Food Quality and Safety Research Group, International Iberian Nanotechnology Laboratory (INL), Av. Mestre José Veiga s/n, 4715-330, Braga, Portugal.
Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain.
Sci Rep. 2021 May 13;11(1):10175. doi: 10.1038/s41598-021-89574-w.
Zebra mussel (Dreissena polymorpha) is considered as one of the 100 most harmful IAS in the world. Traditional detection methods have limitations, and PCR based environmental DNA detection has provided interesting results for early warning. However, in the last years, the development of isothermal amplification methods has received increasing attention. Among them, loop-mediated isothermal amplification (LAMP) has several advantages, including its higher tolerance to the presence of inhibitors and the possibility of naked-eye detection, which enables and simplifies its potential use in decentralized settings. In the current study, a real-time LAMP (qLAMP) method for the detection of Dreissena polymorpha was developed and tested with samples from the Guadalquivir River basin, together with two real-time PCR (qPCR) methods using different detection chemistries, targeting a specific region of the mitochondrial gene cytochrome C oxidase subunit I. All three developed approaches were evaluated regarding specificity, sensitivity and time required for detection. Regarding sensitivity, both qPCR approaches were more sensitive than qLAMP by one order of magnitude, however the qLAMP method proved to be as specific and much faster being performed in just 9 min versus 23 and 29 min for the qPCR methods based on hydrolysis probe and intercalating dye respectively.
斑马贻贝（Dreissena polymorpha）被认为是世界上 100 种最具危害的入侵物种之一。传统的检测方法存在局限性，而基于 PCR 的环境 DNA 检测为早期预警提供了有趣的结果。然而，近年来，等温扩增方法的发展受到了越来越多的关注。其中，环介导等温扩增（LAMP）具有几个优点，包括对抑制剂存在的更高容忍度和可能进行肉眼检测，这使其能够且简化了其在分散环境中的潜在用途。在当前的研究中，开发了一种用于检测斑马贻贝的实时 LAMP（qLAMP）方法，并使用来自瓜达尔基维尔河流域的样本进行了测试，同时还使用了两种针对线粒体基因细胞色素 C 氧化酶亚基 I 特定区域的实时 PCR（qPCR）方法。对所有三种开发方法的特异性、灵敏度和检测所需时间进行了评估。在灵敏度方面，两种 qPCR 方法的灵敏度都比 qLAMP 高一个数量级，然而 qLAMP 方法被证明具有相同的特异性，且速度更快，仅需 9 分钟，而基于水解探针和嵌入染料的 qPCR 方法则分别需要 23 分钟和 29 分钟。
