6× amplification buffer (supplied by the manufacturer) in a final volume of 25 μl reaction mixture. Thermocycling conditions were set at 96 °C for 5 min for initial denaturation, followed by 30 cycles of 1 min at 94 °C, 2 min at 65 °C and 90 s at 72 °C, with a final extension at 72 °C for 7 min. Once the above conditions were standardised for individual primer pairs, all the primer pairs were put together in a single 50 μl reaction mixture for single-tube multiplex PCR

with the same cycling conditions as described above. For two-tube multiplex Talazoparib datasheet PCR, amplifications were conducted separately in two tubes; tube 1 contained the primers for E. acervulina, E. brunetti and E. mitis while tube 2 contained primers for E. maxima, E. necatrix, E. praecox and E. tenella. All the conditions for PCR remained

as described above. The click here amplification of specific PCR products were checked by gel electrophoresis in 2% agarose gels stained with 0.5 μg/ml ethidium bromide. The results of Eimeria species detection for each assay were compared by Chi-square analysis using SPSS version 20 (IBM, US). Results were considered significant when p < 0.05. Triplicate environmental faecal samples were collected from 30 farms and examined microscopically to confirm the presence of Eimeria oocysts (10×/20×). Oocysts were purified, pooled per farm to standardise and split for parallel processing by (i) QIAamp DNA Stool kit, (ii) QIAamp DNA Stool kit plus faecal contamination

and (iii) phenol/chloroform. Using the Eimeria genus 18S rDNA assay 93% (28/30) of the samples processed using the QIAamp DNA Stool kit were PCR positive and 100% of the samples containing ≥5000 OPG at the beginning of the process were positive ( Table 1). The addition of faecal material reduced the PCR positive rate to 30% with only one of 17 samples Phosphoprotein phosphatase containing fewer than 20,000 oocysts found to be positive. Using phenol/chloroform extraction 77% (23/30) samples were PCR positive with a 100% success rate only occurring above 20,000 starting OPG. The protocol found to be most effective (QIAamp DNA Stool kit after oocyst flotation) was subsequently tested on a larger number of field samples to investigate diagnostic sensitivity. In total 139 farms were visited, of which 100 were positive (71.9%) for Eimeria oocysts by microscopic examination with OPG ranging from 0.2 × 103 to 191.3 × 103. All oocyst positive samples were processed. Using the Eimeria genus 18S rDNA assay 96% (96/100) of the samples were PCR positive and 100% of samples containing ≥5000 OPG were positive ( Table 2). Sensitivity dropped below 80% only when samples containing fewer than 500 OPG were processed, although the number of samples tested at this level was very small. Out of 45 poultry farms screened in North India, 37 (82.2%) were positive for Eimeria spp. by microscopic examination with OPG ranging from 0.1 × 103 to 242.5 × 103.