Quantitative real-time PCR assays for detection and quantification of all seven Eimeria species that infect the chicken

Real-time PCR-based quantification ofEimeriagenomes: a method to outweigh underestimation of genome numbers due to PCR inhibition

“…qPCR of the eimerian 5S rDNA has been used for total genome estimation of Eimeria, irrespective of species (Blake et al, 2006), and a variety of additional qPCR assays have been developed for species-specific quantitative identification of different Eimeria species (Blake et al, 2006;Morgan et al, 2009;Vrba et al, 2010). However, the advantage of this method is fully exploited only if truly quantitative estimations (or relative abundance) of each species are obtained (Morgan et al, 2009).…”

“…The advantages of these molecular methods are their reduced requirement for specialist parasitological knowledge, speed, specificity, discriminatory power, objectivity, high throughput and automation potential. Total genome numbers can be estimated using the multicopy 5S rDNA as target (Blake et al, 2006;Vrba et al, 2010) and oocyst numbers calculated using standard or microscopically determined sporulation percentages (Morgan et al, 2009). One of the disadvantages of these techniques in addition to the costs involved is their susceptibility to the presence of PCR inhibitors (Hartman et al, 2005;Sarfo et al, 2011).…”

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of <em>Eimeria</em> that Infect Chickens

“…Supplementary evidence can be gathered through microscopic detection of the environmentally resistant oocyst lifecycle stage in faecal or litter samples, although overlapping morphology can confound all but the expert 6,7 . Molecular alternatives using polymerase chain reaction (PCR), random amplification of polymorphic DNA PCR (RAPD-PCR) and quantitative PCR technologies have been available for up to 20 years [8][9][10] , but to date they have failed to become popular. Relative expense and the requirement for specialist laboratory equipment or processing have limited their uptake, despite the often subjective and technically demanding nature of the older pathology-and microscopybased approaches 10,11 .…”

“…Molecular alternatives using polymerase chain reaction (PCR), random amplification of polymorphic DNA PCR (RAPD-PCR) and quantitative PCR technologies have been available for up to 20 years [8][9][10] , but to date they have failed to become popular. Relative expense and the requirement for specialist laboratory equipment or processing have limited their uptake, despite the often subjective and technically demanding nature of the older pathology-and microscopybased approaches 10,11 . Such limitations can be exaggerated in many of the poorer regions of the world such as South East Asia, where the impact of coccidiosis on poverty may be proportionately greater 12 .…”

“…For this to be applicable each assay must be capable of detecting all strains which may be circulating within each region. While understanding of the genetic diversity prevailing among Eimeria species is limited 26 , the use of target sequences previously validated for use in quantitative PCR with strains from Africa, Asia, Europe and South America provides some evidence of conservation, supporting the utility of these LAMP assays around the world 10 .…”