title: Real-time PCR-based quantification of Eimeria genomes: a method to outweigh underestimation of genome numbers due to PCR inhibition.
abstract: Eimeria species parasites can cause the disease coccidiosis in all livestock species, most notably poultry. Traditional diagnostics such as faecal microscopy have now been supplemented by molecular assays including genus-specific and species-specific quantitative polymerase chain reaction (qPCR), although DNA extracted from faecal samples is commonly affected by PCR inhibition. This was confirmed when genomic DNA extracted from chicken faeces inhibited the threshold cycle value of internal positive control (IPC) DNA amplification by 15.33%. Hence, the objective of the present study was to use IPC qPCR to determine PCR inhibition in a series of experimental samples and use the increase in IPC qPCR threshold cycle value as an individual (sample-specific) correction factor for an established 5S rDNA qPCR used to estimate total Eimeria genome numbers. IPC-corrected genome counts were correlated with conventional oocyst per gram counts and compared with non-corrected counts, revealing a 0.1769 increase in correlation coefficient to outweigh underestimation of oocyst counts. Though the sample size used in this study is small, this limitation would be offset by the sample-specific correction factor determined using the IPC along with each sample.
authors: Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai 600 007, India. dhinakarraj@yahoo.com
authors: G Dhinakar
authors: GD
authors: Raj
authors: S
authors: S
authors: Aarthi
authors: R
authors: R
authors: Selvabharathi
authors: M
authors: M
authors: Raman
authors: D P
authors: DP
authors: Blake
authors: F M
authors: FM
authors: Tomley
mesh_terms: D000818:Animals; D002452:Cell Count / Q000662:veterinary; D003048:Coccidiosis / Q000662:veterinary*; D004539:Eimeria / Q000235:genetics*; D005243:Feces / Q000737:chemistry; D018503:Genome, Protozoan / Q000235:genetics*; D008853:Microscopy / Q000662:veterinary; D033661:Oocysts; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011201:Poultry Diseases / Q000469:parasitology*; D012341:RNA, Ribosomal, 5S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D013045:Species Specificity
chemical_list: D012341:RNA, Ribosomal, 5S