A Field-Friendly Loop-Mediated Isothermal Amplification (FF-LAMP) method for rapid detection of Nosema bombycis in silkworm, Bombyx mori

IF 1.2 4区农林科学 Q4 IMMUNOLOGY ISJ-Invertebrate Survival Journal Pub Date : 2021-06-21 DOI:10.25431/1824-307X/ISJ.V18I1.66-74

V. Sivaprasad, L. Satish, G. Mallikarjuna, N. Chandrakanth, A. Josepha, S. M. Moorthy

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引用次数: 1

Abstract

Pebrine is a destructive disease that exhibits horizontal and vertical transmission and therefore it is the only mandatory quarantine item in sericulture. Here, a field-friendly loop-mediated isothermal amplification (FF-LAMP) method has been developed and validated for the rapid detection of Nosema bombycis, a causative agent of pebrine disease in silkworm, Bombyx mori. FF-LAMP primers were selected and designed for small ribosomal subunit gene and the assay was performed to detect the N. bombycis infection in silkworm. The FF-LAMP reaction was effective at 6 mM MgSO4, 1.4 mM dNTPs at 63 °C. The detection range of LAMP assay was found to be 101 dilutions of N. bombycis spores. Specificity of the primers was tested using DNA isolated from pebrine infected silkworm, pebrine free silkworm and pure N. bombycis by conventional PCR and FF-LAMP assay. Results revealed that the primers were specific to N. bombycis DNA. The FF-LAMP assay was validated in different basic silkworm seed farms with simultaneous microscopic examination of N. bombycis infection. This newly developed method is highly effective, specific, sensitive and rapid in detecting N. bombycis infection, eliminating the DNA purification steps and usage of sophisticated equipment. This method can be used in testing large number of samples making it field friendly method in sericulture industry.

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现场友好环介导等温扩增(FF-LAMP)方法快速检测家蚕微孢子虫

佩布林是一种具有水平和垂直传播的破坏性疾病，因此是蚕桑养殖中唯一的强制检疫项目。本研究开发并验证了一种现场友好型环介导等温扩增(FF-LAMP)方法，用于快速检测家蚕微囊病病原体家蚕微囊病。选择并设计了小核糖体亚基基因的FF-LAMP引物，对家蚕蚕蛹感染进行了检测。FF-LAMP反应在6mm MgSO4, 1.4 mM dNTPs, 63℃条件下有效。LAMP法的检测范围为家蚕孢子的101倍稀释度。采用常规PCR和FF-LAMP法分别对微卤感染家蚕、无微卤家蚕和纯家蚕DNA进行特异性检测。结果表明，引物对家蚕DNA具有特异性。FF-LAMP试验在不同的基础蚕种场进行了验证，同时显微镜检查了家蚕感染情况。该方法有效、特异、灵敏、快速地检测了家蝇感染，省去了DNA纯化步骤和复杂设备的使用。该方法可用于大量样品的检测，是一种适合蚕桑工业现场使用的方法。

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来源期刊

ISJ-Invertebrate Survival Journal IMMUNOLOGY-ZOOLOGY

CiteScore

2.10

自引率

0.00%

发文量

审稿时长

>12 weeks

期刊介绍： Invertebrate Survival Journal (ISJ) is an international and open access journal devoted to prompt and innovative studies on the basic defense mechanisms in invertebrates, in particular with a view to identifying biotechnologies able to act against derived diseases and related economic damage. Contributions will be mainly in the form of Letters to the Editor, Visions and Perspectives, Short Communications, Technical Reports, Research Reports, Review, Minireview and Reports of Meetings. Letters to the Editor can be commentaries or perspectives on invertebrate defence mechanisms or replies to the data published in ISJ.