REAL-TIME FLUORESCENT LOOP MEDIATED ISOTHERMAL AMPLIFICATION FOR DETECTION OF CHANNEL CATFISH VIRUS

Channel catfish virus (CCV) is a highly lethal viral pathogen of channel catfish (Letalurus punetaus), and it is a large DNA virus that can cause lethal hemorrhagic infection in channel catfish. The establishment of a simple, rapid and accurate CCV detection technique is essential for early diagnosis and timely disease control on site. In this study, a real-time fluorescent loop mediated isothermal amplification (RealAmp) was developed for CCV diagnosis and a set of six primers was designed specifically to recognize the target gene ORF77 coding phosphokinase protein of CCV. The results showed that the RealAmp methods can quickly detect the template of pMD18-T-ORF77 within 12min under isothermal condition of 63℃ and the minimum detection limit was 100 copied within 35min. Furthermore, it was found that the method is highly specific without any cross-reactions to other DNA pathogens from aquatic animals including White spot syndrome virus (WSSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Kio herpesvirus (KHV), Red sea bream iridovirus (RSIV), Infectious spleen and kidney necrosis virus (ISKNV) and Singapore grouper iridovirus (SGIV). The intra-assay and inter-assay tests showed that the amplification curves of the parallel samples were substantially coincident, indicating a good reliability. The actual sample results showed that RealAmp method can specifically detect CCV. Overall, these data suggest that the RealAmp method for CCV detection is an effective and low-cost procedure with high specificity and sensitivity, and is expected to become a valuable molecular tool for rapid detection and identification of CCV.