Quantitative detection of Vibrio alginolyticus strain XSBZ14 by a newly developed RT-PCR method

Abstract

PurposeCoral degradation is a worldwide ecological problem. Bacterial diseases are a great danger to coral health. The pathogenic bacterium Vibrio alginolyticus XSBZ14 isolated from diseased coral had been identified as the pathogenic bacterium of Porites andrewsi White syndrome (PAWS) in Xisha Archipelago on transmission experiment. To date, the molecular mechanism by which this pathogen causes disease is unknown, and molecular diagnostics for diseases caused by this bacterium have not been developed. In an effort to restore damaged coral ecosystems in the South China Sea, efforts are underway to transplant flat-branch shore corals. There is therefore an urgent need to further develop specific and rapid detection methods for V. alginolyticus XSBZ14 in order to prevent this epidemic and ensure the successful implementation of compilation transplants.MethodsAt first, a low sequence identity single-copy sequence S2 was selected from the genome by in-house Perl script. Using the designed specific primers, four different types of standard curves were subsequently plotted for the accurate quantification of the strain XSBZ14 in four different samples (DNA, bacterial suspension, coral tissue, seawater). Then, use the strain to infect the Galaxea fascicularis and test the strain in the coral culture water during the week.ResultsThe rapid detection method of pathogenic bacteria by RT-PCR was established. The limit of detection (LOD) of the RT-PCR was 0.88 pg/reaction (0.44 pg/μL) in DNA, 2 CFU/reaction (1000 CFU/mL) in bacterial suspension, 2 CFU/reaction in coral tissue, and 20 CFU/reaction in seawater for the strain XSBZ14, respectively. In addition, according to the detection results of the RT-PCR, the strain XSBZ14 could survive in Galaxea fascicularis for a week, and the strain could also be detected from its reared seawater.ConclusionThese results indicated that the RT-PCR detection method of a coral pathogenic strain XSBZ14 was established. The method is a robust tool for the rapid detection and quantification of the coral pathogen, XSBZ14, and is very useful for PAWS epidemiological survey and specific pathogen-free coral transplantation in the South China Sea. And other coral species and their habitats might act as an important reservoir for the strain XSBZ14 and mediated its horizontal transmission in coral reefs.