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

Apimondia Apimedica-Apiquality International Forum

Oct. 22-25, 2012

Zhenjiang, China

Immunochromatographic Rapid Test “BeeTest” for Detection of Viral and Bacterial Diseases of Honey Bees Apis mellifera

Leonid Kelin（“BeeTest” Ltd, Russian Federation, Novosibirsk, Russia）

Russian company "Beetest" presents a product which is unique in the world - immunochromatographic rapid test "Beetest." As our company produces a preparation “Endoglukin” for prevention and treatment of 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 treatment in order to know exactly what kind of disease should be treated. After several years of researches we finally managed to find specific antigens for each disease that ensures the high sensitivity and delicacy of our test.

With the test “Beetest” you can simultaneously check your hives for the presence of the major bacterial and viral diseases of bees. The test is intended for use in the field, does not require highly trained personnel and can be used directly next to the hive.

Principle of the method

Immunochromatographic rapid test “BeeTest” is a sandwich-type lateral flow assay for simultaneous and differential detection of two 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-step detection of the viral and bacterial proteins in honey bee larvae by Lateral-flow immunochromatographic technology. The principal scheme of the test is on the picture.

The nitrocellulose membrane is coated with specific polyclonal antibodies to proteins of Acute bee paralysis virus or Sacbrood virus or American foulbrood (Paenibacillus larvae) or European foulbrood (Melissococcus plutonius) and specific proteins of these infectious agents.

During the first step disrupt the honey bee larvae in the bottle with Elution buffer and glass balls by vigorous shaking. On the second step drip a couple of drops into the sample window. In this moment specific viral or bacterial proteins (if they are present in the sample) forms the complex with the conjugates of colloidal gold with poly- or monoclonal antibodies.

Further these complex bind with the specific antibodies coated on the membrane thus forming the red line (test line). Conjugates which were not involved in forming the complex with bacterial or viral proteins binds with the specific proteins on the membrane (Picture 2) thus forming another red line (control line). The intensity of red color lines should be assessed visually.

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 any preparations.

1) Extract a larva showing suspicious symptoms with the spatula.

2) Unscrew lid from the Bottle with Elution buffer. Use the spatula to place sample in the bottle. Replace the lid and shake vigorously for about 20 seconds.

Test line Control line

3) Unscrew lid of Bottle and pipe the sample by supplied pipette. One honey bee larvae should be used with all types of cassettes.

4) Hold the Test cassette horizontally and gently squeeze two drops onto the sample well of the each cassette.

5) Keep it horizontal until extract is absorbed (c 30 seconds) and a red dye appears in the viewing window.

6) Wait until the control line appears (labeled C) and read the result (10-15 minutes).

Reading the results

3 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 or no one lines indicates that test doesn’t work.

Analytical sensitivity of the test kit

Analytical sensitivity is 10-50 ng/ml with using of recombinants proteins of Acute bee paralysis virus, Sacbrood virus, American foulbrood (Paenibacillus larvae), European foulbrood (Melissococcus plutonius). 

Shelf life, storage and transportation conditions

Shelf life of the “BeeTest” is 12 month from the date of manufacture. Store at 17-27 °C in the dry dark place.

______________________________________________

*Corresponding author, E-mail: beetest.org@gmail.com

“蜜蜂测试”—免疫色谱法快速检测蜜蜂病毒和细菌疾病

Leonid Kelin （俄罗斯）

（“BeeTest” Ltd, Russian Federation, Novosibirsk, Russia）

俄罗斯公司的“蜜蜂测试”是世界上一种先进独特的快速免疫色谱法。本公司用此法控制和治疗蜜蜂 疾病和刺激蜜蜂种群的发展，以达到更好的制备Endoglukin，先前我们决定研发一种诊断测试法，为了在 蜜蜂疾病治疗前准确定位疾病的种类。经过几年的研究，我们最终找到每一种疾病的特异性抗原，这保证 测试的高效灵敏。有了蜜蜂测试，你可以同时检查你蜂群主要疾病细菌和病毒。本测试不需要专业技术人员，可直接在 蜂箱旁边进行检测。方法原理

蜜蜂测试，快速免疫色谱法，是一种夹心型横流化测试法，可同时对两种蜜蜂疾病病毒和细菌进行示 差检测。

1. 蜜蜂急性麻痹肠道病毒

2. 幼蜂皱萎病病毒

3. 美洲蜜蜂幼虫腐臭病

4. 欧洲蜜蜂幼虫腐臭病

通过横流免疫色谱技术，蜜蜂测试可快速，高效，一步检测出蜜蜂幼虫中病毒和细菌蛋白，它主要的 测试路线如图1 所示：硝酸纤维素膜上涂有蜜蜂急性麻痹肠道病毒，幼蜂皱萎病病毒，美洲蜜蜂幼虫腐臭病毒和欧洲蜜蜂幼 虫腐臭病毒的多克隆抗体以及这些传染源的特殊蛋白。第一步将蜜蜂幼虫放入带有洗脱缓冲液和玻璃球的瓶中，剧烈振荡。第二步滴几滴试剂在采样窗口中，此时特殊病毒和细菌蛋白（如果样品中存在）将与单克隆抗体和胶体金的轭合物形成复合物。图二 测试线和控制线中轭合物和抗原的键合（Au –胶体金和抗体的轭合物, Ag – 抗原) 之后这些复合物和薄膜上的特殊抗体紧紧地结合在一起，形成红线（检测线），那些没有没有和薄膜上 的特殊蛋白结合的复合物（如图二）形成另一条红线（控制线），红色线的强度可以用眼观测出来。试剂和材料

1. 4 个塑料储片夹（一个对应一种疾病）

2.带有玻璃球和洗脱缓冲液的塑料瓶

3.一次性试管

4.一次性药刀

实验步骤 所有试剂都要提前准备好。

1. 用药刀取一只有可疑症状的幼虫

2. 旋开带有洗脱缓冲液的塑料瓶，用药刀把幼虫放入塑料瓶中。盖上瓶盖，剧烈振荡20 秒。

3. 旋开瓶盖，用吸量管吸取洗脱缓冲液。每个幼虫的缓冲液液都要滴入4 个塑料储片夹。

4. 水平拿住测试储片夹，缓慢滴两滴缓冲液在样品室。

5. 保持水平，使提取物被吸收（30 秒），观察窗中出现红线。

6. 等到控制线出现（标记为C）然后读出结果（10-15 分钟）结果

1、阳性结果——出现两条红线（检测线和控制线）—表明目标病原体出现在测试样品中。

2、阴性结果——只出现控制线—表明目标病原体不存在于测试样品中。

3、无效结果——有测检测线没有控制线或者两条都没有表明测试无效

试剂盒的分析灵敏度 用10-50ng/mg 蜜蜂急性麻痹肠道病毒，幼蜂皱萎病病毒，美洲蜜蜂幼虫腐臭病毒和欧洲蜜蜂幼虫 腐臭病毒的重组蛋白质来分析灵敏度。保质期，储存和运输条件 保质期12 个月，储存条件为17-27℃，黑暗干燥处。