SYBR GreenⅠReal-Time Fluorescence Quantitative PCR for Parasitic Boveria labialis Identification from the Sea Cucumber Apostichopus japonicus

Abstract

Boveria disease affects the aquaculture efficiency of sea cucumbers. Boveria labialis causes Boveria disease in Apostichopus japonicus. Owing to the lack of rapid and accurate detection methods, analyzing the transmission route of the pathogen remains complicated. Based on the partial mitochondrial genome sequence of B. labialis obtained using sequencing, primers were designed to amplify nad10 and a SYBR GreenⅠreal-time fluorescent quantitative PCR detection method was established. The concentration of B. labialis in different culture areas and different culture modes of A. japonicus was detected and analyzed. The standard curve established by the designed primers had a good linear relationship in the range of the plasmid standard of 4.05×101–4.05×109 copies/µL, with a reliability (R2) of 0.997. The melting curve showed a single peak, without primer dimers or nonspecific amplification. The minimum detection limit of sensitivity test was 40.5 copies/µL. The designed primers showed specific amplification for B. labialis only and exhibited no cross reaction to Uronema sp., chaenea sp., Colpoda sp. and Paramecium sp. In the repeatability test, the homogeneity of Ct values in the intra-assay and inter-assay of each concentration was high, the CV values of intra- and inter-assay tests were 0.32%–0.82% and 0.40%–0.88%, respectively, indicating good stability. This method was used to detect environmental samples and feeds in different culture areas of four kinds of A. japonicus culture modes. Combined with the comparative analysis of microscopic examination results, there was a moderate positive correlation (R=0.563) between the DNA load of B. labialis in sea water and the infection degree of B. labialis in A. japonicus. A high positive correlation (R=0.931) was found between the DNA load of B. labialis in sediment and attachment samples and the infection degree of B. labialis in A. japonicus. Fresh sea mud was an important carrier for B. labialis transmission. The relevant research results provide reference for the rapid detection, transmission route analysis, and B. labialis prevention and control.

Keywords

• apostichopus japonicus
• parasitic disease
• boveria labialis
• real-time fluorescent quantitative pcr detection
• transmission route