Development of Real-time PCR Method for Detection and Quantification of the Coral Pathogenic Strain XSBZ14

Abstract

Abstract Purpose Coral degradation is a worldwide ecological problem. Bacterial diseases are a great danger to coral health. However the bacterial communities in coral reef ecosystems are complex and variable. Therefore, real-time and accurate detection of coral pathogenic bacteria is necessary. This study aims to develop a detection method for pathogenic bacteria Vibrio alginolytcus XSBZ14, which could cause Porites andrewsi white syndrome (PAWS), is an enormous threat to coral reef ecosystem in the South China Sea. That could provide help for the protection of coral reefs in the South China Sea. Methods At 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, using the strain to infect the Galaxea fascicularis and test the strain in the coral culture water during the week. Results The 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, 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. Conclusion These results indicated that other coral species and their habitats might act as an important reservoir for the strain XSBZ14 and mediated its horizontal transmission in coral reefs. In a word, the RT-PCR detection method of a coral pathogenic strain XSBZ14 was firstly 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.