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豆薯层锈菌

豆薯层锈菌

  • 平台编号:bio-110623
  • 规格:ELISA试剂盒
  • 注意事项:仅用于科学研究或者工业应用等非医疗目的,不可用于人类或动物的临床诊断或治疗,非药用,非食用
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Catalog Number AP 107 Intended Use The ELISA screens for the presence of soybean rust caused by Phakopsora pachyrhizi. The test can detect the presence of the pathogen at the very early stages of infection, from chlorotic lesions (before formation of a pustule) to immature pustules (not releasing spores). During this period it is often difficult and critical to differentiate the soybean rust symptoms from other diseases caused by bacterial, viral or fungal infections and/or insect damage. In addition, the test can also be used to detect advanced rust symptoms with uredinospores and teliospores, complementing visual inspections. In controlled inoculation studies with levels as low as 100,000 spores/mL, this kit has been shown to detect the presence of soybean rust infection before the appearance of visual symptoms. Infection levels in the field may vary depending on environmental conditions. Materials Needed • Tissue Extraction Kit, EnviroLogix Cat. # ACC 002 OR Multi-Wall Mesh Pouch, EnviroLogix Cat. # ACC 021 • pipettes capable of delivering 100 µL • marking pen (indelible) • tape or Parafilm® • timer • distilled or deionized water for preparing Wash Buffer and for diluting 5x Soy Leaf Extraction Buffer • glass bottles or flasks with 250 mL capacity for storage of 1x Soy Leaf Extraction Buffer and 1 liter capacity for Wash Buffer • microtiter plate reader or strip reader • wash bottle, or microtiter plate or strip washer • multi-channel pipette that will measure 100 µL (optional) • racked dilution tubes for loading samples into the plate with a multichannel pipette (optional) • orbital plate shaker (optional) Preparation of Solutions Wash Buffer: Add the contents of the packet of Wash Buffer Salts (phosphate buffered saline, pH 7.4 - Tween 20) to 1 liter of distilled or deionized water, and stir to dissolve. Store refrigerated when not in use; warm to room temperature prior to assay. 1x Soy Leaf Extraction Buffer To prepare, warm 5x Soy Leaf Buffer supplied with the kit to room temperature and mix well before diluting. Add one measure of 5x Buffer to QualiPlate Kit for Soybean Rust Page 2 of 5 Rev. 04-19-07 Punch leaf sample Use pestle to mash leaf tissue Remove unneeded strips Add sample extract Bottle Wash method four measures distilled or deionized water in a suitable container to create 1x Soy Leaf Buffer. Mix thoroughly to dissolve. Store refrigerated when not in use; warm to room temperature prior to assay. 1X Soy Leaf Buffer is stable for two weeks when stored refrigerated at 4-8°C and should be made on an as-needed basis. Please call EnviroLogix Tech Service if you have questions regarding calculating the appropriate amount of buffer to make for the number of samples that will be processed. Sample Preparation Sampling Recommendation: Take a leaf sample that includes a suspect spot or area. The ACC 002 punch cap will result in a leaf sample of approximately 10mm diameter, weighing about 0.01 grams. If using an alternate method, care must be taken to ensure that the sample does not have an excess of non-affected tissue; this may reduce sensitivity. Sample Extraction: 1. Green leaf samples: Take a leaf punch sample by snapping the tube cap of the Disposable Tissue Extractor down on the leaf, encompassing the suspected rust spot. Insert the pestle into the tube and grind the tissue by rotating the pestle against the sides of the tube with twisting motions. Add 500 µl of Soy Leaf Extraction Buffer and continue grinding for 20- 30 seconds or until the leaf tissue is well ground. Use extreme caution to prevent sample-to-sample cross-contamination with plant tissue or exudate. 2. Similar grinding devices (e.g. Multi-Wall Mesh Bags) can be used with a leaf to extraction buffer ratio (weight:volume) of 1:25 – 1:50 (eg. 3 mL to a 2.5 cm diameter leaf section). Using a hard object (e.g. coin) rub across the surface of the mesh bag against a hard surface until the leaf has transparent areas where the inside mesh has been forced through the leaf. After adding Extraction Buffer, massage the exterior of the mesh bag with fingers while holding the top of the bag closed to prevent the buffer from spilling. How to Run the Assay • Read all of these instructions before running the kit. • Allow all reagents to reach room temperature before beginning (at least 30 minutes with un-boxed plates and reagents at room temperature - do not remove plates from bag with desiccant until they have warmed up). • Organize all reagents, sample extracts, and pipettes so that step 1 can be performed in 15 minutes or less. If more than three strips are to be run at one time, the loading time will most likely exceed 15 minutes, and the use of a multi-channel pipette is strongly recommended in steps 1, 5, 8 and 9. • If three or fewer strips are to be run, use a disposable-tip, airdisplacement pipette and a clean pipette tip to add each Standard and sample extract to the wells. Conjugate, Substrate, and Stop Solution may be added in the same manner; alternatively, use a repeating pipette with a disposable tip for these three reagents. • Once all components have reached room temperature, remove the plate from the pouch. If fewer than all twelve strips are used, reseal the remaining strips and the desiccant in the foil pouch, and refrigerate. QualiPlate Kit for Soybean Rust Page 3 of 5 Rev. 04-19-07 Incubate Test can be read visually, or… …complete protocol and add Stop Solution Read plate in a Plate Reader within 30 minutes of the addition of Stop Solution • Use the well identification markings on the plate edge as a guide when adding the samples and reagents. It is recommended that at least two wells each of Blank (Extraction Buffer) and a known-negative soy leaf extract be run on each plate. Additional quality control samples may be added at the discretion of the user. The kit Positive Control is provided to show an example of a strong positive result. Sample extracts may be run in either single or duplicate wells. 1. Add 100 µL of Extraction Buffer Blank, 100 µL of the Soybean Rust Kit Positive Control, 100 µL of any user-prepared negative control leaf extract, and 100 µL of each sample extract to their respective wells. Follow the same order of addition for all reagents. NOTE: It is strongly recommended that a multi-channel pipette be used in steps 1, 5, 8 and 9. 2 Thoroughly mix the contents of the wells by moving the plate in a rapid circular motion on the bench top for a full 20-30 seconds. Be careful not to spill the contents! 3. Cover the wells with tape or Parafilm to prevent evaporation and incubate at ambient temperature for 1 hour. If an orbital plate shaker is available shake plate at 200 rpm. 4. After incubation, carefully remove the covering and vigorously shake the contents of the wells into a sink or other suitable container. Flood the wells completely with Wash Buffer, then shake to empty. Repeat this wash step three times. 5. Add 100 µL of Soybean Rust Enzyme Conjugate to each well. 6. Thoroughly mix the contents of the wells, as in step 2. Cover the wells with new tape or Parafilm and incubate for 1 hour at ambient temperature. Use orbital shaker if available. 7. Wash the wells again as described in step 4. Alternatively, perform four washes (300 µL/well) with a microtiter plate or strip washer. Slap the plate on a paper towel to remove as much water as possible. 8. Add 100 µL of Substrate to each well. Mix thoroughly as in step 2. Cover the wells with new tape or Parafilm and incubate for 20 minutes at ambient temperature. Use orbital shaker if available. NOTE: At this point, results may be scored visually. Test wells that are blue are positive for soybean rust. Very weakly positive (ie very light blue) results may require the use of a plate reader for confirmation. If the plate reader is to be used, continue with step 9. Caution: Stop Solution is 1N Hydrochloric acid. Handle carefully. 9. Add 100 µL of Stop Solution to each well and mix thoroughly. This will turn any positive well contents yellow. NOTE: Read the plate within 30 minutes of the addition of Stop Solution. How to Interpret the Results Visual Inspection After step 8 above, the well containing the Positive Control should show a distinct blue color. If not, that may indicate an invalid assay due to possible improper protocol, and the test should be repeated. QualiPlate Kit for Soybean Rust Page 4 of 5 Rev. 04-19-07 If after step 8 above, a test well is blue, this is interpreted as positive for Phakopsora pachyrhizi. Very weakly positive results may require the use of a plate reader for confirmation. Spectrophotometric Measurement Set the wavelength of the microtiter plate reader to 450 nanometers (nm). (If it has dual wavelength capability, use 600, 630 or 650 nm as the reference wavelength.) Interpreting Results Compare the Optical Density (OD) of the sample extracts to those of the mean Extraction Buffer Blank wells, or preferably, to known-negative leaf extract wells, to determine presence or absence of soybean rust in your sample extract. Samples with absorbances significantly greater than those of the Blank and/or negative leaf extract wells are presumed to be positive for soybean rust. Cross-Reactivity The kit does not cross react with several other rust infections caused by Uromyces, Puccinia and Melampsora species. No cross reactivity was observed with other common fungal genera including Aspergillus, Cercospora kikuchii or C. sojina, Fusarium, Penicillium, Peronospora mansurica, Pseudomonas savastanoi pv. Glycinea, Septoria, Rhizoctonia, Rhizopus, and Xanthomonas campestris pv. Glycinea. No cross reactivity has been observed with similar-looking diseases such as frogeye leafspot, powdery mildew, downy mildew, brown spot, bacterial blight, bacterial pustule. Precautions and Notes • Observe any applicable regulations, federal or state guidelines, or inhouse lab safety protocols when disposing of samples and kit reagents. • Store all QualiPlate components at 4°C to 8°C (39°F to 46°F) when not in use. • Do not expose QualiPlate components to temperatures greater than 37°C (99°F) or less than 2°C (36°F). • Allow all reagents to reach ambient temperature (18°C to 27°C or 64°F to 81°F) before use. • Do not use kit components after the expiration date. • Do not use reagents or test plates from one QualiPlate with reagents or test plates from a different QualiPlate. • Do not expose Substrate to sunlight during pipetting or while incubating in the test wells. • Do not dilute or adulterate test reagents or use samples not called for in the test procedure. • As with all tests, it is recommended that results be confirmed by an alternate method when necessary. QualiPlate Kit for Soybean Rust Page 5 of 5 Rev. 04-19-07 For Technical Support Contact Us At: EnviroLogix 500 Riverside Industrial Parkway Portland, ME 04103-1486 USA Tel: (207) 797-0300 Toll Free: 866-408-4597 Fax: (207) 797-7533 e-mail: info@envirologix.com website: www.envirologix.com LIMITED WARRANTY EnviroLogix Inc. (“EnviroLogix”) warrants the products sold hereunder (“the Products”) against defects in materials and workmanship when used in accordance with the applicable instructions for a period not to extend beyond a product’s printed expiration date. If the Products do not conform to this Limited Warranty and the customer notifies EnviroLogix in writing of such defects during the warranty period, including an offer by the customer to return the Products to EnviroLogix for evaluation, EnviroLogix will repair or replace, at its option, any product or part thereof that proves defective in materials or workmanship within the warranty period. ENVIROLOGIX MAKES NO OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO ANY IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. The warranty provided herein and the data, specifications and descriptions of EnviroLogix products appearing in EnviroLogix published catalogues and product literature are EnviroLogix’ sole representations concerning the Products and warranty. No other statements or representations, written or oral, by EnviroLogix’ employees, agents or representatives, except written statements signed by a duly authorized officer of EnviroLogix Inc., are authorized; they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty. EnviroLogix does not warrant against damages or defects arising in shipping or handling, or out of accident or improper or abnormal use of the Products; against defects in products or components not manufactured by EnviroLogix, or against damages resulting from such non-EnviroLogix made products or components. EnviroLogix passes on to customer the warranty it received (if any) from the maker thereof of such non-EnviroLogix made products or components. This warranty also does not apply to Products to which changes or modifications have been made or attempted by persons other than pursuant to written authorization by EnviroLogix. THIS WARRANTY IS EXCLUSIVE. The sole and exclusive obligation of EnviroLogix shall be to repair or replace the defective Products in the manner and for the period provided above. EnviroLogix shall not have any other obligation with respect to the Products or any part thereof, whether based on contract, tort, strict liability or otherwise. Under no circumstances, whether based on this Limited Warranty or otherwise, shall EnviroLogix be liable for incidental, special, or consequential damages. This Limited Warranty states the entire obligation of EnviroLogix with respect to the Products. If any part of this Limited Warranty is determined to be void or illegal, the remainder shall remain in full force and effect. Parafilm is a registered trademark of American Can Corporation EnviroLogix, the EnviroLogix logo, and QualiPlate are trademarks of EnviroLogix Inc. © EnviroLogix 2007
安瓿瓶冻干管打管说明
 
一、打管说明
1、安瓿瓶开封:用浸过75%酒精的脱脂棉擦净安瓿管,用火焰加热其顶端,滴少量无菌水至加热顶端使之破裂,用锉刀或者镊子敲下已破裂的安瓿管顶端。
2、菌株恢复培养:用无菌吸管吸取0.3--0.5ml适宜的液体培养基,滴入安瓿管内,轻轻振荡,使冻干菌体溶解呈悬浮状。吸取全部菌体悬浮液,移植于1-2支建议的培养基试管中,并在建议的条件下培养。
3、注意事项:菌种活化前,请将安瓿管保存在6-10℃的环境下,某些菌种经过冷冻干燥保存后,延迟期较长,需要连续两次继代培养才能正常生长。
4、复苏后的菌种在传1-2代后使用。
5、暂不启开的安瓿及复苏后需保藏的斜面应于4℃中保藏。
特别注意事项
l、微生物菌种应保藏于低温、清洁和干燥的地方,室温放置时问过长会导致菌种衰退;
2、菌种操作应在无菌条件下进行,防止杂菌污染:
3、斜面菌种保藏时间通常为1-2个月,应根据菌种状况及时转接;冻干菌种保藏时间通常为5~10年;
4、菌种使用过程中如出现杂菌污染或菌种生产性能下降,应及时与中国微生物菌种查询网联系或更换新的菌种。

  西林瓶和甘油冻存管打管说明
 
二、西林瓶菌种打管复活操作步骤
1、用 75%酒精棉擦拭西林瓶表面进行消毒。 
2、在生物安全柜中打开铝制瓶盖和胶盖。 
3、用无菌吸管吸取 0.5ml 左右液体培养基(《菌种说明书》中固体培养基去掉琼脂即可)于西 林瓶中将冻干菌粉全部溶解。 
4、将溶解后的菌悬液转移至盛有 4~5mL 液体培养基的试管中混匀,可将残留在吸管中的 1-2 滴菌悬液转接至固体培养基上。
5、将液体试管和斜面试管于推荐条件下静置培养,以液体培养结果为准。
三、甘油冻存管菌种打管复活操作步骤 
1、准备好推荐的菌种培养基(见《菌种说明书》)。
2、将冻存管下半部浸入 37℃温水中轻轻摇动,使冷冻状态的菌种悬液迅速融化。如果收到菌 种冻存管时菌悬液是融化状态则需要立即进行转接培养。 3、用 75%酒精棉擦拭冻存管表面进行消毒。
4、在生物安全柜中打开冻存管瓶盖,用无菌吸管吸取全部菌悬液,均匀涂布到 2 支试管斜面 或平板上。 
5、将斜面试管或平板于推荐条件下静置培养。对于生长缓慢的菌种应持续培养 2 周至更长的 时间。
四、注意事项 
1、 所有打管操作都需要无菌操作。需由专业微生物技术人员在相应防护设备中进行,生物危 害程度为三类的菌种应在生物安全柜中操作。 
2、 西林瓶和冻存管打开后需一次用完,不能留存。 
3、 菌种经过冻干保藏后,处于休眠状态,需要转接 2-3 代恢复活力。
4、 大部分冻干菌种会在培养几天后生长。有些菌种第一代恢复生长时会有较长的延迟期,培养此类菌种时需要将培养时间延长至正常培养时间的双倍时长,甚至 2 周至更长的时间。