Elandalloussi Laurence M, Leite Ricardo M, Afonso Ricardo, Nunes Patrícia A, Robledo José A F, Vasta Gerardo R, Cancela M Leonor
Molecular Biology and Biotechnology, CCMar, University of Algarve-Campus de Gambelas, 8005-139 Faro, Portugal.
Mol Cell Probes. 2004 Apr;18(2):89-96. doi: 10.1016/j.mcp.2003.09.008.
Perkinsus atlanticus and P. marinus have been associated with mass mortality of bivalve molluscs. Perkinsus infections are routinely diagnosed by histology or the fluid thioglycollate medium (FTM) assay. In this study, we describe the development of a PCR-enzyme-linked immunosorbent assay (ELISA) for amplification and rapid detection of Perkinsus species. The PCR reactions were selected to either amplify an IGS sequence region shared by currently accepted Perkinsus species or to simultaneously amplify IGS regions specific to either P. atlanticus or P. marinus. The specific hybridisation of DIG-labelled amplified products to species-specific capture probes was detected colorimetrically. This assay is able to specifically detect P. atlanticus and P. marinus, and the intensity of the colorimetric signal is dependent upon the amount of amplified product. The PCR-ELISA assay format is 100-fold more sensitive than visualisation of PCR products on ethidium bromide (EtdBr)-stained agarose gels, and as sensitive as Southern hybridisation. The sensitivity limit of PCR-ELISA was 1 pg of DNA from P. atlanticus. No cross-reactivity of the assay was observed against the host DNA. When applied to the detection of P. atlanticus in clams, 39 samples out of 45 yielded concordant results for FTM assay and PCR-ELISA detection.
大西洋派琴虫和海派琴虫与双壳贝类软体动物的大规模死亡有关。派琴虫感染通常通过组织学或巯基乙酸盐流体培养基（FTM）检测来诊断。在本研究中，我们描述了一种用于扩增和快速检测派琴虫属物种的聚合酶链反应-酶联免疫吸附测定（PCR-ELISA）的开发。选择PCR反应来扩增目前公认的派琴虫属物种共有的IGS序列区域，或同时扩增特定于大西洋派琴虫或海派琴虫的IGS区域。通过比色法检测地高辛标记的扩增产物与物种特异性捕获探针的特异性杂交。该检测方法能够特异性地检测大西洋派琴虫和海派琴虫，比色信号的强度取决于扩增产物的量。PCR-ELISA检测形式比在溴化乙锭（EtdBr）染色的琼脂糖凝胶上观察PCR产物灵敏100倍，与Southern杂交一样灵敏。PCR-ELISA的灵敏度极限为来自大西洋派琴虫的1 pg DNA。未观察到该检测方法与宿主DNA有交叉反应。当应用于检测蛤中的大西洋派琴虫时，45个样本中有39个在FTM检测和PCR-ELISA检测中得到了一致的结果。
