1 Introduction

The Apis mellifera (Western honey bee) is a managed honey bee species of high economic importance to agricultural industries globally, generating over AUD $14.2 billion to the Australian economy due to pollination services alone (Karasiński 2018). American foulbrood (AFB) is a lethal, highly contagious, and notifiable bacterial disease affecting managed honey bee larvae and ongoing production profits associated with pollination and bee-related products. The aetiological agent is Paenibacillus larvae, a rod-shaped, gram-positive, spore-forming bacterium which produces highly resistant endospores which are viable for more than 40 years that are found in contaminated feed supplied by nurse bees, with only as little as ten individual spores required to cause disease (Genersch 2010; Genersch et al. 2005; Hoage and Rothenbuhler 1966).

Diagnosis of diseased hives is reliant upon initial visual inspection of clinically symptomatic hives with irregular brood capping that may appear dark and sunken and emit a foul odour and larval remains resembling a brown fluid-like mass deemed “ropy mass” as most common indicators of disease (Agriculture Victoria 2022). However, diagnosis based solely on the appearance of clinically symptomatic hives is unreliable and could lead to misdiagnosis amongst both experienced and hobbyist beekeepers due to the similar appearance of diseased hives and shared symptoms observed in a variety of brood diseases such as European foulbrood (EFB) (De Graaf et al. 2006). Furthermore, the observation of larval remains as indicators of disease, especially in the case of AFB, indicates that the hive and nearby hives will need to be tested and ultimately incinerated to destroy spores, the infectious form of the disease (Genersch 2010). Thus, accurate and rapid diagnosis of sub-clinical infections will prevent further disease transmission to other healthy hives and hive destruction.

Due to the inaccuracy of in-field diagnosis, highly sensitive and specific laboratory-based methods are required for the confirmation of disease presence, usually involving microscopic examination of larval smears, immunological assays, and PCR (Han et al. 2008; Martínez et al. 2010; Olsen et al. 1990). Such methods are restricted in their use, requiring access to expensive laboratory equipment performed by experienced personnel allowing time for further disease transmission amongst susceptible hives due to slow diagnostic turnaround. Point of care (POC) diagnostics allow rapid detection at the site of disease outbreak, which is advantageous in making informed and timely treatment decisions for management in real time.

Only one current molecular diagnostic test is commercially available for in-field detection of AFB, marketed worldwide as a lateral flow device (LFD) that contains P. larvae antibodies that react to P. larvae bacteria in brood reporting results in under 10 min (Tomkies et al. 2009). This lateral flow device is limited in the amount and type of sample processed per test, able to only test one brood per device with the DNA extraction method only optimised for larval samples. Small sample sizes and lack of sample diversity are not representative of the colony disease status, potentially resulting in a high number of false negatives permitting continued disease transmission of a highly virulent and destructive disease. Moreover, using this device on multiple samples to overcome this bias can be quite expensive, priced at $18 AUD per test (Vita Europe Ltd).

Loop-mediated isothermal amplification is a molecular diagnostic method that rapidly amplifies nucleic acids under isothermal conditions utilising a DNA polymerase with high strand displacement ability, amplifying target nucleic acids between 60 and 67 °C (Notomi et al. 2015). Isothermal amplification is highly advantageous for POC applications where resources are limited, eliminating the need for expensive laboratory equipment such as that required for thermocycling in common PCR reactions and amplifying target nucleic acids by utilising cheap and portable equipment. Furthermore, LAMP is a highly robust assay, with its DNA polymerase enzyme more resistant to polymerase inhibitors, emphasising the ideal use of LAMP in the field, where extensive sample purification usually required for most molecular diagnostics is unfeasible (Francois et al. 2011).

The mechanisms of LAMP make it highly specific, utilising two or even three sets of primers and recognising and hybridising to six and eight distinct regions in the target genetic material. These primers make up the four main primers: forward and backward inner primers (FIP and BIP) and the forward and backward outer primers (F3 and B3) (Notomi et al. 2000). Additional primers termed forward and backward loop primers (LF and LB) significantly reduce amplification time, by hybridising to hairpin structures to create loops for cyclic amplification (Nagamine et al. 2002). Final LAMP products represent stem-loops containing inverted repeats of target nucleic acids which hybridise to form cauliflower-like structures generated by the ongoing hybridisation of inverted repeats present within each individual strand of target nucleic acid. The overall mechanisms and characteristics of LAMP make it an ideal molecular diagnostic for field use with reported rapid detection observed in various pathogens such as bacterial (Best et al. 2018; Fu et al. 2021; Khodaparast et al. 2022; Lee et al. 2015; Zhong et al. 2016), viral (Dudley et al. 2020; Sarkes et al. 2020; Shivappa et al. 2008), parasitic (Kong et al. 2012; Matthew et al. 2022; Tran et al. 2022), and fungal pathogens (Müs¸tak et al. 2022; Ohori et al. 2006).

There is an urgent need for improved field-based diagnostics for detecting highly contagious notifiable diseases in intensive farming systems such as those observed in apiculture. This study aimed to develop and optimise a LAMP assay for the detection of AFB from field samples with high sensitivity and specificity for future field applications such as rapid differential diagnostics between AFB and European foulbrood (EFB) and treatment and quarantine for sub-clinical infections of AFB.

2 Methods and materials

2.1 Preparation of synthetic standards

Synthetic DNA representing the region 1321–1773 bp of P. larvae DNA Gyrase B (GyrB) gene (GenBank accession: CP019655) was synthesised by Integrated DNA Technologies (IDT, IA, USA) and cloned into the pCR-Blunt II-TOPO® vector using a PCR™II-Blunt-TOPO® kit (Invitrogen, MA, USA) and transformed into chemically competent DH5α E. coli cells according to manufacturer instructions (Green and Sambrook 2020). Transformants containing the insert were confirmed using colony PCR according to the manufacturer’s instructions (Promega, WI, USA) utilising chemically synthesised M13 forward and reverse sequencing primers (Bioneer, Daejeon, Korea). Total plasmid DNA was extracted using the FastGene Plasmid Mini Kit (Nippon Genetics, Japan) per the manufacturer’s high-copy plasmid protocol. Plasmid DNA quality and quantity were estimated using the NanoDrop™ 2000/2000c Spectrophotometer (Thermo Fisher Scientific, MA, USA) with A260/280 and A260/230 values between 1.8 and 2.0 considered sufficient for DNA amplification. Purified plasmid DNA containing GryB gene was diluted to 5–5 × 10−9 ng/µL and was used as standards to validate the assay and determine analytical sensitivity and as positive controls throughout this study. All samples were stored at − 20 °C until use.

2.2 Design and selection of LAMP assay primers

LAMP primers were designed using the New England Biolabs LAMP primer design tool version 1.0.1 with default parameters (New England Biolabs; https://lamp.neb.com) generating three different sets recognising the 1369–1510 bp region of the GyrB gene (Table I). All generated primers were initially accessed for specificity and potential cross-reactivity before use using an in silico nucleotide BLAST search with GenBank. Primer set 1 was generated with a complete set of LAMP primers, the forward, and backward inner primers (FIP and BIP), outer primers (F3 and B3), and two additional loop primers (LB and LF) (Table I). Due to strict parameters of the primer design software utilised, further complete primer sets for LAMP could not be generated. Therefore, primer sets 2 and 3 were generated missing a single loop primer (FIP, BIP, F3, B3, and LB) (Table I).

Table I Designed LAMP primer sets for AFB-LAMP. Regions complementary to the 5′–3′ sequences are underlined
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2.3 Assay optimisation

All three primer sets were assessed to determine the lowest amplification time with initial primer concentrations of 0.8 µM FIP and BIP, 0.2 µM F3 and B3, and 0.4 µm LB and LF against synthetic standards (5–5 × 10−6 ng/µL). Final LAMP reaction conditions were optimised in 25 µL volumes with 15 µL OptiGene GspSSD2.0 Isothermal Mastermix (ISO-DR004), 5 µL primer mix, and 5 µL of template of GyrB standard (5–5 × 10−9 ng/µL) and nuclease-free water (NFW) replaced template for control reactions. Reactions were performed in duplicates using the Genie II (OptiGene) real-time fluorometer with amplification performed as follows: initial pre-heat at 40 °C/1 min, followed by an amplification step at 65 °C/30 min and anneal step from 98 to 80 °C, at a rate of 0.5 °C/s, with default detection thresholds applied. Results were reported as time to positive (Tp) in minutes: seconds and anneal derivative melting temperature (Tm) reported in degrees Celsius (°C).

Further assay optimisation to improve time to positive amplification time was performed using primer set 1 with 0.4 µm increment for all primer pairs except primers F3 and B3 (0.2 µM) against 0.5 ng/µL of GyrB standards as prepared earlier (Table II).

Table II Assessing AFB-LAMP primer concentrations and their effect on Tp’s produced when using 0.5 ng/µL ofP. larvaecontrol plasmid DNA as a template on LAMP. Concentrations in bold were used throughout the study with a standard deviation of the Tp given in brackets
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2.4 Analytical performance of AFB-LAMP

The analytical sensitivity of the AFB-LAMP assay was determined using a standard curve of a previously prepared synthetic template with ten-fold decreasing concentrations between 5 and 5 × 10−9 ng/µL. Sensitivity was assessed through inter- and intra-assay variation across 10 replicate runs and within duplicate replicates, respectively. The limit of detection was determined according to the accepted % values of inter- and intra-assay variation described later and to amplification time (Tp) < 20-min cutoff threshold as instructed by the manufacturer (OptiGene).

To determine the analytical specificity of the AFB-LAMP assay, honey bee microbiome gDNA from PCR-confirmed AFB negative honey bees (n = 6) was extracted and LAMP assay was performed with honey bee microbiome gDNA template concentration of 5 ng/µL and primer set 1 (1.6 µM FIP and BIP, 0.2 µM F3 and B3, 2.0 µm LB and LF).

2.5 Honey bee sampling

A total of 198 individual worker and drone honey bees were sourced from southern and central Victorian apiaries with unknown infection status. Individual honey bees were collected and kept at 4 °C in 50-mL falcon tubes during transportation and were stored at − 20 °C until processed.

2.6 DNA extraction

Honey bee total genomic DNA was extracted using 50 mg of tissue excised from the abdomen of individual bees (n = 12, 6 with PCR-confirmed AFB infections and 6 PCR-confirmed AFB negative bees) and homogenised with a pellet pestle (Sigma-Aldrich, MO, USA). DNA extraction was performed using the Bioneer AccuPrep® gDNA extraction kit per manufacturer instructions. DNA concentration and quality were assessed on a NanoDrop™ 2000/2000c Spectrophotometer, diluted to 5 ng/µL, and stored at − 20 °C until use (Bioneer, Daejeon, Korea).

2.7 Bacterial 16S PCR from honey bee genomic DNA

All extractions were subject to universal 16S bacterial PCR utilising degenerate primers amplifying different regions of the 16S gene of common gut microbes found in the gastrointestinal tract of honey bees such as but not limited to Enterococcus spp., Salmonella spp., and Enterobacter spp. (Barghouthi 2011). PCR reactions were performed in 25 µL volumes containing 12.5 µL 2 × GoTaq polymerase Mastermix (Promega, WI, USA), 0.1 µM of the G7 degenerate primer mix (Table III), and 5 ng/µL of template. Thermocycling was performed in a C-Master GT thermal cycler (Dynamica, Livingston, UK) under the following conditions: initial denaturation step at 94 °C for 5 min followed by 25 cycles of denaturation step at 94 °C for 1 min, annealing step at 52 °C for 1 min, extension step at 72 °C for 1 min with a final extension step at 72 °C for 10 min.

Table III Primers used in different conventional PCR reactions throughout this study
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2.8 Identification of AFB infection by PCR

All bee samples subject to AFB-LAMP had infection status confirmed with PCR, according to Bakonyi et al. with the following modifications: 0.4 µM of AFB forward and reverse primers that amplify 237 bp fragment of P. larvae 16S RNA with 5 µL of DNA template (Table III). Thermocycling was performed under the following conditions: initial denaturation step at 95 °C for 2 min, followed by 35 cycles of denaturation step at 95 °C at 1 min, anneal step at 63 °C for 1 min, extension step at 72 °C for 1 min, and a final extension step at 72 °C for 5 min.

All PCR products were separated and visualised on 1% (w/v) agarose gel made with 0.5 × Tris–borate ethylenediaminetetraacetic acid (EDTA) buffer (40 mM Tris-HCI, pH [8.3], 45 mM Boric acid, 1 mM EDTA) stained with 0.5 µg/mL ethidium bromide, visualised on a ChemiDoc XRS + Gel Imaging System, and analysed using Image Lab™ software (Bio-rad, CA, USA).

In the verification of AFB-positive bees, the amplicon was subjected to Sanger sequencing performed at the Australian Genome Research Facility (AGRF). Results were confirmed using NCBI BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with samples returning > 95% identity to Paenibacillus larvae 16 s rRNA, strain: ATCC 9545 (GenBank accession: X60619) considered positive for AFB.

2.9 In-field DNA extraction

DNA extraction was initially optimised to mimic sample preparation under field conditions and to assess the effects of different substances within bees that could interfere with DNA amplification both in LAMP and PCR. Briefly, an individual honey bee was added to each 1.5-mL microcentrifuge tube containing 500 µL of one out of the three different extraction buffers to assess which extraction buffer would be suitable for gDNA extraction (Table IV). Each bee and buffer solution were mechanically disrupted using ten 3-mm-diameter stainless steel beads (Qiagen, Hilden, Germany) and hand-shaken to facilitate bead-beating until the solution became turbid. From each neat solution of the different buffers, ten-fold dilutions were made with crude gDNA resuspended in TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH [8]). Ten-fold dilutions ranging from neat, 1/10, 1/20, 1/50, and 1/100 of all buffers were assessed for minimal inhibition required for DNA polymerase to amplify different bacterial species using universal bacterial primers, previously described. Final crude gDNA dilutions of 1/10 in DNA lysis buffer 1 (KOH) were chosen for all field sample DNA extractions using methods previously described and were used throughout the study. This extraction buffer and dilution were selected due to the band brightness of PCR amplicons assessed on gel electrophoresis and assessed against the fastest Tp produced on AFB-LAMP with conditions previously described for both PCR and LAMP reactions.

Table IV DNA lysis buffers used to assess crude DNA extractions from individual honey bees
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2.10 Statistical analysis

All statistical analysis was completed in Microsoft Excel 2020 (version 2312). Inter/intra-assay variation was calculated using the coefficient of variation (CV%) as follows:

$$CV=(\frac{SD}{\chi })\times 100$$

Accepted values for intra- and inter-assay CV are < 10% and < 15%, respectively (Campbell et al. 2010).

3 Results

3.1 LAMP primer design and assay optimisation

AFB-LAMP primer sets were initially assessed by performing a standard curve of P. larvae GyrB plasmid ranging from 5 to 5 × 10−6 ng/µL. Amplification times ranged from 3:30 to 11:45 min, with primer set 1 providing the lowest Tp, and therefore chosen for assay optimisation (Supplementary Table S1). Different primer combinations and concentrations (FIP/BIP 0.8–2.0 µM, F3/B3: 0.2 µM, LF/LB: 0.4–2.0 µM) were assessed for performance utilising 0.5 ng/µL of synthetic standard as the template. The primer combination and concentration in bold were chosen for further assay performance testing due to providing the fastest Tp (Table II). F3 and B3 outer primers remained at a constant concentration of 0.2 µM throughout the study as concentrations > 0.2 µM have demonstrated no significant increase in amplification yield and speed (Foo et al. 2020; Gadkar et al. 2018).

3.2 Analytical performance of AFB-LAMP

Analytical sensitivity was determined by performing a ten-fold serial dilution of P. larvae GyrB standards. The AFB-LAMP assay reliably detected P. larvae starting DNA concentrations of 5 to 5 × 10−7 ng/µL in 2:48–8:51 min (Table V). Starting concentrations of P. larvae plasmid DNA 5 × 10−8 ng/µL or lower amplified intermittently, occasionally amplifying > 20 min. P. larvae plasmid DNA concentrations < 5 × 10−7 ng/µL generated low inter- and intra-assay variation across ten separate AFB-LAMP runs determined by the coefficient of variation (CV%) above cutoff values of 15% and 10%, respectively (Table V). Therefore, 5 × 10−7 ng/µL of P. larvae plasmid DNA was considered the limit of detection (LoD).

Table V Plasmid concentrations of P. larvae controls, inter-assay, and intra-assay coefficient of variation (CV%) for P. larvaeusing the AFB-LAMP assay. Ten-fold serial dilution of P. larvae control DNA was used to establish the limit of detection and inter- and intra-assay coefficient of variations (CV%), suggesting high sensitivity and repeatability of the LAMP assay. The standard deviation of the Tp is given in brackets
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Assay specificity was tested both by in silico analysis and using PCR on AFB-free honey bees. Individual primers did show binding to other species; however, only the entire set of primers was 100% specific to P. larvae (Supplementary Table S2). In addition, no amplification was observed in all bees free from AFB (n = 6), suggesting high specificity (%) due to no observed amplification (Figure 1).

Figure 1.
figure 1

Amplification curve and anneal derivative observed for gDNA extracted from AFB uninfected adult honey bees used in AFB-LAMP. No amplification curves produced from 5.0 ng/µL of PCR confirmed AFB-free honey bees represent no amplification of non-target DNA by AFB-LAMP (A). A single amplification curve was generated using P. larvae control DNA (red) in AFB-LAMP (A). The corresponding anneal peak represents a single product of the positive control, with no anneal peaks generated for uninfected honey bees (B). In duplicate assays, the solid lines represent the 1st repeat, dashed for the 2nd repeat.

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3.3 Performance of AFB-LAMP on field samples

To verify the ability of the designed AFB-LAMP assay to detect P. larvae-infected honey bees, gDNA was extracted from potentially AFB-infected honey bees. AFB-diseased honey bees were reliably detected by the AFB-LAMP assay in 5 out of the 6 individual adult bees returning Tp’s < 20 min (Table VI). Sample two returned a Tp greater than 20 min (28:45 min) and was subsequently considered negative for AFB. PCR and Sanger sequencing was also performed to verify the infection status of the tested bees with 16 s PCR primers from previous literature (Ribani et al. 2020), confirming all six individual honey bees were positive for AFB (Supplementary Figure S1).

Table VI The amplification (Tp) and melting temperatures (Tm) of purifiedP. larvaebacterial DNA extracted from PCR confirmed AFB-positive honey bees on AFB-LAMP assay. The positive control indicated by + ve control is 5.0 ng/µL of P. larvae control DNA. The ( −) is indicative of no Tp or Tm recorded on the Genie II fluorometer (OptiGene) with standard deviation in brackets. Samples with only one duplicate sample amplification resulted in no standard deviation
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3.4 Optimisation of in-field gDNA extraction

To utilise the developed AFB-LAMP assay in the field, a field-suitable gDNA extraction method was developed and optimised. Individual AFB diseased honey bees were lysed by manual bead beating in the presence of three lysis buffers (Table IV). The crude gDNA extracts were diluted from neat solutions to 1/10, 1/20, 1/50, and 1/100 and the presence of P. larvae was tested by LAMP. Of all the dilutions assessed, the 1/10 and 1/100 dilutions of crude honey bee gDNA in lysis buffer 3 (KOH) produced the fastest Tp at 10 and 9.30 min, respectively, with the 1/10 dilution chosen throughout the study for subsequent LAMP and PCR reactions (Figure 2).

Figure 2.
figure 2

Amplification curves and anneal derivative observed for crudeP. larvae gDNA dilutions extracted from an individual AFB-infected honey bee in 3 different common extraction buffers. Amplification curves were produced for different serial dilutions of individual AFB diseased honey bees in gram + ve lysis buffer (A) and KOH (C) and their corresponding anneal peaks (B and D). No amplification curves were generated for AFB diseased bees in PBS + EDTA lysis buffer (E) other than the positive control using P. larvae control DNA (red) and their corresponding anneal peaks (F). In duplicate assays, the solid lines represent the 1st repeat, dashed for the 2nd repeat.

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3.5 Performance of field-optimised AFB-LAMP

To trial the field application of the AFB-LAMP, 183 individual honey bee samples were collected from three different Victorian apiaries with unknown infection status. Genomic DNA was extracted utilising the in-field method and tested by AFB-LAMP. Of 183 samples analysed, the AFB-LAMP assay detected P. larvae in 62 out of the 64 PCR-confirmed AFB-positive bees with Tp’s ranging from 5.45 to 17 min and Tm’s ranging from 84.2 to 84.9 °C with one sample amplifying later than 20 min. The remaining 119 individual bees tested were AFB-negative on LAMP and PCR and thus were determined to be true AFB-negative samples (Table VII). The pilot field study gave an overall AFB-LAMP DSe of 96.88% (62/64) and DSp of 98.32% (2/119) suggesting that AFB-LAMP assay can identify P. larvae infections in the field.

Table VII Comparison of P. larvae detection between AFB-LAMP and conventional PCR on 183 field-optimised extraction samples
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4 Discussion

Primarily, P. larvae detection relies on the subjective expertise of the beekeeper to correctly identify diseased hives, which can be extremely difficult at the sub-clinical stage of infection or for in-experienced beekeepers and may result in misdiagnosis. For example, other brood diseases such as Sacbrood, Chalkbrood, and EFB present similar clinical symptoms in early spring when larvae are still capped, making early diagnosis very difficult for an experienced beekeeper and nearly impossible for a novice (Agriculture Victoria 2020). Therefore, testing for AFB when colonies or larvae are uncapped or symptomatic can inadvertently permit continued disease transmission amongst naïve colonies as asymptomatic or pre-symptomatic colonies have been identified to carry and transmit AFB (D’Alvise et al. 2019; Garrido-Bailón et al. 2013).

Loop-mediated isothermal amplification (LAMP) is a technique that fulfills this requirement of a rapid, highly sensitive, and specific field-applicable molecular diagnostic by detecting low concentrations of target nucleic acids utilising in-expensive and simple equipment. LAMP is able to provide a far more accurate representation of infection status than other field-based diagnostics such as lateral flow devices (LFD) as LAMP test multiple samples per run from an array of sources due to its robust nature ( (human urine) Fernández-Soto et al. 2019; (human stool) Francois et al. 2011; (environmental water) Moehling et al. 2020; (human saliva) Nagura-ikeda et al. 2020; (human blood) Phillips et al. 2019; (shrimp tissue) Sun et al. 2006). Several LAMP assays have already been developed for the detection of many honey bee pathogens including Nosema ceranae, N. apis, N. bombi, Aspergillus flavus, Sacbrood virus (Chinese and Korean), Melissococcus plutonius, Aethina tumida, and Vespa velutina nigrithorax (Cameron et al. 2021; Lannutti et al. 2020; Ma et al. 2011; Ponting et al. 2021). However, most LAMP assays designed and intended for field use have yet to progress to in-field applications due to limitations. Most LAMP assays designed are intended for field use, yet, a majority have not progressed to in-field testing due to a culmination of factors, one of the most important being due to a lack of appropriate sampling methods.

To address sample logistics challenges, this research introduced a proof-of-concept for a field-applicable technique to detect P. larvae in honey bee samples. The method showcased involves bead-based isolation of P. larvae DNA, capable of detecting as little as 5 × 10−7 ng/µL P. larvae DNA in under 10 min. There was no cross-reaction with non-target bacteria present in the gut microbiome of healthy or infected bees. The entire process, from sample preparation to amplification, takes less than an hour to yield results.

To date, there has only been one LAMP assay designed for the detection of P. larvae (Nguyen et al. 2011). This assay developed by Nguyen et al. targeted the Metalloproteinase gene amplifying P. larvae DNA utilising four main primer sequences (FIP, BIP, F3, and B3) with no additional loop primers (Nguyen et al. 2011). The presence of additional loop primers (LF and LB) utilised in this study may have increased amplification speed, exhibited by the difference in Tp’s between the two assays, with this paper able to report a positive detection in as little as 3 min compared to the 45 min reported in Nguyen et al. (Nagamine et al. 2002). In contrast to Nguyen et al., a type II DNA topoisomerase, DNA gyrase B gene (GyrB) was chosen as the target gene for amplification as the gene has shown to be a better molecular marker for taxonomic relatedness by possessing a greater nucleotide evolutionary divergence between bacterial species (Huang 1996; Slack et al. 2006; Subharat et al. 2011; Wang et al. 2007; Watt and Hickson 1994; Yamamoto and Harayama 1996).

Currently, there is only one commercially available field-deployable diagnostic, a lateral flow device (LFD) capable of detecting P. larvae from a single larvae sample via specific monoclonal antibodies. This AFB LFD was modelled on a previously developed and validated LFD for in-field detection of Melissococcus plutonius, the causative agent of European foulbrood (EFB) (Tomkies et al. 2009), now commercially marketed by the VITA (Europe) Ltd. The AFB LFD generates a positive result in less than 10 min indicated by the appearance of a coloured test line. While diagnostic sensitivity has not been reported for AFB LFD, the sensitivity and specificity of the LFD for EFB detection have been (Tomkies et al. 2009). Our AFB-LAMP assay has almost identical diagnostic sensitivity and specificity as the commercial EFB LFD test, reporting under laboratory conditions a 96.4–100% sensitivity and 100% specificity whilst in-field trials reporting a 96.8% sensitivity and 98.9% specificity compared to the AFB-LAMP DSe of 96.88% and DSp of 98.32% (Tomkies et al. 2009).

Secondly, the LFD is limited in the amount and types of samples to be processed, permitting one larval sample per test, omitting other, perhaps more accurate indicators of disease prevalence especially in asymptomatic or pre-symptomatic colonies (Kušar et al. 2021). Bee-related sample types such as hive debris, honey, and individual bees have all shown to contain AFB bacterial and spore contamination despite colonies being identified as asymptomatic which all samples can used in LAMP assays (Bassi et al. 2018; Erban et al. 2017; Kušar et al. 2021).

Bead-beating homogenisation is considered one of the most popular and traditional methods for protein or nucleic acid extraction from tissues, capable of mechanically disrupting the tissue matrix in a time-effective, efficient, and simple way without requiring extensive training or personnel to operate (Verollet 2008). It does this, by achieving a strong multi-directional force, mechanically grinding, hammering, and shearing tissues at great velocities for < 1 min utilising mineral, metallic, or even glass beads with varying diameters in an enclosed container with high throughput and success (Kido 2013).

Bead-beating homogenisation has demonstrated successful mechanical DNA extraction from whole honey bees and abdomens for mostly bee gut microbiome analysis (Bridson et al. 2022; Figueroa et al. 2021; Liberti et al. 2022) and pathogen surveillance (Tsevegmid et al. 2016). However, most honey bee papers published utilising bead-beating for DNA extraction are completed under aseptic conditions and require more vigorous and extensive sample purification steps for downstream applications. No papers to date have adopted a bead-beating approach in-field for extracting DNA from bee-related samples other compare to the now commercially available LFD device (Tomkies et al. 2009).

The combination of bead-beating mechanical lysis and KOH chemical DNA lysis buffer with a simple dilution method produced the best results for amplification on LAMP and PCR than other lysis buffers utilised. These results align with previous papers utilising KOH as a lysis buffer with high success (Cheng et al. 2015; Colombo et al. 2020; Sun et al. 2014), while the omission of EDTA from the lysis buffer did impact DNA extraction. Differing from previous work (Tomkies et al. 2009), individual honey bees were utilised instead of larvae as the bees themselves have been shown to be good indicators of overall colony health as they act as inadvertent reservoirs and carriers of many diseases affecting apiculture (Forsgren and Laugen 2014; Garrido-Bailón et al. 2013; Lindström and Fries 2005). Further development of this assay will facilitate rapid on-site detection and prevention of a highly transmissible and destructive disease for both commercial and hobbyist apiarists.

5 Conclusion

We have developed a rapid, highly sensitive, and specific loop-mediated isothermal amplification (LAMP) assay for the detection of Paenibacillus larvae (P. larvae), the etiological agent responsible for American foulbrood, a bacterial brood disease inadvertently spread by adult bees. Our assay has demonstrated its efficacy in both controlled laboratory settings and field conditions. We envision the utilisation of the AFB-LAMP assay within apiaries, enabling the detection of sub-clinical P. larvae infections. This advancement holds significant promise for enhancing disease management strategies by providing beekeepers with accurate and timely diagnostic tools; our assay stands to facilitate informed decision-making, ensuring the robust health and productivity of bee colonies.