Ultra highly sensitive method for detecting Edwardsiella ictaluri using high-gradient immunomagnetic separation with polymerase chain reaction

Abstract

Edwardsiella ictaluri causes an economically important bacterial disease in farm-raised catfish in the USA and abroad. To elucidate the route of infection for fish bacterial disease, it is important to monitor both fish health and trace bacteria in the water environment. In this study, we applied an ultra highly sensitive method to detect E. ictaluri using high-gradient immunomagnetic separation (HGIMS) with polymerase chain reaction (PCR). HGIMS is a magnetic separation method in which the magnetic force is strengthened by integrating a magnetic gradient between the magnetic filter and nearby column. Immunomagnetic beads were specifically designed to react with the target bacteria, allowing for more efficient collection. The accumulated beads were released from the filter by releasing the magnetic force. After the process, DNA was extracted from the concentrated cells, and PCR was applied to detect E. ictaluri. The HGIMS system had higher detection sensitivity using than the conventional method, and the total assay time, including sample preparation, was about 3.5h. The optimal reaction time of immunomagnetic beads was 15min and the flow rate of the HGIMS system was 10mlmin−1. PCR products of the expected size were obtained from samples concentrated up to 102cfuml−1. The proposed system appears to be suitable for highly sensitive detection of E. ictaluri. Improvements in the bacteria recovery ability of the immunomagnetic beads will further increase the detection limits. Keywords: Edwardsiella ictaluri, Magnetic separation, Polymerase chain reaction, Fish disease, Bacteria detection, Bioanalytical method