Simultaneous Detection and Differentiation of Four Eimeria Species in Chickens (E. tenella, E. maxima, E. necatrix, and E. acervulina) Using a Multiplex TaqMan-MGB qPCR Assay

Chicken coccidiosis is a major parasitic disease in poultry that causes substantial economic losses worldwide. The accurate and simultaneous diagnosis of mixed infections with multiple <i>Eimeria</i> species remains challenging using conventional methods. In this study, we developed a multiplex TaqMan-MGB qPCR assay targeting the ITS1 region for simultaneous detection and quantification of four major pathogenic species: <i>E. acervulina</i>, <i>E. necatrix</i>, <i>E. maxima</i>, and <i>E. tenella.</i> The assay exhibited high specificity without cross-reactivity and achieved a sensitivity 100-fold greater than conventional PCR, with detection limits ranging from 10¹ to 10² copies/μL. Standard curves demonstrated strong linearity (R² ≥ 0.95) and amplification efficiency (95-113%). Repeatability was robust, with intra- and inter-assay CVs below 2%. Applied to 165 clinical samples collected from poultry farms in Fujian Province, China (2022-2024), the assay detected an overall <i>Eimeria</i> infection rate of 93.3%, higher than conventional PCR (89.7%). Epidemiological analysis revealed <i>E. tenella</i> (78.8%, [130/165]) and <i>E. necatrix</i> (78.8%, [130/165]) were dominant, while mixed infections were frequent (83.3%, [136/154]), including dual (45.5%, [70/154]), triple (31.2%, [48/154]), and quadruple (11.7%, [18/154]) infections. This multiplex TaqMan-MGB qPCR provides a rapid, sensitive, and quantitative tool for detecting multiple <i>Eimeria</i> species, greatly improving diagnostic efficiency and supporting clinical diagnosis, surveillance, and control strategies.