Serial dilutions of bovine viral diarrhea virus (BVDV) RNA, as indicated, were amplified and analyzed using the QuantiTect Virus Kit. The steep sigmoidal curves enable accurate C
T value determination, even at low template amounts (10 copies/μl).
[A] Detection of BVDV1 RNA.
[B] Detection of BVDV2 RNA.
NTC: No template control.|Viral RNA was diluted in serial fivefold dilutions and amplified in duplex with an internal control using the QuantiTect Virus Kit.
[A] Amplification plot of the viral targets.
[B] Amplification plot of the internal control.|The QuantiTect Virus Kit provides a simple procedure for quantitative, multiplex, real-time PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, Mg
2+, and
Taq DNA polymerase, even for difficult assays (e.g., assays in which the copy number of the target gene is much smaller than that for the reference gene).|
[A] NH
4+ ions prevent nonspecific primers from annealing to the template.
[B] Synthetic factor MP, an innovative PCR additive, increases the local concentration of primers and probes at the template. Together with K
+ and other cations, synthetic factor MP stabilizes specifically bound primers and probes, allowing efficient primer extension by HotStarTaq
Plus DNA Polymerase.|Viral RNA was diluted in serial fivefold dilutions and amplified in duplex with an internal control using the QuantiTect Virus Kit (see figure "
Reliable detection of viral RNA over a wide linear range") or kits from Suppliers A
II and I. The QuantiTect Virus Kit provided much higher sensitivity than the other reagents tested, enabling reliable analysis of unknown samples.
ND: Not detected after 50 PCR cycles.|Serial tenfold dilutions of RNA standards (in vitro transcribed RNA) were prepared using either QuantiTect Nucleic Acid Dilution Buffer or RNase-free water, as indicated. These dilutions were used as template in one-step RT-PCR either directly (0 test) or after storage for 2 or 4 weeks at -20°C. Using QuantiTect Nucleic Acid Dilution Buffer resulted in lower C
T values and improved stability of the standards.|