To enable higher in-process safety through the correct interpretation of negative results, each QuantiNova Pathogen +IC Kit contains reagents for multiplex, real-time detection of user-defined pathogen targets with internal controls. Amplifying target and control genes in the same reaction increases reliability by minimizing handling errors. The supplied IC can be used to test for successful amplification (e.g., exclusion of PCR inhibitors) or added to the sample prior to nucleic acid purification to control both the efficiency of the purification process and the PCR or RT-PCR amplification. Real-time PCR and one-step RT-PCR can be run simultaneously using the same protocol. The kit also uses a two-phase hot-start procedure (see figure Principle of the novel QuantiNova two-phase hot-start mechanism
). This includes heat-mediated activation of both the HotStaRT-Script reverse transcriptase at 50°C and the PCR polymerase at 95°C. At low temperatures, the HotStaRT-Script forms a complex with an RT-Blocker molecule, leading to inactivation. At 50°C, the complex dissociates and the active HotStaRT-Script enzyme is released. The QuantiNova DNA Polymerase is kept in an inactive state by the QuantiNova Antibody and a novel additive, QuantiNova Guard, which stabilizes the complex. This improves the stringency of the hot-start procedure and prevents extension of non-specifically annealed primers and primer–dimers. Within 2 minutes of raising the temperature to 95°C, the QuantiNova Antibody and QuantiNova Guard are denatured and the QuantiNova DNA Polymerase is activated, enabling PCR amplification. This unique two-phase hot-start enables reaction setup to remain stable for at least 1 hour at room temperature, preventing the creation of artifacts and also facilitating automated reaction setup.
Additionally, the master mix supplied with the kit contains an inert blue dye that does not interfere with the reverse transcription or real-time PCR, but increases visibility in the tube or well. The QuantiNova Yellow Template Dilution Buffer contains an inert yellow dye. When the template nucleic acid, diluted with the QuantiNova Yellow Template Dilution Buffer, is added to the master mix, the color of the solution changes from blue to green, providing a visual indication of correct pipetting and reaction setup (see figure Accurate reaction setup indicated by the built-in pipetting control
). The master mix includes a balanced combination of K+
ions as well as the unique synthetic Factor MP, which together promote stable and efficient annealing of primers and probes to the nucleic acid template, enabling high PCR efficiency even in multiplex reactions (see figure Unique multiplex PCR buffer promotes stable and efficient annealing
). The specially developed PCR buffer contains Q-Bond, which significantly reduces denaturation, annealing and extension times (see figure Mechanism of fast cycling during annealing
). QuantiTect Nucleic Acid Dilution Buffer, supplied with the kit, stabilizes RNA and DNA standards during dilution and reaction setup and prevents loss of nucleic acids on plastic surfaces, such as tubes or pipet tips. The master mix can be stored at 2–8° C for up to 12 months, and the reaction is stable at room temperature for at least one hour, which is convenient for automated procedures.