Detecting Eimeria Species by qPCR

Eimeria species were identified by qPCR using specific primers targeting SCAR markers derived from RAPD fragments for E. acervulina, E. tenella, and E. necatrix, and microneme protein gene 1 for E. maxima [19 (link)]. Oocysts from positive fecal samples were isolated, purified, and concentrated with a saturated salt solution [20 ]. Then, oocysts were washed three times with distilled water by repeated centrifugation [21 (link)]. Genomic DNA was extracted from 0.25 mg fecal sample and 100 μL oocysts pellets using the Isolate Fecal DNA (Bioline) kit according to the manufacturer’s instructions. The qPCR reactions were performed in the CFX96 Touch™ Real-Time PCR detection system (Bio-Rad, London, UK) using IQ Multiplex Powermix (Bio-Rad, London, UK) in a final volume of 20 µL. The amplification conditions were similar to the conditions described by Blake et al. [19 (link)] as follows: 1 × initial denaturation at 95 °C for 20 s; 40 × 95 °C denaturation, 15 s, and primer hybridization combined with 60 °C extension, 30 s. Data were collected at the end of each cycle.