Preparation of bacteria and pearl oysters
The strain Pbs-1 was isolated from diseased Akoya pearl oysters as reported by Sakatoku et al. (2018). Five type strains of the genus Tenacibaculum (T. mesophilum, T. amylolyticum, T. sediminilitoris, T. maritimum, T. ascidiaceicola) were purchased from the National Institute of Technology and Evaluation. Diseased and healthy Akoya pearl oysters, Pinctada fucata, were collected from a pearl oyster farm in Ago Bay, Shima City, Mie Prefecture, Japan. After excision from the shell, the shell pieces from Akoya pearl oysters were transferred into 1.5-ml sterile centrifuge tubes and stored at − 30°C prior to DNA extraction.
Bacterial strains were cultured in marine broth medium (Difco marine broth 2216; Becton, Dickinson and Company, MD, USA) at 25°C for 24 h. After collecting bacteria by centrifugation, bacterial DNA extraction was performed using a Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA). Total DNA was extracted from the shell pieces using a vigorous bead beating DNA extraction kit (FastDNA SPIN Kit for Soil; MP Biomedicals, CA, USA), following the manufacturer’s instructions. Genomic DNA was then quantified by spectrophotometer (Nanodrop 1000 Spectrophotometer; Thermo Fisher Scientific K.K., Kanagawa, Japan) and stored at − 30°C for later use.
LAMP assay primer design
The 16S–23S ISR of all bacteria (strains Pbs-1 and 5 type strains of Tenacibaculum) was sequenced. The ISR was amplified by PCR using primers 5′-ATACGGAGGGTGCAAGCGTT-3′ (forward) and 5′-ACCAGCGGATTTGCCTACCA-3′ (reverse), designed based on the 16S rDNA and 23S rDNA sequences of strain Pbs-1, respectively. PCR amplification of the ISR gene of bacteria was performed in a 20-µL volume containing approximately 5 ng of template DNA, 1 × Ex Taq buffer, 200 µM dNTP mix, 0.25 µM of each primer, and 0.5 U Ex Taq HS (TaqDNA polymerase; Takara Bio Inc., Shiga, Japan). PCR reactions involved an initial denaturation step at 94°C for 3 min, followed by 35 amplification cycles consisting of denaturation at 94°C for 60 s, annealing at 50°C for 60 s, and elongation at 72°C for 60 s. The reaction was terminated with a terminal elongation step at 72°C for 3 min, followed by cooling at 4°C. Amplified products were purified using a QIAquick PCR purification kit (Qiagen K. K., Tokyo, Japan). Purified PCR products were independently ligated into the pGEM-T easy vector system (Promega), following the manufacturer’s protocols. Recombinant plasmids were transformed into Escherichia coli DH5α competent cells using 10 µL of the ligation mixture, and spread on LB plates containing ampicillin. Insert-containing plasmids were purified using the PureYield Plasmid Miniprep System (Promega), amplified using M13 f and M13 r universal primers, then sequenced using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and an ABI Prism 3130xl genetic analyzer (Applied Biosystems). Multiple sequence alignment was performed using Genetyx v14 software (Genetyx, Tokyo, Japan). A set of five primers for LAMP was designed using Primer Explorer v5 (https://primerexplorer.jp/lampv5/index.html) to target the ISR of strain Pbs-1. Primer sets comprised two outer primers (F3 and B3), two inner primers (the FIP primer consisted of the complementary sequences of F1c and F2, and the BIP primer consisted of B1c and B2), and a loop primer (LF) (Table 1).
LAMP reaction condition optimization
The optimal temperature for the LAMP assay was determined at 60°C, 63°C, and 65°C for 35 min. The LAMP was performed in a 25-µL reaction volume containing 1.4 µM each of FIP and BIP, 0.2 µM each of F3 and B3, 1×Reaction mixture (Loopamp DNA amplification reagent kit; Eiken Chemical Co., Ltd. Tokyo, Japan), 8 U Bst DNA polymerase (Eiken Chemical Co., Ltd.), and 50 ng of DNA template. To evaluate the optimal concentration of the two inner primers (FIP and BIP), their concentrations were reacted from 1.0–1.6 µM. The composition of the reaction mixture other than FIP and BIP is the same as above. The LAMP reaction was performed at 63°C for 35 min. To evaluate the optimal concentration of the loop primer (LF), its concentrations were reacted from 0.6–1.2 µM. The composition of the above reaction mixture was used, except that 50 pg of DNA template and LF were used. The LAMP reaction was performed at 63°C for 35 min. The LAMP products were separated by 2% (w/v) agarose gel electrophoresis, stained with ethidium bromide, then photographed using a FAS-Digi PRO (NIPPON Genetics Co., Ltd. Tokyo, Japan).
LAMP assay sensitivity
Ten-fold serial dilutions (50 ng to 50 ag) of DNA extracted from Tenacibaculum sp. strain Pbs-1 were used as templates for LAMP. The LAMP was performed in a 25-µL reaction volume containing 1.4 µM each of FIP and BIP, 0.2 µM each of F3 and B3, 1.0 µM LF, 1×Reaction mixture, 8 U Bst DNA polymerase, and 2 µL of DNA template. The LAMP reaction was performed at 63°C for 35 min or 60 min. LAMP products were detected by 2% (w/v) agarose gel electrophoresis as described above, and visually following addition of 0.008% (w/v) malachite green (Wako Pure Chemical Industries, Ltd., Japan) to the reaction mixture to observe a color change.
Evaluation of the LAMP assay using clinical samples
Three diseased and three apparently healthy pearl oysters were collected from Ago Bay and tested by LAMP assay in a 25-µL reaction volume containing 1.4 µM each of FIP and BIP, 0.2 µM each of F3 and B3, 1.0 µM LF, 1×Reaction mixture, 8 U Bst DNA polymerase, and 2 µL of DNA template. The reaction was performed at 63°C for 60 min. LAMP products were detected by 2% (w/v) agarose gel electrophoresis. The rate for positive detection of strain Pbs-1 in the samples, and assay sensitivity were calculated.