Quantitect probe rrt pcr kit

Viral RNA was extracted from 50 μL of BHI‐immersed swabs by using the Magmax AI/ND 96 Viral RNA kit (Ambion Inc., Austin, TX, USA), which was adapted for semi‐automated extraction using a Kingfisher magnetic particle processor (Thermo Fisher Scientific, Erembodegem, Belgium). Purified RNA was eluted in a final volume of 50 μL. Two microlitres of purified RNA were used as a template for the RRT‐PCR. The Quantitect Probe RRT‐PCR kit (QiagenGmBH, Hilden, Germany) was used for amplification using a Biosystems 7500 Real‐Time PCR Cycler (Applied Biosystems, Lennik, Belgium). RRT‐PCR was performed to amplify the viral M gene, which allowed for detection of all AI virus subtypes (Steensels et al. 2007; Van Borm et al. 2007). RRT‐PCR was run on known dilutions of in vitro synthetised M RNA to establish a standard curve (Van Borm et al. 2007). Quantification of M RNA copy number in each sample was based on the standard curve, and the M RNA copy number per millilitre of BHI‐immersed swabs was calculated.

The viral genomic RNAs of the selected Tracheal swabs were extracted using QiAamp Viral RNA Mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions, rRT-PCR was carried out by a commercial Quantitect Probe rRT-PCR kit (Qiagen, Inc., Valencia CA). The forward, reverse Primers and probe were used after (Wise et al., 2004 (link)). Real-time rRT-PCR was performed in the Stratagene 3005P MXpro Real-Time PCR System (Stratagene, USA). A triplicate of 8.5–10-fold dilutions of challenge NDV were used to generate a standard curve using stock virus dilutions. The point at which the curve crosses the horizontal threshold line is defined as Ct, we plotted virus log10 titers of a sample against the Ct value, where the best fit line was constructed. ND virus quantity in unknown samples was derived by planning the Ct of an unknown against the standard curve and were expressed in log10 EID50/mL equivalents.