At the end of the incubation period, each well was treated for 1 h at 56 °C with 180 μL of ATL buffer (Qiagen, Hilden, Germany) and 20 μL of proteinase K (Qiagen). DNA isolation was performed according to the instructions for the QIAamp DNA mini kit (Qiagen). Each sample was deposited in duplicate in the qPCR plate. The primers and TaqMan probe used for the real-time PCR were positioned inside a specific 452 bp C. parvum sequence reported by Fontaine and Guillot [24]. PCR reactions were carried out in a total volume of 25 μL containing 12.5 µL of iQ™ Supermix (Bio-Rad, Boissy-l’Aillerie, France), primers (forward, 5′-CGCTTCTCTAGCCTTTTCATGA-3′; reverse, 5′-CTTCACGTGTGTTTGCCAAT-3′) and probe (5′ FAM-CCAATCACAGAATCATCAGAATCGACTGGTATC-3′ BHQ1) at 0.4 μM and 0.1 μM (final concentrations), respectively, and 5 μL of DNA template. Assays were run on CFX96™ Thermal Cycler (Bio-Rad) using 96-well hard-shell qPCR plates (BioRad). Cycling conditions were as follows: 95 °C for 3 min, followed by 45 cycles (95 °C for 15 s, 60 °C for min). BioRad CFX Manager Software was used to analyze the amplification curves. The Cq values correspond to the quantification cycle. To evaluate parasite infectivity, the ΔCq was calculated. The ΔCq of a sample is obtained by subtracting the Cq of oocysts inoculated onto HCT-8-free well from the Cq of oocysts inoculated onto HCT-8 monolayer well: ΔCq = (Cqsamplew/o cells − Cqsamplew/cells) for each incubation time. The higher the ∆Cq, the more DNA copies are observed, and thus the more parasitic proliferation there is.

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