Development and utilization of a visual loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay for rapid detection of Echinostomatidae metacercaria in edible snail samples.

Trematodes belonging to the family Echinostomatidae are food-borne parasites which cause echinostomiasis in animals and humans. This is a global public health issue, particularly in East and Southeast Asia. A method to detect the infective stage of Echinostomatidae species is required to prevent transmission to humans. In this study, a loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay was developed for visual detection of the metacercarial stage in edible snails of the genus Filopaludina from local markets in Thailand. The LAMP-LFD method can be performed within 70 min at a consistent temperature of 66 °C, and the results can be interpreted with the naked eye. The detection limits of the assay using Echinostoma mekongi, E. macrorchis, E. miyagawai and Hypoderaeum conoideum genomic DNA were equal between the four species at 50 pg/µL. A specificity evaluation demonstrated that the LAMP-LFD assay had no cross-reaction with another parasite (Thapariella species) or with the snail host species (Filopaludina martensi martensi, F. sumatrensis speciosa, and F. s. polygramma). Clinical test assessments were compared to microscopic examination in 110 edible snail samples. The clinical sensitivity and specificity of the tests were 84.62 % and 100 %, respectively, with a strong level of agreement based on the kappa statistic and the results of both methods were not significantly different (p > 0.05) per McNemar's test. The test successfully developed in this study may be useful for the detection of the metacercarial stage in edible snails for epidemiological investigations, control, surveillance, and to prevent future echinostomiasis health issues.

Assay for the simultaneous detection of Raillietina spp. (R. echinobothrida, R. tetragona, and R. cesticillus) and Ascaridia galli infection in chickens using duplex loop-mediated isothermal amplification integrated with a lateral flow dipstick assay.

Raillietina species and Ascaridia galli are two of the significant intestinal parasites that affect chickens in a free-range system production. They destroy the intestinal mucosa layer, leading to several clinical symptoms such as weight loss, a slowed growth rate, and economic value loss. Thus, the objective of this study was to develop an assay for simultaneously detecting Raillietina spp. (R. echinobothrida, R. tetragona, and R. cesticillus) and A. galli in a single reaction using duplex loop-mediated isothermal amplification (dLAMP) coupled with a lateral flow dipstick (LFD) assay. The analytical specificity of the dLAMP-LFD assay showed a high specific amplification of Raillietina spp. and A. galli without non-target amplification. Regarding the analytical sensitivity, this approach was capable of simultaneously detecting concentrations as low as 5 pg/µL of mixed-targets. To evaluate the efficiency of the dLAMP assay, 30 faecal samples of chickens were verified and compared through microscopic examination. The dLAMP-LFD assay and microscopic examination results showed kappa values of Raillietina spp. and A. galli with moderate (K= 0.615) to high (K= 1) agreements, respectively, while the McNemar's test indicated that the efficiency between assays was not significantly different. Therefore, the developed dLAMP-LFD assay can be used as an alternative screening method to the existing classical method for epidemiological investigation, epidemic control, and farm management, as well as for addressing poultry health problems.

Visualization and development of colorimetric loop-mediated isothermal amplification for the rapid detection and diagnosis of paramphistome infection: colorimetric PAR-LAMP.

Colorimetric detection can be applied to differentiate between positive and negative conditions. It can be coupled with loop-mediated isothermal amplification to diagnose rumen fluke or paramphistome infection, also called colorimetric PAR-LAMP. This study conducted LAMP using three candidate indicator dyes, namely malachite green (MLG), methyl green (MTG), and neutral red (NTR), and the results were observed by the naked eye. The dye concentration was optimized to obtain the most pronounced positive-negative result discrimination. Subsequently, we conducted target sensitivity tests using the DNA of Fischoederius elongatus at different concentrations. To validate the detection accuracy, the result was confirmed by gel electrophoresis. The sensitivity test presented the lowest detectable DNA concentration or limit of detection (LOD), with 1 pg for MLG, 0.5 ng for MTG, and 50 pg for NTR. Different LODs revealed inhibition of LAMP reaction and reduced efficiency of result presentation for colorimetric-based detection, particularly NTR and MTG. For MLG-LAMP, we observed no cross-reaction of non-target DNA and improved reaction with the DNA of Fischoederius cobboldi and Calicophoron sp., with multi-detection. In addition, naked eye observation and agarose gel electrophoresis (AGE) evaluation of the MLG-LAMP results showed a moderate and strong agreement with LAMP-AGE and microscopic examinations. Based on our results, colorimetric PAR-LAMP is a rapid, comfortable, and point-of-care procedure for the diagnosis of paramphistome infection.

Evaluation of semi-quantitative colorimetric assays based on loop-mediated isothermal amplification indicators by using image analysis.

Colorimetric assays are some of the most convenient detection methods, creating discoloration in solutions that is visible to the naked eye. However, colorimetric reactions have some limitations regarding the variability in the color perception of individuals caused by factors such as color blindness, experience, and gender. Semi-quantitative chromatic analysis has been used as an alternative method to differentiate between two colors and accurately interpret the results from a numerical value, with high confidence. Therefore, we developed and determined the optimal model between Red-Green-Blue (RGB) and Commission Internationale de l'Eclairage (CIE) Lab color spaces to establish a semi-quantitative colorimetric assay via image analysis by the ImageJ program for loop-mediated isothermal amplification (LAMP), using the dyes malachite green and phenol red. The semi-quantitative colorimetric assays using the color distance values of the CIELab color space (ΔEab) were more suitable than those using the RGB color space (ΔERGB) for chromatic differentiation between positive and negative reactions in both indicator dyes, demonstrating the feasibility of this assay to be applied in the detection of a wide range of pathogens and infectious diseases.

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.

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 HIGHLIGHTSThis 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.

High-performance triplex PCR detection of three tapeworm species belonging to the genus Raillietina in infected poultry.

Chickens and ducks are important sources of essential proteins and nutrition for global consumption, especially their eggs and meat. Tapeworm infections in chickens and ducks are the cause of serious poultry health and economic problems in the processing of livestock and food production systems. Raillietina are cosmopolitan in distribution and are possibly the most common tapeworm parasites. There are three important species regarding avian infection, with different pathogenicity, including Raillietina echinobothrida, R. tetragona, and R. cesticillus. Co-infection diagnosis of these tapeworms using morphological analysis can be performed, but this is time-consuming and complicated. Therefore, this study aimed to develop a triplex PCR for the detection and discrimination of three Raillietina species. The triplex PCR assay specifically amplified target DNAs with no inter-specific interference and produced a specific band for each species. According to the specificity test, there was no cross-amplification with the DNA template of related parasites and their hosts. The lowest detectable DNA concentrations were evaluated and provided sensitivities of 0.5 pg/µL for R. echinobothrida, 5 pg/µL for R. tetragona, 50 fg/µL for R. cesticillus, and 5 pg/µL for the combination of DNA from all three species. Simultaneous detection limits of egg capsules and gravid proglottids was also performed, with and without feces. The interference of feces in the reaction was related to a decrease in sensitivity, but simultaneous detection of three Raillietina species in amounts lower than one gravid proglottid and ten egg capsules was still successful. Thus, this study is the first triplex PCR assay for Raillietina detection and can be utilized as an alternative diagnostic tool for the detection and discrimination of R. echinobothrida, R. tetragona, and R. cesticillus infection in poultry through the verification of fecal specimens. In addition, it could improve the performance of specific treatments and promote veterinary healthcare.

Molecular detection of three intestinal cestode species (Raillietina echinobothrida, R. tetragona, R. cesticillus) from poultry in Thailand.

Cestodes belonging to the genus Raillietina are a major veterinary health problem affecting the poultry industry, particularly chickens (Gallus gallus domesticus) and ducks (Anas playtrhynchos domesticus). The traditional method for accurately detecting this cestode based on their morphological characteristics is rather difficult due to the large number of morphological similarities. Consequently, this study aimed to develop specific primers for R. echinobothrida, R. tetragona, and R. cesticillus detection that could be used to indicate epidemic areas for protection and infection control. Specific primers were manually designed based on the internal transcribed spacer 2 region and validated, establishing the optimal temperature, final concentration in PCR mixture, specificity, and sensitivity of each primer set. The results showed that the primers amplify specific species without cross-amplifying other parasites and hosts. The PCR products were about 473, 352, and 397 bp long for R. echinobothrida, R. tetragona, and R. cesticillus, respectively. The sensitivity test demonstrated that R. echinobothrida and R. cesticillus-specific primers detect a minimum of 5×10-2 ng DNA, while R. tetragona-specific primers detect a minimum of 0.5 ng genomic DNA. The specific primers successfully developed in this study might be useful for detecting cysticercoids in intermediate hosts or adult stages in poultry for epidemiological surveys, management and control of infection.RESEARCH HIGHLIGHTS This study established specific primers for Raillietina species detection.The ITS2 region is an effective molecular marker for Raillietina identification.

Novel high-performance detection of Raillietina echinobothrida, Raillietina tetragona, and Raillietina cesticillus using loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD).

Cestodes belonging to the genus Raillietina are a major veterinary health problem in the poultry industry, especially in chickens (Gallus gallus domesticus) and ducks (Anas playtrhynchos domesticus). In this study, loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay was established and validated for the detection of R. echinobothrida, R. tetragona, and R. cesticillus in one reaction. The LAMP-LFD assay can be completed in 75 min under isothermal conditions at 66 °C and the results can be obtained by observation with the naked eye. This assay was very specific and had no cross-amplification with other closely related parasites (Cotugnia sp., Diorchis formosensis, Fimbriaria sp., Echinostoma sp., E. miyagawai, Hypoderaeum conoideum, Prosthogonimus cuneatus, and Ascaridia galli) or their definitive hosts (G. g. domesticus, A. p. domesticus). The sensitivity of the LAMP-LFD assay was detected with three Raillietina species at 0.5 ng, which was enough for gravid proglottid DNA detection. The accuracy test showed that the LAMP-LFD assay demonstrated accurate verification results when compared to morphological results. This is a novel LAMP-LFD assay that is highly specific and sensitive for the detection of Raillietina species. It can be applied to detection for epidemiological investigations, monitoring programs, surveillance, control, and to solve veterinary health problems for the poultry industry in Raillietina endemic areas.