Blair D, Waycott M, Byrne L, Dunshea G, Smith-Keune C, Neil K M
School of Tropical Biology, James Cook University, Townsville, Queensland, 4811, Australia.
Mar Biotechnol (NY). 2006 Jul-Aug;8(4):380-5. doi: 10.1007/s10126-005-6121-y. Epub 2006 Apr 25.
This work was prompted by the need to be able to identify the invasive mussel species, Perna viridis, in tropical Australian seas using techniques that do not rely solely on morphology. DNA-based molecular methods utilizing a polymerase chain reaction (PCR) approach were developed to distinguish unambiguously between the three species in the genus Perna. Target regions were portions of two mitochondrial genes, cox1 and nad4, and the intergenic spacer between these that occurs in at least two Perna species. Based on interspecific sequence comparisons of the nad4 gene, a conserved primer has been designed that can act as a forward primer in PCRs for any Perna species. Four reverse primers have also been designed, based on nad4 and intergenic spacer sequences, which yield species-specific products of different lengths when paired with the conserved forward primer. A further pair of primers has been designed that will amplify part of the cox1 gene of any Perna species, and possibly other molluscs, as a positive control to demonstrate that the PCR is working.
这项工作是出于能够使用不单纯依赖形态学的技术在澳大利亚热带海域识别入侵贻贝物种翡翠贻贝（Perna viridis）的需求而开展的。利用聚合酶链反应（PCR）方法的基于DNA的分子方法得以开发，以明确区分贻贝属中的三个物种。目标区域是两个线粒体基因cox1和nad4的部分，以及这些基因之间至少在两个贻贝物种中出现的基因间隔区。基于nad4基因的种间序列比较，设计了一种保守引物，该引物可在针对任何贻贝物种的PCR中用作正向引物。还基于nad4和基因间隔区序列设计了四条反向引物，当与保守正向引物配对时，它们会产生不同长度的物种特异性产物。另外设计了一对引物，可扩增任何贻贝物种以及可能的其他软体动物的cox1基因的一部分，作为阳性对照以证明PCR正在起作用。
