DEVELOPMENT OF SIMPLEX AND MULTIPLEX DETECTION FOR THREE MAJOR PARASITES IN POULTRY BASED ON LOOP-MEDIATED ISOTHERMAL AMPLIFICATION COUPLED WITH A LATERAL FLOW DIPSTICK ASSAY AND MICROFLUIDIC CHIP TECHNOLOGY

Abstract

The study of morphological characteristics, conducted through post-mortem and parasite egg examinations in host’s digestive tract and feces, is a standard set of methods for the diagnosis of gastrointestinal helminth infections. However, these methods require parasitological knowledge and technical skills for species identification, making large-scale sample analysis time-consuming. This study sought to develop the molecular assays using loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) and microfluidic chip technology for the simplex and multiplex detections of three major parasitic groups in poultry: Raillietina (Raillietina echinobothrida, R. tetragona, and R. cesticillus), Ascaridia galli, and Echinostomatidae (Echinostoma miyagawai, E. mekongi, E. macrorchis, and Hypoderaeum conoideum). Seven assays were developed consisting of three simplex LAMP-LFD assays, three duplex LAMP-LFD assays, and one colorimetric LAMP on a microfluidic chip coupled with chromatic analysis. All assays exhibited high specificity in detecting the target species without cross-amplification. Regarding the analytical sensitivities, they showed minimum detectable DNA concentrations ranging from 5×10-2 to 5×10-4 ng/reaction for the LAMP-LFD assays and 40×10-1 to 40×10-3 ng/chip for the colorimetric LAMP on a microfluidic chip. To evaluate the diagnostic performance of each assay, 30 fecal samples were examined. These assays successfully detected parasitic infections in fecal samples, with clinical sensitivities ranging from 66.67% to 100% and clinical specificities from 89.29% to 100%, showing agreement from low to high levels of agreement (K= 0.35-0.93). Moreover, McNemar’s tests indicated that all developed assays were not significantly different when compared to post-mortem examinations. Therefore, these detection tools could serve as a viable alternative assays for a variety of purposes and guide decision-making regarding the use of anthelmintic drugs for treatments and farm management in the future.-