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High Pure Viral Nucleic Acid Large Volume Kit - Viral Detection in Large Sample Volumes

Constanze Stadler, Max Ruhstorfer, Nina Lassonczyk, and Horst Donner*

Roche Diagnostics GmbH, Roche Applied Science, Penzberg, Germany
*Corresponding author

The isolation of nucleic acids from biological samples such as serum, plasma, or whole blood is commonly used for the detection of viral loads. It is therefore desirable to collect this low amount of nucleic acid from large sample volumes in order to measure low viral loads with routinely used qPCR and qRT-PCR principles. The High Pure Viral Nucleic Acid Large Volume Kit enables researchers to isolate viral nucleic acids from up to 2.5 ml of plasma, serum, or whole blood in 25 minutes. The isolation of viral nucleic acids is based on the well-established spin column approach. Nucleic acids are bound to silica under chaotropic conditions and subsequently washed and eluted in 50 µl
volume, ready to use in downstream applications like qPCR or qRT-PCR using a LightCycler® 2.0 Instrument.

Introduction

In recent years, molecular detection of viral loads has become an essential process in research laboratories. Commonly used sample types are serum, plasma, blood, and other body fluids. Such material is easily accessible and generally contains a very low amount of nucleic acid. It is therefore desirable to collect this low amount of nucleic acid from large sample volumes in order to measure low viral loads with routinely used qPCR and qRT-PCR principles.

High Pure Viral Nucleic Acid Large Volume Kit

The High Pure Viral Nucleic Acid Large Volume Kit enables researchers to isolate viral nucleic acids from up to 2.5 ml of plasma, serum, or whole blood in 25 minutes. The kit includes all necessary buffers (binding, washing, and elution buffers), poly(A) RNA as carrier material, and recombinant Proteinase K together with the newly developed High Pure Extender Assembly (Figure 1).

The upper reservoir of the High Pure Extender Assembly, enables researchers to simply load the complete sample–binding buffer solution onto the disposable column avoiding cumbersome sample pre-processing steps. The binding of the nucleic acids to the High Pure spin column is performed by a single spin of the assembly in a table top centrifuge. The nucleic acid containing High Pure column is easily detached from the High Pure Extender Assembly by removing the locking clip a and b (Figure 1). All subsequent steps (washing and elution) are performed using a standard table-top micro-centrifuge. At the end of the isolation process, the viral nucleic acid is eluted in 50 µl elution volume.

Application Data

In order to demonstrate the sensitivity of the High Pure Viral Nucleic Acid Large Volume Kit, 1 ml citrate plasma was spiked with a decreasing amount of HAV viral particles (1x10⁵ to 1x10²). Isolation was performed according to the kit manual, and LightCycler® instrument analysis was performed using the LightCycler® Instrument HAV Quantification Kit*.

As shown in Figure 2, we observed a high sensitivity and good linearity of HAV particles in citrate plasma down to 100 copies per 1 ml of sample volume.

The use of human whole blood is shown in Figure 3. Different amounts of human whole blood research samples (200 µl, 1 ml, and 2.5 ml) were spiked with different amounts of EBV viral particles (1x10⁶, 1x10⁵, and 1x10⁴). Isolation was performed according to the kit manual and quantification was performed using a LightCycler® 2.0 Instrument with the LightCycler® EBV Quantification Kit*.

As shown in Figure 3, we observed a high linearity of crossing-point value in a range from 10⁶ to 10⁴ EBV viral particles independent of the sample volume.

For competitor comparison, 1 ml of negative human serum was spiked with a dilution row (10⁶ to 10⁴) of HAV or EBV particles followed by a subsequent isolation according to the kit manuals.

Isolation efficiency and quality were analyzed by a LightCycler® 2.0 Instrument using the LightCycler® HAV Quantification Kit* or LightCycler® EBV Quantification Kit*, respectively.

As shown in Figure 4, we observed a higher sensitivity and good linearity  of HAV and EBV detection in 1 ml human serum compared with a kit provided by an other supplier.

Conclusions

The High Pure Viral Nucleic Acid Large Volume Kit enables researchers to isolate viral nucleic acids from sample volumes up to 2.5 ml. The well-established spin column approach guarantees high sensitivity and linearity of the isolation process. The newly developed Extender Assembly provides easy handling and isolation of times less than 25 minutes, making it a valuable tool in molecular detection of viral loads.

This article was originally published in Biochemica 2/2008, pages 30-31. ©Springer Medizin Verlag 2008