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 Eimeria 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: E. acervulina, E. necatrix, E. maxima, and E. tenella. 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 Eimeria infection rate of 93.3%, higher than conventional PCR (89.7%). Epidemiological analysis revealed E. tenella (78.8%, [130/165]) and E. necatrix (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 Eimeria species, greatly improving diagnostic efficiency and supporting clinical diagnosis, surveillance, and control strategies.