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首页> 外文期刊>Asia-Pacific Journal of Science and Technology >The efficiency of ELISA technique in Nosema bombycis N. of mulberry silkworm (Bombyx mori L.) detection (Thai)
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The efficiency of ELISA technique in Nosema bombycis N. of mulberry silkworm (Bombyx mori L.) detection (Thai)

机译：ELISA技术在桑蚕Nosema bombycis N.（Bombyx mori L.）检测中的效率（泰国）

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摘要

Pebrine disease caused by Nosema bombycis N. is the most devastating disease of mulberry silkworm (Bombyx mori L.), which transmits continuously through eggs by generation to generation. The infected silkworm “Mae Ling” variety was randomly selected and studied on pebrine symptom development for one generation in laboratory at 25oC (21.5 – 26oC, R.H. 38 – 91%). Numbers of pebrine symptoms containing spores were 15 from total 20 symptoms. The infected samples were observed using phase contrast microscope and found the average spores of 1.77 x 106 – > 2.42 x 108 spores/larva with the average spore size of 2.10 – 2.50 × 4.13 – 4.57 μm. The most appearance infected symptom was the resembled healthy larva; which had 10 percent of infection. The spore suspension of N. bombycis was purified using sucrose density gradient centrifugation (SDGC). Most spores located at sucrose level concentration of 40 – 50 % (w/w). Suitable concentration of antigen for polyclonal antibody production was evaluated and found that the spore concentration of 1 × 109 spores/ml was the most properly. The titer value of this antiserum at 101 – 106 spores/ml of antigen was 1:50 and at 106 spores/ml of antigen the titer value was 1:3,500. In addition, the produced antiserum in this present study was highly specific to protozoa N. bombycis (Mae Ling isolate) but not cross reaction to other silkworm pathogens, fungi: Aspergillus flavus, A. tamarii, Metarhizium anisopliae, bacteria: Bacillus thuringiensis, Serratia marcescens, Staphylococcus aureus, S. sciuri and virus: Nucleopolyhedrovirus (NPV). T his antiserum was tested and developed for N. bombycis detection using indirect ELISA to compare with the previous method (IP 1). The developed detection technique was considered consequently based on the important steps in series. The best result obtained from the beginning steps was used to develop continuously in next steps in series, in order to derive the best final procedure. The result of type and concentration including temperature and time interval for incubation of blocking solution exhibited that the 1% BSA (bovine serum albumin) and 2 hr of blocking time at 37°C were the most effectiveness.
机译：由Nosema bombycis N.引起的白粉病是桑蚕（Bombyx mori L.）中最具破坏性的疾病，它通过卵代代相传。在25oC（21.5 – 26oC，R.H. 38 – 91％）的实验室中，随机选择了感染的蚕“ Mae Ling”品种，并研究了一代人的白蛋白症状。含有孢子的小儿麻痹症症状总数为20，其中有15。使用相差显微镜观察感染的样品，发现平均孢子为1.77 x 106 –> 2.42 x 108孢子/幼虫，平均孢子大小为2.10 – 2.50×4.13 – 4.57μm。外观感染最严重的症状是类似健康的幼虫。其中有10％的感染。使用蔗糖密度梯度离心（SDGC）纯化弹状猪笼草的孢子悬浮液。多数孢子的蔗糖浓度为40 – 50％（w / w）。评估适合多克隆抗体生产的抗原浓度，发现1×109孢子/ ml的孢子浓度最合适。该抗血清在101 – 106孢子/ ml抗原的效价为1:50，在106孢子/ ml抗原的效价为1：3,500。另外，在本研究中产生的抗血清对原生动物N. bombycis（Mae Ling分离物）具有高度特异性，但与其他蚕病原体，真菌：黄曲霉，tamarii，Metarhizium anisopliae，细菌：苏云金芽孢杆菌，沙雷氏菌没有交叉反应。 marcescens，金黄色葡萄球菌，S。sciuri和病毒：核多角体病毒（NPV）。测试了他的抗血清，并开发了用于使用间接ELISA与传统方法（IP 1）进行比对的猪新孢子虫检测。因此，基于一系列重要步骤考虑了开发的检测技术。从开始步骤获得的最佳结果将被用于后续的后续步骤中的连续开发，以得出最佳的最终过程。封闭溶液孵育的温度和时间间隔的类型和浓度结果表明，在37°C下1％BSA（牛血清白蛋白）和2小时封闭时间最为有效。

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