Abi prism r 7500 sequence detection system

The amoA gene’s abundance in AOA and AOB was quantified by real-time PCR using the Arch-amoAF/Arch-amoAR primers for the AOA amoA genes and amoA1F/amoA2R for the AOB amoA genes. All qPCR assays were performed in triplicate with an ABI PRISM R 7500 Sequence Detection System (Applied Biosystems, Waltham, MA, USA) using the SYBR Green I method. The 20 mL qPCR reaction system contained 10 mL FastStart Universal SYBR Green Master (ROX) (Roche, NJ, USA), 0.4 µL 10 µM each forward and reverse primer, 7.2 µL sterilized MilliQ water, and 2 µL standard or extracted soil DNA. Amplification was conducted in a LightCycler® 480 (Roche Applied Science, Penzberg, Germany) as follows: 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C, 45 s at 54 °C and 60 s at 72 °C for AOA, or 40 cycles of 15 s at 95 °C and 2 min at 58 °C for AOB. Standard melting curves were generated using 10-fold serial dilutions of a plasmid containing the AOA or AOB target gene inserts.

At 3 weeks of age, three chicks in each group were randomly chosen and euthanized, and tissues were collected, including the lung, weasand, heart, liver, thymus, spleen, and bursa of Fabricius, for RNA extraction (Omega Bio-Tek, Norcross, GA, USA) to determine the LaSota viral load in different organs according to previously published methods [24 (link)]. Meanwhile, genomic DNA was also extracted from these organs using a DNA extraction kit (Omega Bio-Tek) for the quantification of FAdV and CIAV viral load using published qPCR methods [25 , 26 (link)]. Anticoagulated blood was collected from all chicks at 10, 14, 21, 28, and 35 days post-hatching for DNA extraction to determine the viraemia level of FAdV and CIAV. To make the LaSota, FAdV and CIAV copy numbers in the above tissue conform, viral RNA or DNA concentration (log₁₀) was normalized per 1 µg of total RNA or DNA. The qPCR reactions were set up on ice, and an ABI PRISMR 7500 Sequence Detection System (Applied Biosystems, USA) was used to amplify and detect the reaction products. The qPCR was performed in duplicate, with each sample present in technical duplicate during each run.