Detection of four important Eimeria species by multiplex PCR in a single assay.
The oocysts of some of the recognized species of chicken coccidiosis are difficult to distinguish morphologically. Diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria species. This study reports a multiplex polymerase chain reaction (PCR) assay based on internal transcribed spacer-1 (ITS-1) for the simultaneous diagnosis of the Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix species, which infect domestic fowl. Primer pairs specific to each species were designed in order to generate a ladder of amplification products ranging from 20 to 25 bp, and a common optimum annealing temperature for these species was determined to be 52.5 °C. Sensitivity tests were performed for each species, showing a detection threshold of 1-5 pg. All the species were amplified homogeneously, and a homogenous band ladder was observed, indicating that the assay permitted the simultaneous detection of all the species in a single-tube reaction. In the phylogenic study, there was a clear species clustering, which was irrespective of geographical location, for all the ITS-1 sequences used. This multiplex PCR assay represents a rapid and potential cost-effective diagnostic method for the detection of some key Eimeria species that infect domestic fowl.
Laboratory of Veterinary Parasitology, College of Veterinary Medicine and Bio-safety Research Centre, Chonbuk National University, Jeonju 561-756, Republic of Korea. Electronic address: tick@chonbuk.ac.kr.
Myung-Jo
MJ
You
D000818:Animals; D002645:Chickens; D003048:Coccidiosis / Q000175:diagnosis / Q000453:epidemiology / Q000469:parasitology / Q000662:veterinary*; D003362:Cost-Benefit Analysis; D016054:DNA, Protozoan / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D004539:Eimeria / Q000235:genetics* / Q000302:isolation & purification*; D016786:Eimeria tenella / Q000235:genetics / Q000302:isolation & purification; D015994:Incidence; D060885:Multiplex Polymerase Chain Reaction / Q000191:economics / Q000662:veterinary*; D010802:Phylogeny; D011201:Poultry Diseases / Q000175:diagnosis* / Q000453:epidemiology / Q000469:parasitology; D056910:Republic of Korea / Q000453:epidemiology
D016054:DNA, Protozoan; D021903:DNA, Ribosomal Spacer
Detection; Eimeria; Multiplex PCR