Development of LAMP assay for early detection of Yersinia ruckeri in aquaculture

Introduction

Yersiniosis is a highly infectious disease causing significant economic losses in fish aquaculture worldwide, especially in salmonids (Keeling et al., 2012; Oroji et al., 2021; Yang et al., 2024). Yersinia ruckeri is the etiological agent of enteric red mouth disease (ERM) or Yersiniosis (Keeling et al., 2012). Y. ruckeri is an anaerobic, gram-negative, rod-shaped bacteria that mainly enters the host body through the pavement cells of the gill lamellae, then into the intestine and bloodstream (Keeling et al., 2012; Kumar et al., 2015). Y. ruckeri is a facultative intracellular pathogen, beginning the infective stage in the extracellular space then inhabiting the macrophages during the intracellular phase. The pathogen can also lay dormant in some fish without causing clinical symptoms; however, the asymptomatic hosts transmit disease through defecation to more susceptible fish (Guijarro et al., 2018; Kumar et al., 2015). The common symptoms of the disease include subcutaneous haemorrhage around the mouth, throat, tongue, gills, fins and gums in addition to an enlarged spleen and haemorrhages on the liver’s surface, pancreas, swim bladder or lateral muscles (Kumar et al., 2015). The severity of infections in fish can vary, as some fish naturally recover from infections; however mass mortality has been observed during outbreaks (Yang et al., 2024). While there is no precise assessment of the economic toll caused solely by Y. ruckeri infections, it is one of the significant pathogens in aquaculture, contributing to a combined annual loss of over USD 6 billion (Stentiford et al., 2017). Y. ruckeri is classified into two biotypes (motile and non-motile) and four major serotypes, which have different surface antigens (Carson et al., 2008; Kumar et al., 2015). Serotype O1b biotype I has predominantly been responsible for most outbreaks in the past, which led to the early development of a very effective vaccine; however, recent outbreaks in vaccinated fish caused by biotype II, has raised concerns about the ability to control Y. ruckeri infections (Austin, Robertson & Austin, 2003; Fernández, Méndez & Guijarro, 2007; Guijarro et al., 2018; Kumar et al., 2015; Saleh, Soliman & El-Matbouli, 2008). Without the availability of effective vaccines to control infection in all countries, there is an increased reliance on other control measures such as antibiotics and preventative management practices (Önalan & Çevik, 2020; Valle et al., 2020). Ideally, early pathogen detection will guide on-farm management decisions, enabling a swift response to increasing infection levels and supporting effective pathogen control.

Traditional methods for the detection of Y. ruckeri are bacterial cultures (Rodgers, 1992; Waltman & Shotts, 1984), biochemical tests (Davies & Frerichs, 1989; De Grandis et al., 1988) and serological tests (Olesen, 1991; Stevenson & Airdrie, 1984). Although those methods are adequate for detection, they are laborious, can require particular growth conditions, and may not be applicable to all isolates, which can be challenging to differentiate between closely related species (Austin, 2019; Davies & Frerichs, 1989; Ibrahim et al., 1993). Several molecular techniques, such as restriction fragmentation-length polymorphism (RFLP) (Garcia et al., 1998), polymerase chain reaction (PCR) (Gibello et al., 1999; LeJeune & Rurangirwa, 2000) and real-time PCR (Birkenbach, 2022; Ghosh et al., 2018; Keeling et al., 2012) can successfully detect low levels of the bacteria; however, they require expensive equipment, trained personnel, and are not suitable for point-of-care pathogen detection (Knox, Zerna & Beddoe, 2023).

Loop-mediated isothermal amplification (LAMP) is a method for amplifying DNA at a constant temperature. It uses four to six primers to target six to eight specific regions on a DNA template. The amplification starts with a strand-displacing DNA polymerase that initiates the synthesis of new DNA. Two of the primers form loop structures, which facilitate further rounds of amplification. One of the main benefits of LAMP is that it does not require a specialized thermocycler; instead, the reaction can take place at a stable temperature using either a heat block or a water bath. This makes LAMP a fast, straightforward, and economical choice for field applications, while still achieving the high sensitivity and specificity associated with traditional molecular techniques (Notomi et al., 2000). There has been LAMP assays developed for use in the aquacultural industry, for detection of viral (Caipang et al., 2004; Gunimaladevi et al., 2005), bacterial (Savan et al., 2004; Tsai et al., 2013) and parasitic pathogens (El-Matbouli & Soliman, 2005; Picón-Camacho et al., 2013). Furthermore, there has been a LAMP assay developed for the detection of Y. ruckeri, termed enteric red mouth LAMP (ERM-LAMP), however this test requires extensive sample preparation and DNA extraction within a specialised laboratory (Saleh, Soliman & El-Matbouli, 2008). The existing ERM-LAMP assay utilizes five primers targeting the yruI/yruR gene of Y. ruckeri and can be detected after one hour of incubation at 63 °C using visual inspection, agarose gel electrophoresis or by real-time monitoring of turbidity. Its high sensitivity allows detection of as low as 10 pg of Y. ruckeri genomic DNA. However, the ERM-LAMP method relies on the collection of fish to carry out the initial DNA extraction using tissue samples (Saleh, Soliman & El-Matbouli, 2008). While this is the standard testing method, it is not ideal to sacrifice several expensive, large fish for routine surveillance, nor to require specialists for septic dissection. Therefore, using environmental water for sampling offers a safer and simpler routine point-of-care surveillance method (Khodaparast et al., 2022).

Here, we report the development of a Y. ruckeri-specific LAMP (Yr-LAMP) assay and subsequent DNA extraction method suitable for field use from water, to allow rapid, reliable, and robust detection of Y. ruckeri within 1 h.

Materials and Methods

Yr-LAMP specificity testing

Initial specificity testing was performed using 0.25 ng/µl of target plasmid (pYr) and 100-fold higher concentration (25 ng/µl) of plasmids containing gene homologous from closely related species, pYro or pYf. Reactions were performed and analysed using the Genie^® II and Genie^® III machines, as previously described, with 30 min of amplification.

To further validate the test’s specificity, the Yr-LAMP assay was evaluated against synthetic DNA representing 999 bp of glnA from salmon pathogens, Aeromonas salmonicida subsp. Salmonicida, Flavobacterium psychrophilum, and Renibacterium salmoninarum. This evaluation also included a range of other bacterial species. Total genomic DNA was extracted using Bioneer AccuPrep Genomic DNA extraction kit from a bacterial specificity panel consisting of gram-positive bacteria including, Enterococcus faecalis, Staphylococcus aureus, Streptococcus agalactiae, S. pyogenes, S. salivarius and S. sanguinis and gram-negative bacteria including, Escherichia coli, Pseudomonas aeruginosa, P. fluorescens and Vibrio natriegens species following manufacturer instructions. Total DNA concentrations were determined by Qubit™ 1× dsDNA BR Assay Kit (Invitrogen, Waltham, MA, USA), using Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and were stored at −20 °C until needed.

Furthermore, both fresh and seawater samples were collected and screened. To assess the water collection contaminants, seawater samples and fresh lake water (La Trobe Lake, Victoria, Australia) were filtered using the filtration method outlined in Fig. 1B (Khodaparast et al., 2022). The released genomic DNA was used directly as LAMP template or stored at 4 °C until further use. The presence of bacteria in those concentrated water samples was confirmed using a universal bacterial PCR assay targeting the 16S gene of both gram-positive and gram-negative bacteria (Barghouthi, 2011). The amplification was performed using a primer mixture of seven primers, Golden mixture 7 (Table S1). The PCR reaction mixture totalled 25 µl, consisting of a final concentration of 1X Promega GoTaq^® Green Master Mix, 0.1 µM of each of the seven primers and 5 µl of concentrated seawater or lake water as a template. The PCR product was observed using 1.5% (w/v) agarose gel with 0.2 μg/mL ethidium bromide, with electrophoresis performed at 90 v for 70 min (Barghouthi, 2011).

LAMP was completed following the isolation of genomic DNA, plasmids containing gene homologous of closely related species, synthetic DNA of salmonid pathogens and the concentration of water samples. Using the optimised concentration of the selected Yr-LAMP primer set at 65 °C, the LAMP reaction was run for 30 min to detect the amplification. Reactions were performed in triplicates and tested by the Genie^® II and Genie^® III machines; as previously described, the annealing temperature of the assay was used to confirm the correct product.

Results

Primer screening

The target gene in this study, glutamine synthetase (glnA) from Y. ruckeri, was chosen for its divergent DNA sequence following alignment comparisons with similar genes within the national center of biotechnology information (NCBI) database. When manually identifying the optimal region of the glnA sequence for designing LAMP primers, an alignment of the glnA gene from Y. ruckeri revealed that the region between 70 and 585 bp showed over 99% similarity among Y. ruckeri strains and less than 90% similarity with various Yersinia species. Using the sequence mentioned above as a template for designing primers resulted in four primer sets with Yr#1 and Yr#2 software generated and Yr#3 and Yr#4 manually designed (Table 1). Manually designed LAMP primers allowed for greater flexibility in targeting specific areas of glnA that had lower percentage similarity. The Yr#1, Yr#2, Yr#3 and Yr#4 were designed to amplify regions with approximate similarity 88%, 89%, 90% and 89%, respectively, to non-target sequences. Initial testing of the four different primer sets for Y. ruckeri-glnA resulted in varying time to positive amplification (Tp), ranging from 05:37 ± (7 s) to 17:47 ± (92 s) min (Table 2, Fig. S1). Primer set Yr#1, which consists of five primers designed to amplify 209 bp region from 219 to 427 bp of the Y. ruckeri glnA gene (Fig. 2), gave the fastest Tp (05:37 min ± 7 s) and was chosen for all future experiments.

Table 2:

Amplification times for the four suggested LAMP primer sets with their time to positive (Tp) and standard deviation values.

Primers set	Yr#1*	Yr#2	Yr#3	Yr#4
Tp (mm:ss)	05:37 ± 7 s	06:30 ± 15 s	07:42 ± 80 s	17:47 ± 92 s

DOI: 10.7717/peerj.19015/table-2

Note:

* Primer set selected to be used throughout Yr-LAMP study.

Specificity assessment of Yr-LAMP

Further specificity was performed using an optimized primer set against a range of gram-positive and gram-negative bacteria (Fig. 3). Presence of bacteria in fresh and seawater samples was confirmed by PCR using universal bacterial primers targeting 16S gene. Agarose gel then confirmed that bacteria were present in the filtered water sample, as a multiple band profile was observed (Fig. 4). Seawater sample #8 and lake water sample #4 were selected to resemble the environmental water in further experiments. A single amplification peak specific to pYr amplification was detected approximately at 6 min with Tm 88.2 °C. There was no amplification for any of the non-target samples during the 30 min LAMP amplification time (Fig. 3), indicating that the Yr-LAMP assay was specific for Y. ruckeri and did not display cross-reactivity with any of the other panel bacteria or other microorganisms which naturally inhabit seawater or lake water.

Discussion

In this study, we utilized competent E. coli cells integrated with synthetic DNA encoding the Y. ruckeri glutamine synthetase gene for DNA extraction and sensitivity testing. The use of E. coli was necessitated by the unavailability of Y. ruckeri cultures or clinically infected samples. Our DNA extraction method, which employed KOH, proved effective even on gram-positive bacteria, indicating its robustness and potential applicability across different bacterial types (Ackerly, Tran & Beddoe, 2024). Initial assessments demonstrated that while E. coli served as a useful surrogate due to its gram-negative nature, it is imperative to validate these findings with Y. ruckeri bacterial cells to ensure accurate sensitivity values. To advance this validation, efforts should be made to acquire Y. ruckeri samples and replicate the DNA extraction and sensitivity tests on Y. ruckeri cells and compare the results with those derived from E. coli.

While the Yersinia genus encompasses a range of species, including Y. enterocolitica and Y. frederiksenii, some of which have been found in fish, not all are associated with fish diseases (Carson et al., 2008). Therefore, it is essential to identify a distinct target gene that ensures high specificity for detecting Y. ruckeri amidst other related and unrelated bacteria present in the fish environment (Carson et al., 2008). The 16S rRNA gene, which is approximately 1,500 base pairs in size, is one of the most conserved genes in bacteria due to its critical role in cell function. It is also the most extensively represented gene in the GenBank database, with over 90,000 sequences available (Clarridge, 2004; Hao, Pei & Brown, 2017; Janda & Abbott, 2007). The aforementioned reasons make 16S rRNA the ideal candidate for designing specific PCR primers however, challenges may occur when determining eight distinct regions for designing LAMP primers (Carson et al., 2008; Notomi et al., 2000). The glnA gene is a promising marker for identifying Y. ruckeri because it is conserved among various strains. A real-time PCR assay targeting the glnA gene of Y. ruckeri demonstrated 100% specificity in detecting ERM infection (Keeling et al., 2012; Kotetishvili et al., 2005). The conservation of this gene sequence within Y. ruckeri makes the assay a versatile diagnostic tool for identifying new bacterial variants globally (Kotetishvili et al., 2005).

The previously published ERM-LAMP assay utilized five primers targeting the yruI/yruR gene of Y. ruckeri in tissue samples. While the assay demonstrated high specificity for Y. ruckeri, it required one hour for amplification (Saleh, Soliman & El-Matbouli, 2008). The newly developed Yr-LAMP assay comprises five LAMP primers targeting the glnA gene of Y. ruckeri, which was conserved in all tested Y. ruckeri strains and varied from other species within the same genus (Kotetishvili et al., 2005). The assay was optimised to use primer concentrations of 1.6, 0.2, 0.8 µM of FIP & BIP, F3 & B3 and LF, respectively, of the Yr#1 primer set to achieve high specificity and sensitivity. All the amplicons had a similar melting temperature of approximately 88 °C, even at a concentration of 0.5 × 10⁻⁸ ng/µl. However, this concentration resulted in stochastic amplification and was therefore considered inconsistent and excluded. While the existing ERM-LAMP and real time PCR has analytical sensitivity of 1 pg and 5 fg respectively, our newly developed Yr-LAMP assay can detect 0.5 × 10⁻⁷ ng/µl of plasmid DNA which is equivalent to 11.3 copies/µl in under 20 min. This is a shorter detection time compared to both of those assays (Keeling et al., 2012; Saleh, Soliman & El-Matbouli, 2008). Accurate sensitivity comparison among the three assays is not possible since our assay was tested on plasmids with the target gene, while the others used genomic DNA. Nonetheless, our Yr-LAMP assay is highly sensitive, three-fold more sensitive than the corresponding cPCR and with a detection limit comparable to other published LAMP assays (Agarwal et al., 2022).

Previous studies tracked the route of Y. ruckeri in the host fish, the bacteria enter the fish via gills and shortly it reaches the intestine although it was not observed in kidney until the third day post infection and a week later was observed in the brain and other internal organs (Méndez & Guijarro, 2013; Ohtani et al., 2014). Those findings support the concept of early detection of pathogen is a key factor for containing infection and mitigating the excess use of antimicrobial treatment (Yu et al., 2022). Sampling and extraction can be considered the most critical steps in diagnostics, they can promote the assay to be field-deployable or not. The current assays for detecting Y. ruckeri are either invasive using fish tissues (Keeling et al., 2012; Saleh, Soliman & El-Matbouli, 2008) or non-invasive using faeces (Carson et al., 2008; Ghosh et al., 2018) or blood samples (Altinok, Grizzle & Liu, 2001; Bastardo, Ravelo & Romalde, 2012). Although the published Y. ruckeri detection methods are rapid (approximately one hour) for LAMP and real time PCR, the extraction step itself was long. Extracting genomic DNA from tissue samples, blood, or faecal samples with commercial extraction kits required several hours and purifying them from PCR inhibitors took even longer. Therefore, to address these challenges for our Yr-LAMP assay application, we developed field-tolerant sampling and extraction methods from environmental water (Carson et al., 2008; Keeling et al., 2012; Saleh, Soliman & El-Matbouli, 2008). For testing the field performance of Yr-LAMP assay, environmental water spiked with serial dilutions of E. coli bearing the target gene was filtered and lysed using KOH. The Yr-LAMP assay could successfully detect as low as 0.08 cells/µl (3.9 × 10⁻¹⁰ ng/µl of pYr) of the initial collected water in less than 15 min. Relating these results to the sensitivity test, we can suggest that approximately 1.13 × 10² copies/µl of pYr were recovered and extracted from the initial 4,000 cells spiked into the 50 ml water. On the other hand, cPCR could only detect until 8.0 cells/µl of the initial 50 ml water sample, two-fold less sensitive than the Yr-LAMP assay.

It was confirmed that Yr-LAMP only amplified the positive control and no other bacteria, suggesting that Yr-LAMP is specific. As the assay will be used on environmental samples there is potential for cross reactivity with preexisting microorganisms present in the water habitat, water samples from the beach and lake were collected, filtered, and concentrated using the filtering method. Although a diverse range of microorganisms exists in both freshwater and seawater, the microbiome extracted from these samples did not result in amplification by our developed LAMP assay. This outcome demonstrates the assay’s high specificity (Baharum et al., 2010; Wang et al., 2012). Although using KOH for direct extraction of DNA in-field was successful and sensitive in previous work (Ackerly, Tran & Beddoe, 2024; Khodaparast et al., 2022), it was observed to have a slight inhibitory effect on LAMP represented in a minor decline in Tm than the positive control. Those observations are consistent with other findings regarding the effect of inhibitors on the melting temperature of LAMP (Nwe, Jangpromma & Taemaitree, 2024; Shirshikov & Bespyatykh, 2022). However, despite this effect on Tm, the inhibition did not impact the assay’s sensitivity, and therefore it was not considered problematic.

Studies have demonstrated that Y. ruckeri can survive for up to 90 days or more in both fresh and marine waters (Romalde et al., 1994). Therefore, detecting Y. ruckeri in water samples can inform real-time management strategies. For instance, the presence of Y. ruckeri in sea cages may indicate the need for treatment or the delay of introducing new fish into the sea cages.

The data reported in our Yr-LAMP assay was obtained by using real-time LAMP instruments, Genie^®ll or Genie^®lll (OptiGene, Horsham, England) real time fluorometer. Genie is a lightweight, portable device that can work with batteries. Despite the widespread use of the Genie instruments in numerous studies globally (Enicks, Bomberger & Amiri, 2020; Fowler et al., 2021; Neeraja et al., 2015; Saar et al., 2021), they are constrained by the limited number of chambers, with Genie^®lll offering eight and Genie^®ll providing 16 wells. This restricts the number of samples available for testing to six and 14, respectively, excluding positive and negative controls. Furthermore, the capacity is reduced by half if duplicate tests are necessary, making these instruments unsuitable for high-throughput detection. For wide range surveillance, other detection alternatives can be used, such as lateral flow dipstick (LFD) or visualise colour detection. For example, Yu et al. (2022) developed an impressive LAMP assay capable of detecting Singapore grouper iridovirus (SGIV) directly from fin samples that were boiled in 50 µl of 0.02 N NaOH. The results of the LAMP assay were indicated by a colour change from yellow to pink, with the entire process taking approximately an hour and was able to detect less than 6 copies/µl of the virus. The straightforward extraction process combined with the rapid and sensitive nature of the assay makes it an effective on-site method for efficiently managing SGIV infections (Yu et al., 2022).

LFD has gained recent popularity due to its versatility in detecting various pathogens, including bacteria (Thongkao et al., 2015), virus (Ding et al., 2010) and parasites (Ding et al., 2010). Its ease of use, exemplified by the accessibility of COVID-19 test strips even for non-specialists, underscores its practicality (Yüce, Filiztekin & Özkaya, 2021). Looking ahead, exploring the feasibility of adapting the Yr-LAMP assay into an LFD format presents an opportunity to circumvent the testing limitations inherent in Genie devices. By integrating swabbing or the straightforward filtration and extraction methods demonstrated in our study, we aim to develop a high-capacity, device-free, point-of-care detection method suitable for deployment by untrained personnel.

Conclusions

Yersinia ruckeri poses significant economic risks in the aquaculture industry, necessitating regular surveillance to pre-emptively manage infections and reduce reliance on antimicrobial agents, thereby preventing outbreaks. The Yr-LAMP primers exhibit high specificity, effectively discriminating against organisms naturally occurring in fish environments and closely related Yersinia species. This method identifies the pathogen at extremely low levels, with an analytical sensitivity of 0.08 cells/µl in less than 20 min. Integrating these specific LAMP primers with filtration and KOH extraction enhances the system’s speed, reliability, affordability, and simplicity, making it feasible for farm workers to detect Y. ruckeri infections early and effectively control outbreaks.

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