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.