A loop-mediated isothermal amplification method for the detection of members of the genus Ranavirus

Abstract

A loop-mediated isothermal amplification (LAMP) method was developed for detection of members of the genus Ranavirus. The optimum reaction mixture contained 2.5 μL of each inner primer, RV-FIP (20pmol/μL) and RV-BIP (20pmol/μL), 0.5 μL of each outer primer, RV-F3 (10pmol/μL) and RV-B3 (10pmol/μL), 1.25 μL of each loop primer, RV-LF (20pmol/μL) and RV-LB (20pmol/μL), 3.5 μL dNTP mix (10mM each), 8 μL MgSO4 (25mM), 1 μL of Bst DNA polymerase (8 U/mL, large fragment; New England Biolabs Inc., Beverly, MA, USA), 2.5 μL 10×supplied buffer, and 1 μL of template DNA in a final volume of 25 μL. The optimum reaction conditions were 63 °C for 60min. This LAMP method could detect Andrias davidianus iridovirus (ADIV), soft-shelled turtle iridovirus (STIV), and epizootic hematopoietic necrosis virus (EHNV), all of which belong to the genus Ranavirus, but it could not detect other viruses such as koi herpes virus (KHV), channel catfish virus (CCV), infectious spleen and kidney necrosis virus (ISKNV) and white spot syndrome virus (WSSV). The detection limit of the LAMP method was 100 copies of STIV DNA segment, and the sensitivity was 10 times higher than that of the polymerase chain reaction (PCR) assay. The results could be estimated visually by eye when calcein was added.