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Fast and Sensitive Detection of Viral Targets in Quantitative RT-PCR Using the Universal One-Step qRT-PCR Master (ROX)

Conny Falter, Alexander Bartes, Daniel Heisswolf, Erhard Fernholz, and Cordula Jany*

Roche Applied Science, Penzberg, Germany

*Corresponding autor: cordula.jany@roche.com

Introduction

For qRT-PCR assays it is a common procedure to perform two individual work steps. First, RNA is transcribed into cDNA, and then the target to be quantified is amplified separately. This two-step approach is believed to result in highest sensitivity of target detection. However, the sensitivity of nucleic acid amplification assays is determined by multiple factors and differs from target to target. In contrast, a one-step protocol in qRT-PCR benefits from short overall reaction times, shorter hands-on-times and low contamination risks. Here we show the highly sensitive quantitative detection of viral target RNA in real-time PCR to a copy number fewer than 10.

Materials and Methods

Real-time PCR instruments offer different modes of d detection, and in many cases the released signal is detected in relationship to the reference dye ROX. The new Universal One-Step qRT-PCR Master (ROX) is a ready-to-use mix for quantitative real-time RNA detection in hydrolysis probe format. Since it contains a special ROX the mix can be used without adjustments on a variety of real-time instruments such as ABI 7500 Real-Time PCR System, ABI 7500 Fast Real-Time PCR System, ABI 7900 HT Fast Real Time PCR System, ABI StepOnePlus Real-Time System, and Stratagene Mx3000P qPCR System.

In this study the Universal One-Step qRT-PCR Master (ROX)  was used to reversely transcribe and amplify sequences from hepatitis A virus (HAV) and influenza A virus. As a source of viral RNA, internally isolated HAV and influenza A virus standard RNA was used. HAV- and influenza A virus-specific primers had a final concentration of 0.5 µM each. The corresponding hydrolysis probes were used in a final concentration of 0.25 µM. The size of the detected amplicons was 246 nucleotides for HAV and 96 nucleotides for influenza A virus. A serial dilution from 105 to 1 copy per reaction was investigated as described in the instruction manual.

Results and Discussion

The sensitivity of the Universal One-Step qRT-PCR Master (ROX) was investigated using two different viral targets on two different real-time instruments.

Figure 1 shows the detection of influenza A viral RNA on the Applied Biosystems 7900 HT Fast Real Time System. Even a single copy of viral RNA was detected without difficulty, proving a highly efficient reverse transcription and amplification reaction even when using a reaction time of just 10 minutes for the reverse transcription step and a rapid cycling protocol for the PCR amplification. Similar results were obtained with HAV RNA using the Applied Biosystems StepOnePlus™ Real-Time PCR System (Figure 2). In summary, the consistent results would allow reliable quantification of the investigated targets. For both viral targets, the amplification process showed linearity over six orders of magnitude.

This article was originally published in Biochemica 2/2009, pages 25-26. ©Springer Medizin Verlag 2009