Establishment of the real-time fluorescent quantitative PCR method for detecting Aeromonas in sediment environment of aquaculture

Abstract:

Aeromonas is one of the pathogens causing skin ulcer syndrome in cultured Apostichopus japonicas. It is important to monitor the population of these bacteria in the culture environment for disease control. Unfortunately, current real-time quantitative PCR detection method is mostly based on the pure culture and cannot truly reflect the bacteria population in the sediment environment. Therefore, we established a real-time fluorescent quantitative PCR method to improve the pathogen detection. The simulative sediment samples were made by adding known-concentration bacteria into the sterilyzed sediment; the DNA extraction method was selected by comparing three improved DNA extraction methods; genus-specific primers were selected based on the GyrB gene of Aeromonas. Meanwhile, PCR reaction conditions and systems were optimized and a standard curve based-on extracted sediment DNA was utilized to examine the bacteria in the sediment. The results indicated that the modified lysozyme-SDS gentle lyse method and genus-specific primers (IAF and IAR based on GyrB gene of Aeromonas) were efficient and unique to detect Aeromonas. In addition, the standard curve was set up by using the extracted DNA from the simulated sediment samples as template. The new real-time fluorescent quantitative PCR protocol was highly sensitive and specific to detect different species of Aeromonas from the sediment with the quantification limit of 103 CFU/g. The coefficient of variation fell between 0.21% and 0.80% by statistical analysis (less than 5%), which demonstrated the good repeatability of the method. Thus, we have established a highly sensitive, specific, and reproducible method to measure Aeromonas in sediment environment of aquaculture even with low quantification.