Calcein

M2-induced leakage of calcein entrapped in DPPG liposomes was investigated at 25 °C, as described in a previous study [10 (link)]. The fluorescence intensity of calcein was monitored at 515 nm using a fluorescence spectrometer (FP-8300, Jasco, Tokyo, Japan) with an excitation wavelength of 490 nm. The extent of calcein release was calculated according to F = (I_f − I₀)/(I_max − I₀), where I₀ and I_f are the initial and final (approximately 40 min after the initial manipulation) intensities of fluorescence, respectively, and I_max is the maximal fluorescence intensity obtained by adding Triton X-100 (all the entrapped calcein was released). calcein-loaded DPPG liposomes were prepared by the extrusion method using a solution containing 60 mM calcein and 10 mM HEPES (pH 7.0). Untrapped calcein was separated from the liposomes by gel filtration using a bio-spin chromatography column (BIO-RAD, Hercules, CA, USA) with Sephadex G-75. This step was performed above the lipid phase-transition temperature. calcein was purchased from Tokyo Chemical Industry (Tokyo, Japan). The lipid concentration (400 μM) was determined using a phosphorus assay [37 (link)].

A set of specific primers, composed of FIP, BIP, LF, LB, F3, and B3, for LAMP detection of DIV1 was designed to target the gene of the second largest subunit of DNA-directed RNA polymerase II in the DIV1 genomic sequence (GenBank accession No. MF599468), using PrimerExplorerV4 (http://primerexplorer.jp/elamp4.0.0/index.html). These primers compared with the database in NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to analyze sequence similarities. The primers were synthesized by Sangon Biotech (Shanghai, China). Each LAMP mixture contained 1.6 µM each of inner primers FIP and BIP, 0.8 µM each of loop primers LF and LB, 0.2 µM each of outer primers F3 and B3, 1.4 mM of dNTP mix (TaKaRa, Dalian, China), 1.2 M betaine (Solarbio, Beijing, China), 25 µM calcein (Sigma, St. Louis, MO, USA), 500 µM MnCl₂, 6 mM MgCl₂, 8 U Bst 2.0 DNA polymerase (New England Biolabs Inc., Beverly, USA), 1× supplied buffer and the specified amount of template DNA in a final volume of 25 µL. The procedure was 60 cycles for 60 °C, following 5 min at 85 °C on the CFX-96 Quantitative Fluorescence Instrument (BioRad, Hercules, CA, USA) using calcein fluorescent channel. Detection specificity of the LAMP primers were examined using 100 ng of the total DNA extracted from uninfected prawns and shrimp infected with other pathogens, including WSSV, Vp_AHPND, IHHNV, and EHP.