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利用环引物内切酶切割环介导的等温扩增实时检测与左旋咪唑耐药相关的捻转血矛线虫 S168T 变异体。

Real-time single-base specific detection of the Haemonchus contortus S168T variant associated with levamisole resistance using loop-primer endonuclease cleavage loop-mediated isothermal amplification.

机构信息

School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow, Glasgow, Scotland, United Kingdom; Kreavet, Kruibeke, Belgium.

Molecular Diagnostics Research Group, School of Biological and Chemical Sciences, University of Galway, Galway, Ireland.

出版信息

Mol Cell Probes. 2024 Feb;73:101946. doi: 10.1016/j.mcp.2023.101946. Epub 2023 Dec 19.

Abstract

Haemonchus contortus is a parasitic haematophagous nematode that primarily affects small ruminants and causes significant economic loss to the global livestock industry. Treatment of haemonchosis typically relies on broad-spectrum anthelmintics, resistance to which is an important cause of treatment failure. Resistance to levamisole remains less widespread than to other major anthelmintic classes, prompting the need for more effective and accurate surveillance to maintain its efficacy. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) is a recently developed diagnostic method that facilitates multiplex target detection with single nucleotide polymorphism (SNP) specificity and portable onsite testing. In this study, we designed a new LEC-LAMP assay and applied it to detect the levamisole resistance marker S168T in H. contortus. We explored multiplexing probes for both the resistant S168T and the susceptible S168 alleles in a single-tube assay. We then included a generic probe to detect the acr-8 gene in the multiplex assay, which could facilitate the quantification of both resistance markers and overall genetic material from H. contortus in a single step. Our results showed promising application of these technologies, demonstrating a proof-of-concept assay which is amenable to detection of resistance alleles within the parasite population, with the potential for multiplex detection, and point-of-care application enabled by lateral flow end-point detection. However, further optimisation and validation is necessary.

摘要

捻转血矛线虫是一种寄生性血食线虫,主要影响小反刍动物,并对全球畜牧业造成重大经济损失。旋毛虫病的治疗通常依赖于广谱驱虫药,但对这些药物的耐药性是治疗失败的一个重要原因。与其他主要驱虫药相比,左旋咪唑的耐药性不太普遍,因此需要更有效和准确的监测来维持其疗效。环引物内切酶切割环介导等温扩增(LEC-LAMP)是一种新开发的诊断方法,可实现具有单核苷酸多态性(SNP)特异性的多重靶标检测和便携式现场测试。在这项研究中,我们设计了一种新的 LEC-LAMP 检测方法,并应用它来检测捻转血矛线虫中的左旋咪唑耐药标记 S168T。我们探索了在单个管中同时检测耐药 S168T 和敏感 S168 等位基因的多重探针。然后,我们在多重检测中包含了一个通用探针来检测 acr-8 基因,这可以在单个步骤中方便地定量检测耐药标记和捻转血矛线虫的总遗传物质。我们的结果表明这些技术具有有前景的应用,证明了一种概念验证检测方法,该方法适用于检测寄生虫群体中的耐药等位基因,具有多重检测的潜力,并通过侧流终点检测实现了即时护理应用。然而,还需要进一步优化和验证。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ee7e/10884526/19b4a0d021b8/gr1.jpg

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