QuantiNova Pathogen +IC Kit

The QuantiNova Pathogen +IC Kit enables simultaneous detection of viral RNA or DNA targets and the QuantiNova Internal Control (IC) over a wide linear range without loss of sensitivity when multiplexing. The protocol has been developed to run ultrafast cycling experiments with high reliability on most cyclers. Combined amplification of pathogen targets and the IC RNA or DNA increases in-process safety by ensuring the correct interpretation of negative results. The included IC can be used to monitor the nucleic acid purification and/or amplification process, while the visual pipetting control allows a visual check after manual or robotic pipetting (see figure Accurate reaction setup indicated by the built-in pipetting control). With the unique two-phase hot-start procedure it is possible to perform manual or automated reaction setup at room temperature without compromising performance (see figure Principle of the novel QuantiNova two-phase hot-start mechanism). The QuantiNova Pathogen +IC Kit is robust even in the presence of inhibitors and allows the use of degenerated primers/probes. The kit offers ultrafast, in-process controlled multiplex qPCR, increasing workflow efficiency and reliability.

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⁺ and NH₄⁺ 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.

The QuantiNova Pathogen +IC Kit provides a simple procedure for the detection of user-defined pathogen targets and the IC. It contains a ready-to-use master mix for simultaneous real-time detection of viral RNA (1-step RT-PCR) or viral, bacterial and fungal DNA (PCR). There is no need to optimize reaction and cycling conditions, simply mix the supplied master mix with the pathogen assay (primers and probe), the supplied Internal Control Assay and the IC DNA or RNA. Alternatively, the IC can be added to the sample purification procedure. Then add your sample DNA or RNA and start the reaction on any cycler. The kit handbook contains optimized protocols for use with TaqMan probes on a wide range of cyclers. The visual pipetting control minimizes human errors by allowing visual checks after manual or robotic pipetting. The unique two-phase hot-start procedure allows manual or automated reaction setup at room temperature without compromising performance. Each QuantiNova Pathogen +IC Kit includes the Internal Control Assay and IC DNA or RNA for use as an amplification control via direct addition to the reaction mix. Alternatively, the IC can be added to the purification procedure to control both the purification process and amplification. The Internal Control Assay provided in the kit uses MAX as a reporter dye. Alternatively, an Internal Control Assay labeled with a Cy5 analog is available as an accessory.

The QuantiNova Pathogen +IC Kit provides sensitive real-time PCR or one-step RT-PCR using sequence-specific probes for simultaneous detection of pathogen DNA and/or RNA, including an IC. The kit allows automated reaction setup at room temperature and can be used on a wide range of real-time cyclers.