Apimedica: New ‘BeeTest’ for Detection of Viral, Bacterial Diseases of Honey Bees – “蜜蜂测试”—免疫色谱法快速检测蜜蜂病毒和细菌疾病

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Oct. 22-25, 2012Zhenjiang, ChinaImmunochromatographic Rapid Test “BeeTest” for Detection of Viraland Bacterial Diseases of Honey Bees Apis melliferaLeonid Kelin（“BeeTest” Ltd, Russian Federation,Novosibirsk, Russia）Russian company "Beetest" presents a product whichis unique in the world - immunochromatographic rapid test "Beetest."As our company produces a preparation “Endoglukin” for prevention and treatmentof viral diseases of honey bees and stimulation of the bee colony development,we decided to develop a diagnostic test as a prior stage for diseases treatmentin order to know exactly what kind of disease should be treated. After severalyears of researches we finally managed to find specific antigens for eachdisease that ensures the high sensitivity and delicacy of our test.With the test “Beetest” you can simultaneously check yourhives for the presence of the major bacterial and viral diseases of bees. Thetest is intended for use in the field, does not require highly trainedpersonnel and can be used directly next to the hive.Principle of the methodImmunochromatographic rapid test “BeeTest” is asandwich-type lateral flow assay for simultaneous and differential detection oftwo viral and two bacterial honey bee diseases:· Acute bee paralysis virus;· Sacbrood virus;· American foulbrood (Paenibacillus larvae);· European foulbrood (Melissococcus plutonius)“BeeTest” was developed for rapid, quality and one-stepdetection of the viral and bacterial proteins in honey bee larvae byLateral-flow immunochromatographic technology. The principal scheme of the testis on the picture.
The nitrocellulose membrane is coated with specificpolyclonal antibodies to proteins of Acute bee paralysis virus or Sacbroodvirus or American foulbrood (Paenibacillus larvae) or European foulbrood (Melissococcusplutonius) and specific proteins of these infectious agents.During the first step disrupt the honey bee larvae in thebottle with Elution buffer and glass balls by vigorous shaking. On the secondstep drip a couple of drops into the sample window. In this moment specificviral or bacterial proteins (if they are present in the sample) forms thecomplex with the conjugates of colloidal gold with poly- or monoclonalantibodies.Further these complex bind with the specific antibodiescoated on the membrane thus forming the red line (test line). Conjugates whichwere not involved in forming the complex with bacterial or viral proteins bindswith the specific proteins on the membrane (Picture 2) thus forming another redline (control line). The intensity of red color lines should be assessedvisually.
Reagents and materials:1) 4 plastic cassettes (one cassette for one disease);2) Plastic bottle with a glass balls and Elution buffer;3) Single-use pipette;4) Single-use spatula.Assay procedure:All reagents are ready-to-use and doesn’t need in anypreparations.1) Extract a larva showing suspicious symptoms with thespatula.2) Unscrew lid from the Bottle with Elution buffer. Use thespatula to place sample in the bottle. Replace the lid and shake vigorously forabout 20 seconds.Test line Control line3) Unscrew lid of Bottle and pipe the sample by suppliedpipette. One honey bee larvae should be used with all types of cassettes.4) Hold the Test cassette horizontally and gently squeezetwo drops onto the sample well of the each cassette.5) Keep it horizontal until extract is absorbed (c 30seconds) and a red dye appears in the viewing window.6) Wait until the control line appears (labeled C) and readthe result (10-15 minutes).Reading the results3 Positive result - two lines show up (Test and Control) -indicates that the target pathogen is present in the test sample.4 Negative result - Control line shows up only, no Test line- indicates that the target pathogen has not been detected in the test sample.5 Indefinite result - one Test line without Control line orno one lines indicates that test doesn’t work.Analytical sensitivity of the test kitAnalytical sensitivity is 10-50 ng/ml with using ofrecombinants proteins of Acute bee paralysis virus, Sacbrood virus, American foulbrood (Paenibacillus larvae),European foulbrood (Melissococcus plutonius). Shelf life, storage and transportation conditionsShelf life of the “BeeTest” is 12 month from the date ofmanufacture. Store at 17-27 °C in the dry dark place.______________________________________________*Corresponding author, E-mail: beetest.org@gmail.com“蜜蜂测试”—å…�疫色谱法快速检测蜜蜂病毒和细è�Œç–¾ç—…Leonid Kelin （ä¿„ç½—æ–¯）（“BeeTest” Ltd, Russian Federation,Novosibirsk, Russia）俄罗斯公å�¸çš„“蜜蜂测试”是世界上一ç§�先进独特的快速å…�疫色谱法。本公å�¸ç”¨æ­¤æ³•æŽ§åˆ¶å’Œæ²»ç–—蜜蜂疾病和刺激蜜蜂ç§�群的å�‘展，以达到更好的制备Endoglukin，å…ˆå‰�我们决定研å�‘一ç§�诊断测试法，为了在蜜蜂疾病治疗å‰�准确定ä½�疾病的ç§�ç±»。ç»�过几年的研究，我们最终找到æ¯�一ç§�疾病的特异性抗原，è¿™ä¿�è¯�测试的高效ç�µæ•�。有了蜜蜂测试，ä½ å�¯ä»¥å�Œæ—¶æ£€æŸ¥ä½ èœ‚群主è¦�疾病细è�Œå’Œç—…毒。本测试ä¸�需è¦�专业技术人员，å�¯ç›´æŽ¥åœ¨èœ‚ç®±æ—�边进行检测。方法原ç�†èœœèœ‚测试，快速å…�疫色谱法，是一ç§�夹心型横æµ�化测试法，å�¯å�Œæ—¶å¯¹ä¸¤ç§�蜜蜂疾病病毒和细è�Œè¿›è¡Œç¤º 差检测。1. 蜜蜂急性麻痹肠é�“病毒2. 幼蜂皱è�Žç—…病毒3. 美洲蜜蜂幼虫è…�臭病4. 欧洲蜜蜂幼虫è…�臭病通过横æµ�å…�疫色谱技术，蜜蜂测试å�¯å¿«é€Ÿ，高效，一步检测出蜜蜂幼虫中病毒和细è�Œè›‹ç™½，它主è¦�çš„ 测试路线如图1 所示：ç¡�酸纤维素膜上涂有蜜蜂急性麻痹肠é�“病毒，幼蜂皱è�Žç—…病毒，美洲蜜蜂幼虫è…�臭病毒和欧洲蜜蜂幼虫è…�臭病毒的多克隆抗体以å�Šè¿™äº›ä¼ æŸ“æº�的特殊蛋白。第一步将蜜蜂幼虫放入带有洗脱缓冲液和玻璃ç�ƒçš„瓶中，剧烈振è�¡。第二步滴几滴试剂在采样窗å�£ä¸­，此时特殊病毒和细è�Œè›‹ç™½（如果样å“�中存在）将与å�•å…‹éš†æŠ—体和胶体金的轭å�ˆç‰©å½¢æˆ�å¤�å�ˆç‰©。图二测试线和控制线中轭å�ˆç‰©å’ŒæŠ—原的键å�ˆ（Au–胶体金和抗体的轭å�ˆç‰©, Ag – 抗原) 之å�Žè¿™äº›å¤�å�ˆç‰©å’Œè–„膜上的特殊抗体紧紧地结å�ˆåœ¨ä¸€èµ·，å½¢æˆ�红线（检测线），那些没有没有和薄膜上 的特殊蛋白结å�ˆçš„å¤�å�ˆç‰©（如图二）å½¢æˆ�å�¦ä¸€æ�¡çº¢çº¿（控制线），红色线的强度å�¯ä»¥ç”¨çœ¼è§‚测出æ�¥。试剂和æ��æ–™1. 4 个塑料储片夹（一个对应一ç§�疾病）2.带有玻璃ç�ƒå’Œæ´—脱缓冲液的塑料瓶3.一次性试管4.一次性è�¯åˆ€å®žéªŒæ­¥éª¤æ‰€æœ‰è¯•å‰‚都è¦�æ��å‰�准备好。1. 用è�¯åˆ€å�–一å�ªæœ‰å�¯ç–‘症状的幼虫2. 旋开带有洗脱缓冲液的塑料瓶，用è�¯åˆ€æŠŠå¹¼è™«æ”¾å…¥å¡‘料瓶中。盖上瓶盖，剧烈振è�¡20 秒。3. 旋开瓶盖，用å�¸é‡�管å�¸å�–洗脱缓冲液。æ¯�个幼虫的缓冲液液都è¦�æ»´å…¥4 个塑料储片夹。4. 水平拿ä½�测试储片夹，缓慢滴两滴缓冲液在样å“�室。5. ä¿�æŒ�æ°´å¹³，使æ��å�–物被å�¸æ”¶（30 秒），观察窗中出现红线。6. 等到控制线出现（标记为C）然å�Žè¯»å‡ºç»“æžœ（10-15 分钟）结果1、阳性结果——出现两æ�¡çº¢çº¿（检测线和控制线）—表明目标病原体出现在测试样å“�中。2、阴性结果——å�ªå‡ºçŽ°æŽ§åˆ¶çº¿—表明目标病原体ä¸�存在于测试样å“�中。3、无效结果——有测检测线没有控制线或者两æ�¡éƒ½æ²¡æœ‰è¡¨æ˜Žæµ‹è¯•æ— æ•ˆè¯•å‰‚盒的分æž�ç�µæ•�度用10-50ng/mg蜜蜂急性麻痹肠é�“病毒，幼蜂皱è�Žç—…病毒，美洲蜜蜂幼虫è…�臭病毒和欧洲蜜蜂幼虫è…�臭病毒的é‡�组蛋白质æ�¥åˆ†æž�ç�µæ•�度。ä¿�质期，储存和è¿�输æ�¡ä»¶ ä¿�质期12 个月，储存æ�¡ä»¶ä¸º17-27℃，黑暗干燥处。