing sites were detected in infected potatoes. 20. the infected area at least three the next vegetation period after infection detection service that rule 3.1. and 3.2. checks referred to in point. 21. The infected area where surface water is definitely about infected or potentially infected, service: 21.1. carry out regular annual inspections, taking water and solanaceous host plants and by testing in accordance with the provisions of 1, 2, 3, and 4;
21.2. irrigation and watering systems and, where appropriate, define water-use restrictions to prevent spraying of host plants risk of the organism spreading. You can cancel the prohibition, on the basis of the annual results. Water to which the prohibition applies, may be used if taken measures and destruction of the organism is not possible spread of the organism;
21.3. the watch collection with a host of materials related to the manufacturing or packaging companies that have discovered the organism infected liquid waste. 22. If the person has not complied with the service specified phytosanitary measures and infected or potentially infected areas are planted a_few years previously infected or potentially infected or non-certified potatoes, service enforced can chemically or mechanically destroy potato plantations. With the destruction of potato related costs shall be borne by the person. III. The European Commission and the Member States of the European Union information 23. If according to this provision, 1, 2, 3, and 4. the laboratory referred to in annex methods of a positive test result and suspect that the organism is infected host plants used for surface irrigation water, which flows from another Member State of the European Union or source to another Member of the European Union, the service in each of these cases shall inform the Member States of the European Union. 24. If the suspected infected potatoes are imported into or exported from a Member State, the service immediately of such event shall inform the European Commission: 24.1. submit notification indicating the following information: 24.1.1. potato or tomato lot, denomination of the variety;
24.1.2. consignor and recipient's name and address;
24.1.3. the potato or tomato lot date of delivery;
24.1.4. delivered in the potato or tomato lot size;
24.1.5. plant passport number or the manufacturer's or dealer's registration number for plant-health control exposed the plants and plant products circulating in the registry of parties;
24.2. the notification shall be accompanied by a copy of the plant passport or a copy of the delivery note. 25. the service shall inform the European Commission and the Member States of the European Union for each capture of the organism. The notification shall specify the following infor lag: 25.1. date in accordance with the provisions of paragraph 5 of a positive test result of sampling and detection of the organism, a description of the investigation to identify the source of contamination and the possible spread of the infection (also indicate to what extent carried out sampling);
25.2. the 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, defined as infected, and the infection level description, production sites and lots of potatoes. Specifies the names of the varieties of potatoes, seed potatoes-categories;
25.3. the infected host plants shipment or lot attached to the phytosanitary certificate or plant passport number;
25.4. the infected seeds or other purposes (food processing, fodder) potatoes varieties and categories;
25.5. the infected area and in particular the phytosanitary measures;
15.9. the rules referred to in point 8 of the test result of extract, prepared the slide immunofluorescence testing, infected Eggplant (Solanum melongen) material and pure cultures of the organism;
25.7. infected water bodies recognised description, name and location, as well as barring the infected areas of the irrigation. 26. If the Department intends to apply other phytosanitary measures of infected and potentially infected potatoes for use, before service of the application shall inform the European Commission and the Member States of the European Union. 27. the service every year until 1 June shall notify the European Commission and the other Member States of the European Union in the previous year, performed the survey details and results. On the same farm for planting potatoes selected checks, notifications sent every year until 1 September. IV. final question 28. Be declared unenforceable in the Cabinet of 26 July 2005, the provisions of no. 568 "potato brown rot of eradication and control arrangements" (Latvian journal, 2005, nr. 122). Informative reference to European Union directives, the regulations include provisions resulting from: 1) of the Council of 20 July 1998 Directive 98/57/EEC on Ralstonia solanacearum (Smith) Yabuuc et al.; 2) Commission 14 July 2006 Directive 2006/63/EC amending annexes II and VII to Council Directive 98/57/EC on Ralstonia solanacearum (Smith) yabuuchi et al.. Prime Minister a. Halloween Minister of Agriculture m. Roze Ministry of Agriculture presented version of annex 1 of the Cabinet of Ministers of 29 May 2007 by Regulation No 364 of the potato tuber sampling and detection and identification of the organism they shall: 1. sample preparation 1.1. standard sample size is 200 tubers per test. Intense look in sampling tubers gu sample is larger, the test results can be difficult to obtain or to interpret. However, this procedure can easily be used for samples with less than 200 tubers are not available in the quantity required. All validation methods described below is based on sample testing of 200 tubers. The potato extract described below can also be used for detection of the organism; 1.2. additional pre-treatment prior to sample preparation: samples before testing 1.2.1 stand up to 25-30 0 c temperature for up to two weeks to encourage multiplication of all population of the organism; 1.2.2. wash the tubers. Between each sample test to use appropriate disinfectants (when PCR-test, chlorine compounds are used to clear the potential pathogen DNA) and detergents. Allow the tubers to dry. Washing process is particularly useful (but it is not mandatory) for samples with excess soil and if test or PCR test for the direct isolation; 1.3. with a clean and disinfected scalpel or vegetable knife the skin at each remove heel (heel) to reveal the vascular. Carefully cut small pieces of potato from the heel end and try to cut out the non-vascular tissue to a minimum; 1.4. collect the heel end cores or new disposable containers which can be closed or severely (in case containers are reused, they are cleaned and disinfected using chlorine compounds). The heel end recommended process immediately. If this is not possible, they can be stored in a container, adding a buffer is no longer than 72 hours refrigerated for not longer than 24 hours at room temperature. Process the heel ends or pieces, using one of the following procedures: 1.4.1. base or transfuse pieces with sufficient quantity (approximately 40 ml) of extraction buffer (Appendix 6) and shuffle the Rotary shaker (50 to 100 rpm) for four hours at a temperature of no lower than 24 0 c, and 16-24 hours refrigerated; 1.4.2. homogenizing heel ends with a sufficient quantity of (approximately 40 ml) of extraction buffer (Appendix 6) with mixer (Waring or ultra Thurax) or by crushing in a sealed disposable maceration bag (e.g. Stomacher or strong polyethylene Bioreba, 150 mm x 250 mm; sterilized with ionizing radiation) using a rubber mallet or appropriate grinding (e.g. Homex). 1.5. the top layer of the clear supernatant. If excessively cloudy, clarify either by slow speed centrifugation (at not more than 180 g for 10 min at 4-10 ºc temperature) or by vacuum filtration (40-100 µm), washing the filter with additional (10 ml) extraction buffer; 1.6. the bacterial fraction to focus, 15 minutes 4-10 ºc temperature centrifugation at 7000 g (or 10 min at 10000 g.), and not mixing the residue, discard the supernatant liquid top layer; 1.7. resuspend the sediment 1.5 ml pellet buffer (Appendix 6). Use 500 µ l 500 µ l of the organism, Clavibacter michiganensis subsp. sepedonicus and 500 µ l reference needs. Obtain a final concentration of 10-25% (v/v) sterile glycerol add 500 µ l of the reference aliquot part and the remaining test aliquot, Vortex and store at-16 to-24 0 c (weeks) or-68 to-86 0 c (months). An aliquot of the test during the test store 4-10 ºc temperature. Repeated freezing and thawing is not recommended. If the extract is required to transport, ensure the transportation of refrigerator 24 hours; 1.8. the positive control organism materials and samples required treated separately to avoid contamination. This applies to IF slides and to all tests; 2. IF test: 2.1 use Multiwell microscope slides with preferably 10 Windows of at least six millimeters in diameter. Test control material like sample; 2.2. prepare the test slides by one of the following procedures: 2.2.1. Pellet with relatively little starch sediment into the first box, transfer the potato pellet dilution 1/100 (15 µ l box with six millimeter diameter-quantity for larger Windows in proportion to the increase). Then a similar volume of undiluted Pellet (1/1) to transfer the remaining boxes in the row. The second row can be used as duplicate or for a second sample; 2.2.2. other pellet preparing the resuspended pellet decimal dilutions (1/10, 1/100) pellet buffer. Pipette into each box in a row into the same quantity of re-suspended pellet appliance (15 µ l is appropriate for 6 mm window diameter-for larger Windows increase proportionally to the quantity). The second row can be used as duplicate or for a second sample; 2.3. dry the droplets at ambient temperature or heated up to 40-45 ºc. The bacterial cells to the slide by heating (15 minutes at a temperature of 60 0 c), flaming, with 95% ethanol or according to the suppliers of the antibodies for specific instructions. If necessary, fixed slides before further testing can be stored in the frozen form of drying box for the shortest possible period of time (no more than three months); 2.4. IF procedure: 2.4.1. preparation of the test slide according to this annex to point 2.2.1. Prepare a double dilution. In the first box, must be ½ of the titre (T/2), the other-¼ titre (T/4), ½ of the titre (T/2), the titre (T) full and double the titre (2T) (Figure 1); Figure 1 Test slide preparation of resuspended pellet dilution 1/100 1/1 1/1 1/1 1/1 [] of resuspended pellet dilution (T = titre) T/2 T/3 T/2 T 2T [] double dilution of antiserum/antibody 1. sample • 1 • 2 • 3 • 4 • 5 1. duplicate samples or sample 2 • 6 • 7 • 8 • 9 • 10 2.4.2. preparation of the test slide according to paragraph 2.2.2 of this annex. Prepare the working dilution of the antibodies (DA) in IF buffer (Figure 2). The working dilution affects the specificity of determination; Figure 2 Test slide preparation of Antiserum/antibody working dilution 1/1 1/1 10/100 blank empty [] of resuspended pellet decimal 1. sample • 1 • 2 • 3 • 4 • 5 1. duplicate samples or sample 2 • 6 • 7 • 8 • 9 • 10 2.4.3. slides to put on wet paper napkins. Each test box full overlay with the dilution of antibodies. The volume of antibody applied to each box must be at least equal to the volume of the extract. If there is no antibody suppliers of specific instructions, the following procedure should be carried out: 2.4.3.1. slides to treat wet napkins covered way 30 minutes at ambient temperature (18-25 0 c); 2.4.3.2. shake off droplets of each slide and Rinse carefully with IF buffer. Wash for five minutes in IF buffer-Tween dipping (annex 6) and then IF buffer. To avoid aerosol formation or droplet transfer that may result causing contamination. Carefully take excess liquid gently wipe; 2.4.3.3. slides to put on wet paper napkins. Cover the test Windows with the dilution of FITC conjugate used to determine the titre. The volume of conjugate applied on the box must be equal to the volume of antibody used; 2.4.3.4. slides shall pass to the wet napkins covered way 30 minutes at ambient temperature (18-25 0 c); 2.4.3.5. shake off droplets of conjugate slide. Before rinse and wash. Carefully wipe the excess liquid; 2.4.4.7. pipette on each window 5-10 µ l 0.1 M phosphate-buffered glycerol (Appendix 6) or a commercially available color stability enabling the binder and apply a coverslip; 2.5. reading the IF test: 2.5.1. examine test slides on an epifluorescence microscope with filters suitable for working with FITC, water or oil immersion at a magnification of 500 to 1000. The boxes look across two diameters at right angles and around the perimeter. Samples of the cells do not see or appear in small amounts, to examine at least 40 microscope fields; 2.5.2. in the test Windows of the test slides for bright Fluorescing cells with characteristic morphology of the organism. The fluorescence intensity must be equivalent to the positive control strain at the same antibody dilution. Cells with incomplete staining or with weak fluorescence is not taken into account; 2.5.3. take into account the specific size only Fluorescing cells with typical morphology at the titre of the antibodies or the working dilution, as defined in paragraph 2.4 of this annex; 2.6. IF test reading interpretation: 2.6.1. If found bright Fluorescing cells with characteristic morphology, identify specific cell average number of microscope field and calculate the number of typical cells per ml of resuspended pellet in accordance with the provisions of annex 7. IF the test is positive for samples with no less than 5 × 103 typical cells per ml of resuspended Pellet. The following sample is considered potentially infected and needs further testing; 2.6.2. IF test is negative for samples with less than 5 × 103 typical or non-typical cells per ml of resuspended pellet and the sample is considered negative. Further testing is not carried out; 3. PCR test: 3.1 use of validated PCR reagents, respectively, and protocols in accordance with the provisions of Annex 8. It is recommended that you choose a method of internal control. Use appropriate precautions to avoid contamination of the sample with target DNA. The PCR test is carried out in specially equipped molecular biology laboratories, in order to decrease the risk of sample contamination with target DNA. Negative control samples (DNA extraction and PCR) is always used as the definitive procedure to determine whether the DNA transfer has taken place. Include the following in the PCR test negative controls: 3.1.1. sample extract that previously tested negative for the presence of the organism; 3.1.2. buffer controls used for extracting the bacterium and the DNA from the sample; 3.1.3. PCR reaction mix; 3.1.4. If possible, also use the PCR DNA from positive control samples. To avoid potential contamination, prepare positive controls prepare separate from the test look for maritime industries and services. Extract the sample as purified from soil. Therefore, in certain cases, if intended for use in PCR Protocol, it is recommended to prepare extracts from washed potatoes; 3.2. Dna purification methods. Target DNA purification from complex sample substrates are available in a variety of methods, thus detaching the PCR and other enzymatic reaction inhibitors and the sample concentration of target DNA. For use with validētaj-PCR method, described in annex 8 of these regulations, is optimized for the following method: 3.2.1. Pastrik (2000) method: 3.2.1.1. pipette 220 l to µ lizēšan buffer (100 mM NaCl, 10 mM TRIS-HCl [pH 8,0], 1 mM EDTA [pH 8,0]) 1.5 ml microvial; Add 100 µ l 3.2.1.2. sample extract and place for 10 minutes in a heating block or water bath at 95 ºc temperature; 3.2.1.3. the tube 5 minutes put the ice; 3.2.1.4. add 80 µ l lysozyme stock solution (50 mg lysozyme per ml in 10 mM TRIS HCl, pH 8.0) and incubate at 37 0 c 30 min. temperature; 3.2.1.5. Add 220 µ l Easy DNA ® solution A (Invitrogen), mix well by vortexing and incubate at 30 min 65 ° C; 3.2.1.6. Add 100 µ l of Easy DNA ® solution B (Invitrogen), Vortex, Vortex vigorously until the sample is uniformly viscous; 3.2.1.7. Add 500 µ l of chloroform and stir up the viscosity decreases and the mixture becomes homogeneous; 3.2.1.8. centrifuge for 20 min at 4 0 c temperature at 15 000 g to separate phases and form the interphase; 3.2.1.9. transfuse top phase pure microvial; 3.2.1.10. Add 1 ml of 100% ethanol (-20 ° C), mix and 10 min to hold ice; 3.2.1.11. centrifuge for 20 min at 4 0 c temperature at 15 000 g and remove ethanol from pellet; 3.2.1.12. Add 500 µ l 80% ethanol (-20 0 c) and stir flipping; 3.2.1.13. centrifuge for 10 minutes at a temperature of 0 c 15 4 000 g, save the pellet and remove ethanol. 3.2.1.14. allow the pellet to dry in the environment or DNS vakuumžāvētāj; 3.2.1.15. resuspend the pellet in 100 µ l of sterile ultra pure water (UPW) and leave at least 20 min at room temperature; 3.2.1.16. to use PCR to store-20 0 c temperature; 3.2.1.17. spin down any white precipitate by centrifugation and use 5 µ l of the PCR in the clear top layer containing DNA; 3.2.2. other methods. Other DNA extraction methods, such as Qiagen DNeasy plant Kit, may be applied provided that it is demonstrated effectiveness equivalent to DNA from control samples in the treatment that contains 103 to 104 1 ml pathogenic bacteria. 3.3. PCR: 3.3.1. prepare the PCR test and control templates in accordance with the test protocols (annex 8). Prepare one decimal sample DNA extract (1:10 in upw); 3.3.2. in accordance with the published protocols prepare the appropriate PCR REACTION mix in a contamination-free environment (annex 8). If possible, we recommend using a multiplex PCR protocol that also incorporates an internal PCR control; 3.3.3. Add 2-5 µ l DNA extract to 25 µ l PCR reaction in sterile PCR tubes according to PCR Protocols (annex 8); 3.3.4. add negative control containing only PCR reaction mix and add the same grade water originating in what was used in the PCR mix in place of the sample; 3.3.5. Insert tubes in the same either, which was used for prior checking, the Guide and implement the optimized PCR program (annex 8). 3.4. the analysis of the PCR product: 3.4.1 split the PCR Amplicon with agarose gel electrophoresis. Electrophoresis is made with 5-8 cm at least 12 µ g/l DNS propagation reaction mix of each sample mixed with 3 µ l ballast buffer (Appendix 6) in 2.0% (w/v) agarose gels in TRIS-actet-EDTA (Tae) buffer (annex 8). Use the appropriate DNA marker, e.g., 100 bp ladder; 3.4.2. reveal DNA bands, 30b-60 min. dyeing with ethidium bromide (0.5 mg/l), with the mutagenic substances. taking appropriate precautions; 3.4.3. painted with UV gel short-wave transillumination (λ = 302 nm for example) seen stained PCR products size match the expected (annex 8), process document; 3.4.4. for all new finds or cases of authenticity of the PCR Amplicon by performing enzymatic restriction analysis with the remaining amplified DNA samples, the optimum time for optimum temperature can be with an appropriate enzyme and buffer (annex 8). Identify the sections that cracked agarose gel electrophoresis, and the methods listed above and after staining with ethidium bromide to observe characteristic restriction fragment, using UV transillumination. Compare the positive testimony before and after cleavage reactions. 3.5. the PCR test result interpretation: 3.5.1 the PCR test is negative if the body to specific PCR Amplicon of expected size in that pattern is not found, but it detects all in the positive control (case of multiplex PCR with plant specific internal control primers: a second PCR-product of expected size must be amplified with the sample in question); 3.5.2. The PCR test is positive if it is found the special body to PCR Amplicon of expected size with characteristic and restriction (if necessary), provided that it is not derived from any of the negative control samples. Reliable confirmation of a positive result can also be obtained by repeating the test with a second set of PCR primers (annex 8). Contamination may be suspected if the expected Amplicon is obtained from one or more of the negative control samples; 4. FISH test: 4.1. potato extract fixation: 4.1.1. prepare fixative solution in accordance with the provisions of Annex 9; 4.1.2. pipette 100 µ l of each sample extract into an Eppendorf tube and centrifuge for seven minutes with 7 000 grams; 4.1.3. remove the supernatant and dissolve the pellet in 200 µ l of fixative prepared