Development and evaluation of a rapid, specific, and sensitive loop-mediated isothermal amplification assay to detect Tenacibaculum sp. strain pbs-1 associated with black-spot shell disease in Akoya pearl oysters

Black-spot shell disease decreases pearl quality and threatens pearl oyster survival. Establishment of a rapid, specific, and sensitive assay to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease is of commercial importance. We developed a rapid, specific, and highly sensitive loop-mediated isothermal amplification (LAMP) assay to detect Tenacibaculum sp. Pbs-1 in Akoya pearl oysters Pinctada fucata. A set of five specific primers (two inner, two outer, and a loop) were designed based on the 16S–23S internal spacer region of strain Pbs-1. The optimum reaction temperature was 63 °C, and concentrations of the inner and loop primers were 1.4 and 1.0 µM, respectively. The LAMP product can be detected using agarose gel electrophoresis, and the color change in the reaction tube can be detected visually (by the naked eye) following the addition of malachite green. Our assay proved to be specific for strain Pbs-1, with no cross-reactivity with five other species of Tenacibaculum. The detection limit of the LAMP assay at 35 min is 50 pg, and at 60 min it is 5 fg. We evaluated the LAMP assay using diseased and healthy pearl oysters. The results demonstrate the suitability and simplicity of this test for rapid field diagnosis of strain Pbs-1.


Introduction
Aquaculture production in Japan during the period 1993-2017 has been relatively stable at 1 × 10 6 t per year (Matsuura et al. 2019). However, according to the Ministry of Agriculture, Forestry and Fisheries statistics in Japan, the domestic production of pearl oysters has decreased year after year, peaking at approximately 125 × 10 3 t in 1967 and decreasing to approximately 16 × 10 3 t in 2020. This decrease is mainly attributed to mass mortality events of pearl oysters caused by viral or bacterial infection, such as Akoya oyster disease caused by Candidatus Maribrachyspira akoyae (Matsuyama et al. 2017), an unidentified virus in 2019 and 2020 (Matsuyama et al. 2021), and black-spot shell disease caused by Tenacibaculum sp. strain Pbs-1 (Sakatoku et al. 2018). Of these, black-spot shell disease-characterized by localized blackish shell discoloration-is a serious problem in cultured pearl production (Sakatoku et al. 2018), as it diminishes pearl quality and threatens pearl oyster survival. Although this disease was first reported in the 1950s, no treatment and prevention strategies are yet available. Tenacibaculum sp. strain Pbs-1, the causative agent of black-spot shell disease in Pinctada fucata, is recognized as a major problem in the cultured pearl industry. To prevent or monitor disease outbreaks, a rapid, specific, convenient and sensitive method is needed to detect strain Pbs-1. Onsite detection would allow for emergency treatment, such as removal of infected individuals from the farm, which would also circumvent the cost of specialized inspection. Moreover, a rapid, specific, and sensitive method of pathogen detection will be helpful to more fully understand this disease and to develop treatment for it.
Various pathogens in aquaculture can be detected using immunological techniques such as ELISA (Swain and Nayak 2003;Hu et al. 2020), immunofluorescence (McCarthy 1975;Liu et al. 2020), immunohistochemistry (Ekman and Norrgren 2003;Khimmakthong et al. 2013), and lateral flow immunoassay (Seo et al. 2020;Su et al. 2015); molecular techniques such as PCR (López et al. 2011), real-time PCR (Halaihel et al. 2009;Adamek et al. 2016), and loopmediated isothermal amplification (LAMP) (Gahlawat et al. 2009;Xu et al. 2010); and nanotechnology-based methods such as MALDI-TOF-MS (Piamsomboon et al. 2020) and flow cytometry (Ryumae et al. 2010). Of these, the LAMP method was developed to amplify specific genes using a designated primer set and a DNA polymerase with strand displacement activity (Notomi et al. 2000). The advantages of LAMP are that DNA can be specifically amplified under isothermal conditions, and that gene amplification can be detected visually (by the naked eye) following addition of a dye such as malachite green to the reaction mixture (Lucchi et al. 2016;Fuertes-Perez et al. 2020). Therefore, it is possible to detect the pathogen in the field using minimal equipment to maintain a constant temperature (e.g., water bath or heat block). In addition to isothermal gene amplification and visual detection of amplification, LAMP is faster (within 60 min) and more sensitive than conventional PCRbased methods.
Although 16S rRNA genes are widely used in bacterial taxonomy because they contain variable regions, the internal spacer region (ISR) between the 16S and 23S rRNA genes is known to be more variable among bacterial species than ribosomal genes themselves (Barry et al. 1991;Hassan et al. 2003a, b;Osorio et al. 2005). We developed a rapid, specific, and sensitive LAMP method targeting the ISR of Tenacibaculum sp. strain Pbs-1 and provide the first report on the diagnosis of Tenacibaculum sp. Pbs-1 associated with black-spot shell disease.

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. amylolyticum, T. ascidiaceicola, T. maritimum, T. mesophilum, T. sediminilitoris) were purchased from the National Institute of Technology and Evaluation. Diseased and healthy Akoya pearl oysters Pinctada fucata (average body weight 50.2 g) were collected from an aquaculture farm in Ago Bay, Shima City, Mie Prefecture, Japan, in July of 2016. After excision from the shell, blackened or normal shell pieces were transferred into 1.5 ml sterile centrifuge tubes and stored at − 30 °C prior to DNA extraction.

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, Irvine, 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 (i.e., strain Pbs-1 and the five type strains of Tenacibaculum) was sequenced. The ISR was amplified by PCR using primers 5′-ATA CGG AGG GTG CAA GCG TT-3′ (forward) and 5′-ACC AGC GGA TTT GCC TAC CA-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 (Taq DNA 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 then 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:// prime rexpl orer. 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; Fig. 1).

LAMP reaction condition optimization
The LAMP assay was optimized using different temperatures (60 °C, 63 °C, and 65 °C) for 35 min, and then at a constant temperature of 63 °C for different durations (25 min, 35 min, and 60 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 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 to 1.6 µM. The composition of the reaction mixture other than FIP and BIP was the same as described 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 to 1.2 µM. The composition of the reaction mixture was the same as described above except that 50 pg of DNA template and LF were used. Finally, 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, and then photographed using a FAS-Digi PRO gel imaging system (NIPPON Genetics Co., Ltd, Tokyo, Japan).

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 the assay sensitivity were calculated.

Optimization of Pbs-1 LAMP reaction conditions
The LAMP reaction was performed using DNA from genus Tenacibaculum (strain Pbs-1 and five related species) as a template to determine the optimum reaction temperature, reaction time, and concentrations of the inner and loop primers. Reaction temperatures were examined at 60 °C, 63 °C, and 65 °C. Electrophoresis produced clear ladder bands at 60 °C and 63 °C, but not at 65 °C (Fig. 2a). Because the specificity increased at higher temperatures, we regard 63 °C to be the optimal reaction temperature for the detection of strain Pbs-1 with the LAMP assay. To evaluate the optimal reaction time for the LAMP assay, it was first determined at 63 °C for 25 min, 35 min, and 60 min. No amplification was detected in any of the samples in the reaction for 25 min, but good amplification of strain Pbs-1 DNA was detected in reactions for ≥ 35 min (Fig. 2b). To evaluate the optimal concentration of the two inner primers, their concentrations were reacted from 1.0 to 1.6 µM (Fig. 3). Only DNA from strain Pbs-1 was detected at all tested primer concentrations. Amplified products at 1.0 or 1.2 µM were visualized as weak bands, whereas clear ladder-like bands were produced at 1.4 and 1.6 µM. Because a low concentration of the reagent could reduce the running cost, we regard the optimal concentration of inner primers to be 1.4 µM for this LAMP assay.
To evaluate the optimal concentration of loop primer, its concentrations were reacted from 0.6 to 1.2 µM (Fig. 4). Electrophoresis produced nonspecific ladder-like bands at 1.2 µM, whereas DNA only from strain Pbs-1 was amplified at 0.6-1.0 µM. Among them, the amplification product obtained by the reaction using 1.0 µM loop primer was the clearest, we regard the optimal loop primer concentration to be 1.0 µM for this LAMP assay. All tests were performed three times on the same sample to confirm its reproducibility. Thus, the optimum conditions for this LAMP reaction determined to be a reaction temperature of 63 °C, reaction time of ≥ 35 min, and concentrations of inner and loop primers of 1.4 and 1.0 µM, respectively.

Detection limits of LAMP
LAMP reactions were performed at 63 °C for 35 min or 60 min, and their respective detection limits were compared using tenfold serial dilutions (50 ng-50 ag) using DNA from strain Pbs-1 as the template. Detection limits of Pbs-1 DNA by LAMP assay for 35 min or 60 min were 50 pg (Fig. 5a) and 5 fg (Fig. 5c), respectively. Additionally, by adding malachite green to the reaction mixture, a positive LAMP reaction (dark blue) could be detected visually (a negative reaction corresponded to light blue) (Fig. 5b, d).
These visual detection results correlated with those of gel electrophoresis. The detection limit of Pbs-1 DNA by PCR assay was 0.5 ng (Fig. 5e). The LAMP assays for 35 min and 60 min, respectively, were 10 times and 100,000 times more sensitive than PCR.

Evaluation of the LAMP assay using DNA from pearl oysters
Evaluation of the LAMP assay was performed using DNA samples extracted from three diseased and three healthy pearl oysters. All diseased oysters tested positive for the presence of strain Pbs-1 when using the LAMP assay (Fig. 6). The Pbs-1 strain was detected in one otherwise healthy-looking oyster.

Discussion
Tenacibaculum sp. strain Pbs-1, the causative agent of blackspot shell disease in P. fucata, is recognized as a major problem for the cultured pearl industry. A method of rapid, specific, convenient, and sensitive detection of strain Pbs-1 is important to prevent or monitor disease outbreaks. Although many highly optimized, highly sensitive, and specific PCRbased assays exist, they are time-consuming and require levels of technical expertise and specific instruments (such as thermal cyclers) to perform, rendering them impractical for field detection of strain Pbs-1. In contrast, the LAMP reaction can be performed at 60-65 °C within a relatively short period of time and requires only a water bath or a heating block to maintain a constant reaction temperature. We designed specific primers for the LAMP assay based on the ISR between the 16S and 23S rRNA genes of Tenacibaculum sp. Pbs-1. ISR nucleotide sequences were determined from strain Pbs-1 and five related bacterial strains (Accession Nos.LC724053-LC724058). The homologies between Pbs-1 and the related species (T. mesophilum, T. amylolyticum, T. sediminilitoris, T. maritimum, T. ascidiaceicola) were 96.5, 39.2, 76.9, 85.9, and 73.0%, respectively. From the alignment results of these sequences, we designed five specific primers to detect Pbs-1 using the default conditions in PrimerExplorer v5 (Table 1, Fig. 1).
Our LAMP method reacted well at 60-63 °C. Although we conducted our analyses in the laboratory, we suggest that this method could be used in the field using a common water bath and/or an inexpensive heating block, and is therefore suitable for field use where precise temperature control is difficult. The optimum concentration of LF was 1.0 μM, but a higher concentration (1.2 μM) confirmed the amplification of T. sediminilitoris DNA; this is because the B2 or B3 sequences that bind to the products produced by adding LF are the same or very similar. Excluding the time for electrophoresis, a conventional PCR assay takes 2-3 h  (Hassan et al. 2008;López et al. 2011;Wiklund et al. 2000), whereas the LAMP assay can be performed within 1 h. To our knowledge, the LAMP assay of swine vesicular disease virus in pigs is the most rapid method at 25 min (Blomström et al. 2008). In aquaculture, to our knowledge, the shortest reaction times using the LAMP method to detect viruses or bacteria are 35 min to detect lymphocystis disease virus in Japanese flounder Paralichthys olivaceus (Li et al. 2010), and 30 min to detect Vibrio parahaemolyticus in artificially contaminated shellfish (Chang et al. 2011). The relatively short time required for our Pbs-1 LAMP assay (35 min) is comparable with other tests, making the technique especially suitable for on-site detection on pearl oyster farms.
The detection limits of Pbs-1 DNA by LAMP assay for 35 min or 60 min were 50 pg (Fig. 4a) and 5 fg (Fig. 5c), respectively. LAMP assay durations of 35 min or 60 min were respectively 10 times and 100,000 times more sensitive than detection of Pbs-1 with a well-established molecular method PCR (Fig. 5e). Furthermore, the LAMP reaction at 60 min was 10,000 times more sensitive than it was at 35 min. This result indicates that amplification can be accomplished within 35 min if speed is required, and in 60 min if high sensitivity is required. To our knowledge, the most sensitive level of bacterial LAMP is 0.3 fg (Fall et al. 2008). Although the sensitivity of Pbs-1 LAMP reacted at 60 min was slightly inferior, it was sufficiently sensitive to detect strain Pbs-1. Additionally, by adding malachite green to the reaction mixture, the positive LAMP reaction changed to a dark blue, which could be discerned visually. The correlation between results judged visually with those observed after gel electrophoresis is consistent with previous reports (Sun et al. 2006;En et al. 2008;Ren et al. 2010;Xu et al. 2010). We regard the visual inspection of LAMP products using malachite green to be an advantage because it eliminates the need for time-consuming electrophoresis and subsequent staining with the carcinogenic ethidium bromide. These results further support potential use of the LAMP assay in the field.
Many nucleic acid amplification techniques with isothermal amplification have been developed, such as nucleic acid sequence-based amplification (Compton 1991), self-sustained sequence replication (Fahy et al. 1991), helicase-dependent amplification (Vincent et al. 2004), exponential amplification reaction (Reid et al. 2018), strand displacement amplification (Walker et al. 1992), recombinase polymerase amplification (Lutz et al. 2010), rolling circle amplification (Fire and Xu 1995) and ladder-shape melting temperature isothermal amplification (Wang et al. 2021). However, these methods have their own limitations, such as lengthy processing times, nonspecific amplification, and the need for a detector to gather fluorescence signals (Bodulev and Sakharov 2020;Zhao et al. 2015;Wang et al. 2021). The LAMP method developed in this study has solved all those limitations of nucleic acid isothermal amplification techniques because it was rapid, specific and visually detectable.
Evaluation of the LAMP assay was performed with DNA samples extracted from three diseased and three healthy pearl oysters. When the LAMP assay was used, all three diseased oyster samples tested positive for Pbs-1 (as expected). Interestingly, one seemingly healthy oyster also tested positive for Pbs-1 (Fig. 6). LAMP products were also digested using the restriction enzyme MseI, and the size of the digested fragment was consistent with the expected size of 136 bp (data not shown). This result indicates that LAMP amplification was performed by Pbs-1 DNA, not by oyster genomic DNA. LAMP products of positive samples were directly sequenced using F1c primer, and all samples matched the sequence of Tenacibaculum sp. strain Pbs-1. Furthermore, no cross-reactions with DNA from other Tenacibaculum species were observed using the Pbs-1 LAMP assay, indicating that LAMP is highly specific for target DNA sequences (Figs. 3 and 4). From these results, no false positives have so far been confirmed by the LAMP method developed in this study. However, more samples are needed to examine the false-positive rate in detail. Future research should endeavor to confirm the detection limit of this new LAMP assay and to analyze the relationship between the number of bacteria and the detection limit in detail. To do so, we must develop a real-time PCR method specific for the strain Pbs-1. The current results strongly suggest that the likelihood of false-positive detections is low and that Pbs-1 at low densities can also be detected from asymptomatically infected oysters. Use of the LAMP assay developed in this study will allow for emergency treatment of pearl oysters by prompt removal of infected individuals from the aquaculture farm, while data on the distribution of strain Pbs-1 and the relationship between its growth and environmental factors might be more easily obtained. Although this study clearly reveals the effectiveness of the new technique, it will be necessary to confirm its efficiency by increasing the sample number, as positive samples were detected among both diseased and healthy-looking pearl oysters. For example, we can remove bacterium-infected oysters that will die due to the operating stress during the post-operative care or will produce low-quality pearls by examining small amounts of the hemolymph drawn from the pre-operative individuals using the LAMP assay. Indeed, there is a development of new post-operative method in which oysters are immersed in low salinity seawater to increase the production of high-quality pearls because of suppression of physiological responses including an inflammatory reaction (Atsumi et al. 2014), which might still provide physiological stresses to the post-operative oysters resulting from its suppression of defense mechanisms, followed by promoting bacterial growth in oysters, suggesting worthwhile treatment for pearl culture by removing infected oysters before the operating. In the preliminary experiment, strain Pbs-1 was Fig. 5 Sensitivities of LAMP and PCR for detection of Tenacibaculum sp. strain Pbs-1. The LAMP reaction was performed at 63 °C for 35 min, and the products were detected using a agarose gel electrophoresis, and b visually by adding malachite green. M, marker. The LAMP reaction was performed at 63 °C for 60 min, and the products were detected using c agarose gel electrophoresis, and d visually by adding malachite green. e PCR products detected using agarose gel electrophoresis. M marker, N.C. negative control also detected from hemolymph of diseased oysters using the LAMP assay developed in this study (data not shown).
Until now, only PCR/DGGE (denaturing gradient gel electrophoresis) or culture methods were able to detect the pathogen Tenacibaculum sp. strain Pbs-1 (Sakatoku et al. 2018). Black-spot shell disease caused by this bacterium also occurs on pearl farms in other parts of Asia, such as Vietnam (Sakatoku et al. 2018). Regarding cost and simplicity, our assay is a rapid, simple, and economically viable alternative to perform for large-scale shellfish screening, on both national and international scales.

Conclusions
The LAMP-based assay described herein represents a specific, rapid, and simple diagnostic method to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease. Our report is the first to detail the LAMP method for molecular diagnosis of strain Pbs-1. Reactions are performed in a single tube, incubated for 35 min or 60 min, in a water bath or on a heating block at 63 °C. In addition to its high specificity, the detection limit of the LAMP assay is 50 pg at 35 min and 5 fg at 60 min, which is respectively 10 times and 100,000 times more sensitive than that achieved with conventional PCR. The LAMP products can be detected by agarose gel electrophoresis or, more conveniently, detected visually by adding malachite green to the reaction mixture. The LAMP assay developed in this study facilitates the field detection of strain Pbs-1 in terms of isothermally reactive (no thermal cycler required), rapid, and visual detection. This technique has considerable potential for routine diagnosis of Pbs-1 in less well-equipped oyster hatcheries and/or pearl farms.