An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goats.

IF 0.5 4区农林科学 Q4 VETERINARY SCIENCES Veterinaria italiana Pub Date : 2022-12-30 DOI:10.12834/VetIt.2426.15340.2

Gnanavel Venkatesan, Anand Kushwaha, Amit Kumar, D P Bora, P Sasikumar

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Abstract

The orf virus (ORFV) is an epitheliotropic virus causing a highly contagious skin disease mainly in sheep and goats. Several diagnostics including molecular tools like Loop mediated isothermal amplification (LAMP) assay are available to detect ORFV in affected species. However, the carry-over contamination associated with LAMP as open tube format prevents the assay applicability as point of care test in field diagnostic settings. In this study, the B2L gene based LAMP assay was optimized in a closed tube format using hydroxynaphthol blue (HNB) and calcein as pre-addition dyes and it has shown a clear positive and negative signal at 60 °C using 4 and 5 mM concentrations of MgSO4 respectively for these dyes. Optimitimzed assay that could reveal the result within one hour is highly specific and senstive with a limit of detection at 12.5 femtogram of viral genomic DNA or ~85 virus genome equivalent. This improved method prevented the cross-contamination of future LAMP reactions in the laboratory without compromising diagnostic sensitivity (100%) and specificity (100%) when compared to open tube system. This closed tube LAMP method has potential to act as a simple visual detection assay for the rapid and specific diagnosis of ORFV in sheep and goats.

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改进的肉眼闭管环介导等温扩增(LAMP)法快速鉴定绵羊和山羊口蹄疫病毒。

口蹄疫病毒(ORFV)是一种致上皮性病毒，主要在绵羊和山羊中引起高度传染性皮肤病。包括环介导等温扩增(LAMP)测定等分子工具在内的几种诊断方法可用于检测受感染物种中的ORFV。然而，与LAMP作为开管格式相关的携带污染阻碍了该分析在现场诊断设置中作为护理点测试的适用性。在本研究中，以羟基酚蓝(HNB)和钙黄蛋白为预添加染料，在封闭试管中优化了基于B2L基因的LAMP检测，在60°C下，分别使用4和5 mM浓度的MgSO4对这两种染料显示出明显的阳性和阴性信号。优化后的检测方法可在1小时内显示结果，具有高度的特异性和敏感性，检测限为病毒基因组DNA的12.5飞图或约85个病毒基因组当量。与开管系统相比，这种改进的方法在不影响诊断灵敏度(100%)和特异性(100%)的情况下，防止了未来实验室LAMP反应的交叉污染。这种封闭管LAMP方法有潜力作为一种简单的视觉检测方法，用于快速和特异性诊断绵羊和山羊的ORFV。

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来源期刊

Veterinaria italiana VETERINARY SCIENCES-

CiteScore

1.50

自引率

0.00%

发文量

审稿时长

>12 weeks

期刊介绍： The journal was created as the Croce Azzurra in 1950. A quarterly peer-reviewed journal devoted to veterinary public health and other aspects of veterinary science and medicine, Veterinaria Italiana is published by the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ (Istituto Zooprofilattico Sperimentale dell''Abruzzo e del Molise) in Teramo, Italy. The goal of the journal is to provide an international platform for veterinary public health information from Italy and other countries, particularly those in Eastern Europe and Africa, Asia and South America. Veterinarians and veterinary public health specialists are encouraged to share their knowledge and experience on this platform.