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开发并评估环介导等温扩增(LAMP)检测方法,用于检测中国捻转血矛线虫种群同工型 1 β-微管蛋白基因 E198A SNP。

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection of the E198A SNP in the isotype-1 β-tubulin gene of Haemonchus contortus populations in China.

机构信息

State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, PR China.

State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, PR China; School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, 442000, PR China.

出版信息

Vet Parasitol. 2020 Feb;278:109040. doi: 10.1016/j.vetpar.2020.109040. Epub 2020 Jan 23.

Abstract

Haemonchus contortus is one of the most important gastrointestinal nematodes of small ruminants around the world, seriously hampering the healthy development of the sheep industry. The control of this parasite mainly depends on anthelmintics, however, drug resistance of H. contortus has become a serious problems worldwide. Previous studies demonstrated that the E198A (GAA to GCA), a single nucleotide polymorphism (SNP) in the isotype-1 β-tubulin gene is associated with benzimidazole resistance in H. contortus. However, only PCR-RFLP and ARMS-PCR methods have been previously used for the detection of the E198A mutation. In the present study, a loop-mediated isothermal amplification (LAMP) assay was established for rapid detection of the E198A SNP in H. contortus. The results showed that optimization of LAMP reaction reagents and conditions could achieve this. The resulting amplicons were visualized by adding hydroxynaphthol blue dye (HNB) prior to amplification. The color of LAMP products amplified without DNA or from DNA from worms with the E198A homozygous susceptible genotype was still violet, but the products with DNA from worms with the E198A heterozygous genotype or the E198A resistant homozygous genotype changed to sky blue. The specificity of this method was further verified by sequencing, which confirmed the successful LAMP detection of the E198A mutation with high specificity. In conclusion, the developed LAMP method has high specificity and good reproducibility for screening the E198A SNP of isotype-1 β-tubulin gene of H. contortus of field samples without using sophisticated equipment, providing useful technique for the rapid detection and thus prevention and control of benzimidazole resistant H. contortus infections.

摘要

捻转血矛线虫是全世界反刍动物最重要的胃肠道线虫之一,严重阻碍了绵羊养殖业的健康发展。该寄生虫的控制主要依赖于驱虫药,但捻转血矛线虫的耐药性已成为全世界的严重问题。先前的研究表明,β-微管蛋白基因同种型 1 中的 E198A(GAA 到 GCA)单核苷酸多态性(SNP)与捻转血矛线虫对苯并咪唑类药物的耐药性有关。然而,以前仅使用 PCR-RFLP 和 ARMS-PCR 方法来检测 E198A 突变。在本研究中,建立了一种用于快速检测捻转血矛线虫 E198A SNP 的环介导等温扩增(LAMP)检测方法。结果表明,可以通过优化 LAMP 反应试剂和条件来实现。在扩增前添加羟基萘酚蓝染料(HNB)可可视化扩增产物。没有 DNA 的 LAMP 产物或来自 E198A 纯合敏感基因型蠕虫的 DNA 的产物仍为紫色,但来自 E198A 杂合基因型或 E198A 抗性纯合基因型蠕虫的 DNA 的产物变为天蓝色。通过测序进一步验证了该方法的特异性,测序结果证实了该方法能够成功地高特异性检测 LAMP 检测 E198A 突变。总之,该方法具有特异性高、重复性好的特点,无需使用复杂的设备即可筛选现场样本中捻转血矛线虫的β-微管蛋白基因同种型 1 的 E198A SNP,为快速检测和预防控制苯并咪唑耐药捻转血矛线虫感染提供了有用的技术。

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