Study on the development of carbon nanotube enhanced biosensor for gender determination of fish

To efficiently produce seedlings in aquaculture farms, it is essential to control the ratio of males and females by sex distinguishing. On the other hand, the concentration of 11-ketotestosterone (11-KT), which is contained in the plasma, would be an excellent indicator that is significantly different between males and female fish.However, the concentration of 11-KT is challenging to measure due to its low concentration. In this study, we attempted to develop a high-sense biosensor system enhanced by single-walled carbon nanotubes (SWCNTs) with high conductivity and improved the efficiency and accuracy of fish gender determination. First, a self-assembled monolayer (SAM)was formed on the surface of a disk-type gold electrode, and an anti-rabbit IgG was immobilized on the SAM end.Next, an anti-11-KT antibody was immobilized on this IgG antibody, and then SWCNTs were immobilized on the electrode surface to fabricate the biosensor.Then, we attempted to quantify 11-KT by analyzing the minor electrochemical changes on the electrode surface before and after the reaction of an anti-11-KT antibody with 11-KT as an antigen by cyclic voltammetry.Next, after examining the influence of various conditions (temperature, pH, reaction time) on the response value of this sensor, the specificity of the sensor using other hormones was confirmed.Furthermore, as an application of this sensor to the actual sample, the 11-KT concentration in a Nile tilapia (Oreochromis niloticus) plasma was measured and correlated with the measurement value obtained by the conventional method (ELISA) was compared

Molecular cloning of cDNA encoding a 20S proteasome 2 subunit from goldfish (Carassius auratus) and its expression analysis

publishe

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

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 metho

Viral nervous necrosis (VNN) as a critical infectious disease of orange-spotted grouper, Epinephelus coioides, in the Philippines

Orange-spotted grouper, Epinephelus coioides, is a valuable commodity in the Philippines. In 2001, mass mortality occurred in the grouper larvae at Aquaculture Department, Southeast Asian Fisheries Development Center (SEAFDEC/AQD) and the disease was identified as viral nervous necrosis (VNN). Since then, the disease has been observed every year and the grouper hatcheries have been devastated. In this paper, recent studies of VNN which were conducted at the SEAFDEC/AQD from 2001 to 2006 are reviewed. 1) Susceptibility to the VNN virus was tested among fish species that were cultured in mangrove brackish are. Five representative cultured fish species including orange-spotted grouper, Asian sea bass (Lates calcarifer), mangrove red snapper (Lutjanus argentimaculatus), milkfish (Chanos chanos) and rabbitfish (Siganus guttatus) were used in the test where the virus was intraperitoneally injected into the juveniles. Although low or no mortality occurred in the challenge test, histopathological changes were observed in the brain and retina where the virus was re-isolated. The results were the same among the species except for rabbitfish which had no evidence for the infection. It was verified that the virus has a wide host range. 2) To estimate the possible risk of viral spread by vertical transmission, virus distribution was determined in asymptomatic groupers including 7 broodstock and 17 juveniles with body weights ranging from 4 to 12 kg and 2 to 9 respectively. The virus was detected by PCR method. The highest detection rate was in the brain, and the virus was also detectable in other organs such as the gills, heart, spleen, kidney, blood, esophagus, stomach, intestine, liver, gonad, swim bladder and/or skin. 3) As a possible VNN vaccine, a DNA p;asmid encoding the capsid protein of the virus was evaluated. After the challenge, the mortalities between the native and DNA-injected fish appeared significantly different (P<0.05).This study was supported by funds from Japan International Research Center for Agricultural Sciences (JIRCAS) Research Project "Studies on Sustainable Production Systems of Aquatic Animals in Mangrove Brackish Areas"