Costa Cátia, Ferreira Guilherme D, Simões Marco, Silva Joana L, Campos Maria J
MARE-Marine and Environmental Sciences Centre, 2520-630 Peniche, Portugal.
MARE-Marine and Environmental Sciences Centre, ESTM, Polytechnic of Leiria, 2520-630 Peniche, Portugal.
Microorganisms. 2022 Oct 18;10(10):2060. doi: 10.3390/microorganisms10102060.
Vibriosis, an often-fatal disease induced by pathogenic members of the Vibrionaceae family, causes severe economic losses in aquacultures. To mitigate/avoid vibriosis outbursts, it is vital to detect and quantify these pathogens as early as possible. However, standard microbiological methods are time-consuming and often underestimate cell counts, which calls for the development of valid alternatives. In this study, real-time polymerase chain reaction (qPCR) was employed to detect the pathogenic species , , and using a new primer pair targeting the gene. In addition, the DNA extraction efficiency of three methods, two commercial kits and the boiling method, was compared. The most efficient method was the DNeasy Blood and Tissue kit, with a detection limit ranging between 154 and 600 CFU mL in the case of and , and 48 CFU mL for . Thus, this study presents the development and evaluation of a method for the early quantification of all three species in saline suspensions. However, the results obtained by spiking a microalgae sample with emphasize the importance of adjusting the DNA control's standard curve to the relevant extraction matrices, as it affects the DNA extraction efficiency and may hamper an accurate quantification with qPCR.
弧菌病是由弧菌科致病成员引起的一种常致命的疾病，在水产养殖中造成严重经济损失。为减轻/避免弧菌病爆发，尽早检测和定量这些病原体至关重要。然而，标准微生物学方法耗时且往往会低估细胞数量，这就需要开发有效的替代方法。在本研究中，采用实时聚合酶链反应（qPCR），使用针对 基因的新引物对来检测致病性物种 、 和 。此外，还比较了三种方法（两种商业试剂盒和煮沸法）的DNA提取效率。最有效的方法是DNeasy Blood and Tissue试剂盒，对于 和 ，检测限在154至600 CFU/mL之间，对于 为48 CFU/mL。因此，本研究展示了一种用于早期定量盐悬液中所有三种物种的方法的开发和评估。然而，用 对微藻样品进行加标所获得的结果强调了根据相关提取基质调整DNA对照标准曲线的重要性，因为这会影响DNA提取效率，并可能妨碍用qPCR进行准确的定量。
