QIAcuity OneStep Advanced EG Kit

• For dye-based RT-digital PCR reactions using EvaGreen
• HotStart RT feature enabling simultaneous Nanoplate setup for QIAcuity Four and Eight
• Reaction setup at room temperature thanks to HotStart RT feature
• 4x concentrated PCR Master Mix for loading more sample

The QIAcuity OneStep Advanced EG PCR Kit enables sensitive quantification of RNA or RNA+DNA targets in one combined RT-PCR reaction on the QIAcuity Digital PCR System. The kit contains a 4x concentrated Master Mix and a 100x concentrated RT Mix optimized for microfluidic use in the QIAcuity Nanoplates. The kit improves the specificity and efficiency of dye-based digital PCR to provide accurate quantification analysis. As an intercalating dye, EvaGreen binds to double-stranded DNA and enhances the quantitative accuracy of gDNA or cDNA measurements on the QIAcuity dPCR instruments.

The new kit is equipped with a HotStart Reverse Transcription (RT) Enzyme. Thanks to the RT Enzyme, you can work at room temperature to simultaneously set up your reaction and nanoplate for loading into QIAcuity Four or Eight instruments.

The kit works in combination with the QIAcuity Digital PCR System and the QIAcuity Nanoplates.
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Advanced reverse transcription performance

The new QIAcuity Advanced Reverse Transcription Mix includes a new patent-pending HotStart reverse transcription technology. The new HotStart RT enzyme is non-functional at room temperature and prevents the generation of off-target cDNA even when left to incubate at 37°C for many hours. With this enzyme, you can set up your reactions  at room temperature. Importantly, with the HotStart RT enzyme you can assemble and load multiple nanoplates in parallel, at ambient temperatures, without risking  unwanted and potentially unspecific RT activity. The HotStart RT enzyme is blocked while nanoplates remain inside the QIAcuity instrument before transfer to the thermal cycler, where the RT step of the protocol occurs.

Dye-based detection with EvaGreen

The special Master Mix in the QIAcuity OneStep EG PCR Kit enables accurate double-stranded DNA target amplification and quantification. The kit includes an optimized reference dye needed for counting analyzable partitions in Nanoplates and dPCR analysis. Moreover, EvaGreen provides a higher fluorescence signal than SYBR Green at the same concentrations and delivers maximum amplification efficiency, specificity and sensitivity in dPCR.

The QIAcuity OneStep Advanced EG Kit contains a ready-to-use 4x PCR Master Mix, eliminating the need to optimize reaction and cycling conditions. With the OneStep chemistry you can perform the RT and amplification for viral RNA of interest in a single step. In the OneStep reaction, RNA and DNA targets can also be detected together, for example, viral RNA with bacterial DNA.

The principle of the dPCR reaction in the nanoplates is described here.

The QIAcuity OneStep Advanced EG Kit contains a ready-to-use 4x PCR Master Mix, so you don’t need to optimize reaction and cycling conditions. Simply add the 100x RT mix, template RNA and primer-probe sets to the Master Mix and follow the procedure outlined in the Quick-Start Protocol to get fast and reliable results on the QIAcuity Digital PCR System. With the new HotStart RT enzyme, the parallel setup and simultaneous loading of multiple nanoplates into QIAcuity Four and Eight instruments are possible (see figure  Nanoplate setup and loading).

The QIAcuity OneStep Advanced Probe Kit, combined with the QIAcuity Digital PCR System and the QIAcuity Nanoplates, enables quantitative analysis of RNA targets for various applications, including viral RNA or mRNA detection. Applications that require a simultaneous detection of RNA and DNA targets are also possible, for example, in wastewater testing.