Investigator Quantiplex Kit

The validation of Investigator Quantiplex Kit showed that it yielded robust and reproducible results within the normal range of conditions expected in forensic casework and human identity testing.The kit enables a significantly fasterPCRthan similar kits from other suppliers,yielding results in just 48 minuteson QIAGEN"s Rotor-Gene Q thermal cycler (see the figure "Superior reaction time").

The marker of the Investigator Quantiplex Kit has been validated and no copy-number variation has been detected between genders or across populations, making this marker an ideal real-time PCR target for DNA quantification (see the figure "No copy-number variation in the marker").

Furthermore, the assay provides sensitivity down to <1 pg/µl, with accurate quantification of 4.9 pg/µl, where the standard curve shows linearity (see the figure "The Investigator Quantiplex kit retains excellent sensitivity down to 1 pg/µl").

Human identification is commonly based on the analysis of short tandem repeats (STRs), single nucleotide polymorphisms (SNPs), or deletion insertion polymorphisms (DIPs), depending on the demands of an examination or on the sample quality. These multiplex assays are complex systems that require a defined range of template input.

The Investigator Quantiplex Kit contains reagents and a DNA polymerase for specific amplification of a 146 bp autosomal multi-copy region of the human genome and for direct detection of the specific PCR product on the Rotor-Gene Q or Applied Biosystems 7500 Real-Time PCR Systems. The proprietary multi-copy region was selected in order to give high sensitivity with high reliability within different genders and populations. The target is detected using the green channel on the Rotor-Gene Q or the FAM dye channel on Applied Biosystemsinstruments.

In addition, the Investigator Quantiplex Kit contains a balanced internal amplification control that is used to test successful amplification and detects PCR inhibitors. This heterologous amplification system is detected as a 200 bp internal control (IC) in the yellow channel on the Rotor-Gene Q or in the VIC dye channel on Applied Biosystemsinstruments.

Detection of amplification is performed using Scorpions primers and a novel, fast PCR chemistry. Scorpion primers are bifunctional molecules containing a PCR primer covalently linked to a probe (see thefigure "Scorpions primers and their function"). The fluorophore in this probe interacts with a quencher, also incorporated into the probe, which reduces fluorescence. During PCR, when the probe binds to the amplicon, the fluorophore and quencher become separated. This leads to an increase in fluorescence in the reaction tube.

The reaction chemistry was carefully optimized to further support the rapid mechanism. The novel PCR chemistry and enzyme, which were specifically developed for human identification, enable high sensitivity and speed. Furthermore, the specially developed PCR chemistry contains the patent-pending additive Q-Bond, which significantly reduces denaturation, annealing, and extension times.

The kit is a ready-to-use system for the detection and quantification of human DNA using real-time PCR. See the table "Technical specifications" for more information. A set of template files has been developed to streamline the instrument setup and the analysis of the results on the Rotor-Gene Q (see table "Template files available for the Rotor-Gene Q").

The Investigator Quantiplex Kit is for quantifying human DNA, and for determining if PCR inhibitors are present and if further sample purification is needed. It is suitable for:

• Forensic database and casework samples
• Paternity testing samples
• Disaster victim identification and anthropological work
• Other human identity testing applications