Feasibility of a DNA biosensor assay based on loop-mediated isothermal amplification combined with a lateral flow dipstick assay for the visual detection of Ascaridia galli eggs in faecal samples

ABSTRACT Ascaridia galli is an important nematode that causes ascaridiasis in free-range and indoor system chicken farms. Infection with A. galli may damage the intestinal mucosa and inhibit nutrient absorption, leading to a reduced growth rate, weight loss and a decreased egg production. Consequently, A. galli infection is a significant health problem in chickens. In this study, we developed a loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay for the visual detection of A. galli eggs in faecal samples. The LAMP-LFD assay consists of six primers and one DNA probe that recognize the internal transcribed spacer 2 (ITS2) region; it can be performed within 70 min and the results can be interpreted with the naked eye. Using the LAMP-LFD assay developed in this study, A. galli DNA was specifically amplified without any cross-reactions with other related parasites (Heterakis gallinarum, Raillietina echinobothrida, R. tetragona, R. cesticillus, Cotugnia sp., Echinostoma miyagawai) and definitive hosts (Gallus gallus domesticus, Anas platyrhynchos domesticus). The minimum detectable DNA concentration was 5 pg/μl, and the detectable egg count was 50 eggs per reaction. The assay can be performed in a water bath, without the need for post-mortem morphological investigations and laboratory instruments. It is therefore a viable alternative for the detection of A. galli in chicken faeces and can replace classical methods in field screening for epidemiological investigations, veterinary health and poultry farming management. 
 RESEARCH HIGHLIGHTS
 This is the first study using the LAMP-LFD assay for Ascaridia galli detection. The results can be observed by the naked eye. The developed assay can be used to detect Ascaridia galli eggs in faecal samples.


Introduction
Ascaridiasis is a parasitic disease caused by nematodes of the genus Ascaridia (Shane, 2005;Zada et al., 2015;McDougald, 2020). Among them, A. galli is one of the most common nematodes affecting poultry health and food production from organic free-range, conventional free-range (housing system) and indoor system chicken farming, with a cosmopolitan prevalence of 10.4-96.7% in Austria, Italy, Germany, the Netherlands, Tunisia, Nepal and Thailand (Slimane, 2016;Thapa et al., 2015;Grafl et al., 2017;Sharma et al., 2018;Subedi et al., 2018;Wuthijaree et al., 2019). Infection with A. galli may disrupt the intestinal mucosa and cause diarrhoea, anorexia, haemorrhages, blood loss, anaemia, inhibited nutrient absorption and obstruction of the intestinal lumen, leading to a reduced growth rate, weight loss and decreased egg production (Permin et al., 2006;Lalchhandama, 2010;Zada et al., 2015;Sharma et al., 2018;Biswas et al., 2021). Hence, a high-performance diagnostic method is essential for the control, prevention and treatment of A. galli infections in chickens.
Conventional detection methods can be used for direct identification and diagnosis, based on the morphological characteristics of adult or egg stages by post-mortem investigations and the examination of eggs in faeces or mucosal scraping from an intestine (Martín-Pacho et al., 2005;Permin et al., 2006;Zada et al., 2015;Zloch et al., 2021). However, the conventional methods require personnel with scientific abilities and professional skills to distinguish among eggs from A. galli and other co-infecting species (Tarbiat et al., 2021). In addition, the egg stage in faeces from the laying of mature female worms cannot be detected in early infection or male worm infection, which is a limitation of the faecal examination (Oladosu et al., 2022). To overcome this problem, molecular techniques have been used as an alternative method as they can detect DNA in tissue from the moulting of larvae during growth or specific antibody responses against A. galli antigens in blood before the presence of eggs in faeces (Marcos-Atxutegi et al., 2009). Several recent studies have indicated that molecular techniques can be applied to detect A. galli based on DNA amplification, including specific primer PCR (Qazaz, 2020;Biswas et al., 2021;Watcharakranjanaporn et al., 2021) and droplet digital PCR (ddPCR) (Tarbiat et al., 2021). Nevertheless, these methods require unmovable instrumentation and gel electrophoresis analysis, which is a limitation for laboratories with a lack of specialized equipment and point-of-care screening (Sriworarat et al., 2015;Wang et al., 2019).
To assess the applicability of molecular methods in field detection, a loop-mediated isothermal amplification (LAMP) principle uses at least two pairs of primers that specifically recognize six regions on the target DNA under only one constant temperature, depending on the strand displacement activity of Bst DNA polymerase (Notomi et al., 2000). Efficiency has been improved by adding an optional primer pair (loop F, loop B) to enhance the amplification of the quantity of the DNA produced during LAMP (Nagamine et al., 2002;Niessen & Vogel, 2010;Tang et al., 2011;Shang et al., 2020). Also, LAMP can be performed using a simple device, such as a water bath or a portable heater, without an expensive thermal cycler machine, and the amplified product can be verified after combination with post-amplification analysis techniques, namely agarose gel electrophoresis, SYBR Green I fluorescence examination and turbidimetry (Iseki et al., 2007;Tang et al., 2011;Zhao et al., 2017). However, these techniques cannot differentiate between specific and nonspecific products. To solve this problem, restriction enzyme digestion was used for specific product confirmation, although this approach is time-consuming (Tang et al., 2011).
As an alternative technique, a lateral flow dipstick (LFD) assay can be integrated with LAMP to detect specific LAMP products via specific probe hybridization. Furthermore, this technique allows observations with the naked eye, without laboratory equipment (online supplementary Figure S1); it is simpler, more rapid and more accurate than the techniques mentioned above. The LAMP-LFD assay has been used to detect pathogenic parasites such as Babesia spp., Meloidogyne spp., Raillietina spp., Toxoplasma gondii, Paragonimus westermani and Plasmodium falciparum (Niu et al., 2011;Yang et al., 2016;Yongkiettrakul et al., 2017;Lalle et al., 2018;Xunhui et al., 2019;Panich et al., 2021;Xue et al., 2021). However, A. galli detection using LAMP-LFD has, so far, not been reported.
The aim of this study was to develop a novel LAMP-LFD assay for A. galli detection to easily interpret the results using the naked eye. Moreover, the LAMP-LFD assay was used to diagnose A. galli infection via faecal samples of chickens, using a simple water bath. This method may be used as an alternative assay in epidemiological investigations, control and risk reduction regarding A. galli epidemics in chickens.

Parasite collection and morphological identification
Adults of A. galli and other related parasite species were obtained from the intestines of chickens (Gallus gallus domesticus) purchased from local markets in Bangkok, Chumphon and Maha Sarakham Provinces, Thailand. Specimens were examined under a stereomicroscope (Nikon SMZ445, Japan), fixed in 70% ethanol for morphological identification, and preserved in absolute ethanol for DNA extraction. For morphological identification, the fixed specimens were identified to the species level using taxonomic keys (Tanveer et al., 2015;Butboonchoo et al., 2016;McDougald, 2020).

DNA extraction and molecular confirmation
Total genomic DNA was extracted using a GF-1 Tissue DNA Extraction Kit (Vivantis, Shah Alam, Malaysia), according to the manufacturer's instructions. Briefly, this kit relies on proteinase K as a main chemical reagent for cell lysis and a mini spin column to purify DNA. After extraction, the DNA concentration was measured using a NanoDrop Lite spectrophotometer (Thermo Scientific, Waltham, MA, USA) and adjusted to a final concentration of 5 ng/μl with deionized water. Finally, the extracted DNA samples were stored at −20°C until further use. For molecular confirmation, the internal transcribed spacer 2 (ITS2) regions of A. galli (n = 6) were amplified using primers ITS3 (5 ′ -GCATCGATGAAGAACGCAGC-3 ′ ) and ITS4 (5 ′ -TCCTCCGCTTATTGATATGC-3 ′ ) (Barber et al., 2000). The PCR products were separated and estimated using 1.5% agarose gel electrophoresis in TBE buffer (Tris base, boric acid, EDTA, pH 8, 0.5 M), with in-gel staining using ViSafe Green (Vivantis). The PCR products (approximately 450 bp) were sequenced and compared with sequences published in the GenBank database for molecular confirmation. The DNA sequences of A. galli were submitted to the GenBank database (accession numbers ON024768-ON024773).

LAMP primer and probe design
The A. galli LAMP primers and DNA probe were manually designed using our ITS2 sequences (representative sequences: ON024768-ON024773), which were aligned with other sequences from the GenBank database, ). Details of the LAMP primers are shown in Table 1.

LAMP reaction optimization
The LAMP assay was performed as previously described by Notomi et al. (2000). The LAMP reaction mixtures contained 1x isothermal amplification buffer (New England Biolabs, Ipswich, MA, USA), comprising 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 6 mM MgSO 4 , 0.1% v/v Tween 20, pH 8.8 at 25°C, 0.2 μM each of F3 and B3 primers, 0.8 μM each of loop F and loop B primers, 1.6 μM each of FIP and BIP, primers 1.4 mM of each dNTP, 1.0 M betaine (Sigma-Aldrich, Burlington, MA, USA), 1 μl of template DNA and 4U Bst DNA polymerase (New England Biolabs). Each reaction mixture was adjusted to a total volume of 12.5 μl with deionized water. To investigate the optimal conditions for LAMP amplification, gradient temperatures were initially set at 60-66°C. Subsequently, the LAMP reaction was optimized using various amplification times of 10, 20, 30, 40, 50 and 60 min. The LAMP reactions were performed in a thermal cycler machine (Analytik Jena AG, Jena, Germany) and selected according to the strong ladder-like pattern. To preliminarily confirm the target LAMP products, 1 μl of the LAMP product was digested with DraI enzyme (Vivantis) at 37°C for 180 min and separated using 1.5% agarose gel electrophoresis.

LFD development
The FIP primer and DNA probe were conjugated with biotin and carboxyfluorescein (FAM) at the 5' end, respectively. The quantity of the FAM-probe and the incubation time were varied to determine the optimal hybridization condition. For the hybridization procedure, the optimal FAM-probe concentration was added to the LAMP products, followed by incubation at 66°C for the appropriate duration. After hybridization, 8 μl of hybridized LAMP products were mixed with 90 μl of the assay buffer in a 1.5-ml tube and dipped with the LFD strip for 5 min (Milenia Genline HybriDetect, Giessen, Germany). For result interpretation, the presence of test and control lines indicated a positive result, whereas the presence of only the control line demonstrated a negative result ( Figure S1).

Analytical specificity and sensitivity of the developed LAMP-LFD assay
To evaluate the analytical specificity of the LAMP-LFD assay, genomic DNAs of A. galli and eight other species (parasites: H. gallinarum, R. echinobothrida, R. tetragona, R. cesticillus, Cotugnia sp. and E. miyagawai; hosts: G. g. domesticus and Anas platyrhynchos domesticus) were used. The analytical sensitivity of the assay was tested with the initial 5 ng of A. galli DNA and diluted 10-fold with serial dilutions (5-5 × 10 −7 ng/μl) to determine the lowest detectable concentration. As a control, the LAMP products were analysed by 1.5% agarose gel electrophoresis and visualized using a gel documentation system (Ingenius, Syngene, Frederick, MD, USA) before visual inspection of LFD development.

Detection limit in the egg stage
Different numbers of non-embryonated eggs (1, 10, 20, 30, 40, 50, 100, 150 and 200 eggs) from the uterus of female A. galli were extracted using the same extraction kit as mentioned above and verified under the optimal conditions described above to assess the detection limits of the LAMP-LFD assay.

Faecal sample validation
To assess the efficacy with clinical samples, faeces from the cloaca of dead chickens (n = 18) were individually collected (approximately 0.25-0.42 g) to use with LAMP-LFD assay. To further confirm the infection status, the intestine of each chicken was longitudinally dissected and examined by direct macroscopic methods and under a stereomicroscope to confirm the presence or absence of adult worms. DNA from chicken faeces was extracted using a GF-1 Tissue DNA Extraction Kit as mentioned above and then  -AACGAGCTATTATAAAACGCTTTTATCA AGTAG-TTTT-TGTATGTGTATGTGCGTATGC  58  BIP-AG  CAGGTACTACAATTAGATGATGATGATGATG-TTTT-AGCATTAGAGCTGCATTCATG  56  Loop F-AG  TACAACGTAAACAAAC  16  Loop B-AG  TTTTGTTGCATATTTCTTT  19  Probe-AG  FAM-GCGTATGCGTTTGTTTACGTTG  22 tested with the LAMP-LFD assay using a water bath (as field detection protocol) at 65.7-66.4°C for 60 min and paraffin oil to prevent evaporation of the mixture. In addition, sensitivity and specificity of the LAMP-LFD assay were calculated following the formula of Lalkhen and McCluskey (2008).

Optimization of temperature and amplification time for the LAMP assay
Two parameters, temperature and amplification time, were optimized to achieve maximum amplification of the specific target regions. The results showed that the extent of amplification in the temperature range of 60-66°C was similar (data not shown). Thus, a temperature of 66°C was selected for the LAMP assay to determine the optimal amplification time. Regarding the LAMP amplification time, the ladder-like pattern products were first observed at 50 min, and were weakly patterned products; the strong pattern products peaked at 60 min (data not shown). Therefore, the optimal LAMP conditions were 66°C and 60 min. To further confirm the amplified target products, 1 μl of LAMP products yielded under the optimal conditions was digested using DraI enzyme, which produced one band of 123 bp (online supplementary Figure S2). Based on the results, amplification of specific target regions could be achieved using the LAMP assay.

Visualization of the LAMP-LFD assay
To determine the appropriate quantity of the FAMprobe, the FAM-probes at 2, 20 and 200 pmol were individually hybridized with the LAMP products at 66°C for 5 min. The results revealed that 2 pmol provided the most intense colour at the test line. Consequently, the FAM-probe at 2 pmol was chosen to validate the incubation time at 5, 10 and 15 min, with each incubation time differing slightly. Thus, FAM-probe at 2 pmol, an incubation temperature of 66°C and an incubation period of 5 min were the optimal hybridization conditions ( Figure 1) and used in the subsequent experimental steps.

Evaluation of the analytical specificity and sensitivity for the LAMP-LFD assay
The analytical specificity of the LAMP-LFD assay was tested with eight different species, including parasites and hosts. Based on the results, only A. galli DNA was amplified, without cross-reaction with other species (Figure 2). Regarding analytical sensitivity, LAMP-LFD was conducted using 5-5 × 10 −7 ng/μl of A. galli DNA; the lowest detectable concentration was 5 × 10 −3 ng/μl or 5 pg/μl (Figure 3). Moreover, the LFD interpretation based on observations with the naked eye was consistent with the gel electrophoresis analysis results. This indicates that the LAMP-LFD assay was highly specific for A. galli detection and yielded an analytic efficiency equal to that of the standard technique, but without the requirement for specialized laboratory devices.

Determination of the detection limit for the LAMP-LFD assay
The detection limit of A. galli eggs was determined using 1-200 eggs. The minimum egg number detectable by LFD verification was 50 eggs per reaction, which corresponded with the results using gel electrophoresis (Figure 4).

Application of the LAMP-LFD assay to faecal samples
The sensitivity and specificity of the LAMP-LFD assay was verified using faecal samples from chickens (n = 18). Overall, seven out of 18 samples were identified to be infected the adult worms of A. galli in the intestine using microscopic examination. Using the LAMP-LFD assay, we detected four out of seven samples that were positive by microscopic examination. The other three samples were false negatives, whereas false positives did not occur in the LAMP-LFD assay ( Figure 5, online supplementary Figure  S3). Consequently, the LAMP-LFD assay had clinical sensitivity and specificity of 70% and 100%, respectively, when applied to clinical samples.

Discussion
Ascaridia galli is an important intestinal roundworm with the highest prevalence in chickens (Tarbiat et al., 2021). The species has a simple and direct life cycle that does not require an intermediate host. Moreover, embryonated eggs can resist and survive for up to 66 weeks in the environment, making this species a dominant parasitic species in the poultry industry, including various production systems (Grafl et al., 2017;McDougald, 2020). Ascaridiasis can show as weight loss, reduction in growth and reduced egg production rates, leading to economic losses (Zada et al., 2015;Biswas et al., 2021). Therefore, the development of a simple, rapid and cost-effective diagnostic method is essential for screening, prevention and control in epidemic areas. Generally, the diagnosis of A. galli infection requires examination of the morphological characteristics of the adult stage in the intestine or of eggs in faeces under a light microscope, which relies on the expertise and training of the personnel and on the investigation period (Martín-Pacho et al., 2005;Permin et al., 2006;Zada et al., 2015). In this study, we developed a LAMP-LFD assay for A. galli detection to interpret the results by the naked eye without specialized instrumentation. Although the detection time (70 min) is longer than that of the conventional faecal egg count technique (i.e. Mini-FLOTAC) (Barda et al., 2013), our assay is easy to perform and more convenient when numerous samples are investigated simultaneously. Specific primers for PCR (Biswas et al., 2021) and ddPCR (Tarbiat et al., 2021) have been successfully established for A. galli detection based on the ITS region. This shows that the ITS region can be used to develop a molecular assay for A. galli. Hence, we designed LAMP primers targeting the ITS2 region for A. galli detection. Analytical specificity indicated that the LAMP primers based on the ITS2 region provided highly specific amplification, without any cross-reaction with other parasites and hosts. Regarding the LFD assay, a DNA probe was designed to recognize the unique region in biotinylated LAMP products, which facilitated the analysis of target LAMP products, making this technique superior to others, such as gel electrophoresis (Ji et al., 2020), turbidimetry (Mori et al., 2004), fluorescence measurement (Fan et al., 2018), melting temperature analysis (Tone et al., 2017) and colourimetric indicators (Thapa et al., 2019). Additionally, the visual inspection of the LFD assay is similar to that in the gel electrophoresis analysis, but without requiring costly laboratory instruments.
Regarding analytical sensitivity, the LAMP-LFD assay had a DNA detection limit of 5 pg/μl, which is better than that obtained in a previous study by Watcharakranjanaporn et al. (2021), where the minimum DNA detectable was 156.3 pg/μl from targeting the NADH dehydrogenase subunit 4 (ND 4) gene using the PCR technique. Furthermore, the detection limit of the LAMP-LFD assay was 50 eggs, which is more efficient than that described previously (Watcharakranjanaporn et al., 2021). The faecal egg-counting techniques, including Kato-Katz, Mini-FLOTAC and McMaster, can detect 10 eggs per gram of faeces (Barda et al., 2014;Cools et al., 2019). Nevertheless, several conventional methods (i.e. post-mortem detection, mucosal scraping, simple flotation and faecal egg counting) are not able to diagnose 100% of A. galli infection yet (Zloch et al., 2021). Previous studies have reported that an average quantity of 1207 eggs per gram of faeces, without clinical signs of infection, has no impact on chicken health, indicating the developed assay can be used in a low infection or a low worm burden (Gauly et al., 2005(Gauly et al., , 2007Feyera et al., 2022). Therefore, the developed LAMP-LFD assay is a new alternative method for A. galli detection or for the confirmation of diagnostic results on a molecular level.
We further validated the LAMP-LFD assay on faecal samples obtained from naturally-infected chickens by comparisons with a microscopic examination. The results revealed that the LAMP-LFD assay had a high level of specificity for A. galli detection because of the absence of false positive results. However, there were three false negatives in the LAMP-LFD assay (Figure

5).
To find the cause of the problem, the LAMP-LFD assay was used to test faecal samples spiked with varying numbers of A. galli egg quantity (50, 100, 150, 200, 250, 300, 350, 400, 450 eggs) in 0.25 g of uninfected individual chicken faeces (the minimum weight of faeces used in the faecal sample validation step) that were confirmed as being worm-free in the intestines by post-mortem examination of the intestines. Based on the results, 50 and 250 eggs could not be detected, and the intensity of the ladder-like pattern products was not in accordance with the gradually increased number of eggs (online supplementary Figure S4). This may be explained by the different inhibitors in the faeces, which lead to decreased efficacy of the LAMP amplification. Previous studies reported other factors that lead to false negative results, such as (i) the unstable distribution of eggs in faeces, (ii) earlystage infections with larvae, and (iii) male worm infection (Oladosu et al., 2022). To improve the efficiency of the LAMP-LFD assay, we suggest that the inhibitors in faeces should be reduced as much as possible, for example by using simple filtration via gauze before DNA extraction.
Our developed LAMP-LFD assay can deliver results within 70 min, using a simple water bath or portable heater instead of a thermal cycler machine to maintain the temperature. As the results can be observed with the naked eye, this approach does not need expensive and unportable equipment like the conventional PCR method and microscopic examination, making it feasible for use on-site or as point-of-care screening to guide the decision of anthelmintic treatment in birds. Although the LAMP-LFD assay is frequently applied to detect parasites, this is the first report of the detection of A. galli with this technique.

Conclusions
We successfully developed the first DNA biosensor using the LAMP-LFD assay for A. galli detection in faecal samples within 70 min, without preparation and DNA extraction. The LFD assay can be performed immediately after completion of the LAMP reactions without laboratory devices and the results can be observed with the naked eye. This assay may be used as an alternative method and may be applied in the field in context of epidemiological studies, surveillance, control and poultry farming management.

Ethical approval
All experimental methods related to birds were proceeded by the National Research Council of Thailand and approved by the Committee for Animal Care and Use Committee Srinakharinwirot University (License No. SWU-A-025-2562).

Funding
We considerably acknowledge Srinakharinwirot University, Thailand [grant number 209/2565], National Research Council of Thailand (NRCT) [grant number N24B650301], the Science Achievement Scholarship of Thailand (SAST) for providing research funding.