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Comparing Two Automated Methods for Viral Total Nucleic Acid Purification

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LabFact #1

We highly recommend the use of barrier pipette tips for assembling components for PCR or RT-PCR to prevent contamination of reactions and reagents with template DNA, RNA or primers.

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Abstract

In this article, we compare two automated methods for isolating viral total nucleic acid from human serum and plasma: The Maxwell® RSC Viral Total Nucleic Acid Purification Kit and the Qiagen EZ1 Virus Mini Kit. DNA purified from CMV was tested in qPCR while RNA purified from HIV was analyzed for yield and amplified in RT-qPCR. The nucleic acid purified by the Maxwell® RSC System had lower Cq values and higher yield compared to the Qiagen system.

Samantha Lewis

Promega Corporation

Publication Date: May 2015; tpub_163

Introduction

Isolating viral nucleic acid from blood or serum is the first step in detection and analysis for microbiology or virology research. An extraction method for viral DNA or RNA needs to purify across a range of virus titers and provide high sensitivity for downstream applications. Purified nucleic acids need to be free from inhibitors for use in applications such as qPCR and qRT-PCR. Being able to automate nucleic acid extraction means less hands-on time and reduces cross-contamination.

The Maxwell® Rapid Sample Concentrator (RSC) Instrument is an automated system that will process up to 16 samples in a single run. Using prefilled reagent cartridges like those in the Maxwell® RSC Viral Total Nucleic Acid Purification Kit, the Maxwell® RSC Instrument can purify DNA or RNA from a wide range of sample types, and the integrated Quantus™ Fluorometer means you can collect purification and quantification data in one report.

Using human blood and plasma with known quantities of either cytomegalovirus (CMV) or human immunodeficiency virus (HIV), performance of the Maxwell® Viral Total Nucleic Acid Purification Kit on the Maxwell® RSC Instrument was compared with the Qiagen EZ1 Virus Mini Kit automated on the BioRobot® EZ1™ instrument by analyzing nucleic acid yield and amplification reactions.

Materials

  • Human serum and plasma (BioreclamationIVT)
  • CMV inactivated virus (Zeptometrix Cat.# NATCMV-0005)
  • AgPAth-ID™ One-Step RT-PCR Kit (Life Technologies Cat.# AM1005)
  • EZ1 Virus Mini Kit (Qiagen Cat.# 955134)
  • Armored RNA® Quant HIV Subtype B (Asuragen Cat.# 42100)
  • Maxwell® RSC Viral Total Nucleic Acid Kit (Cat.# AS1330)
  • GoTaq® Probe qPCR Master Mix (Cat.# A6101)
  • Maxwell® RSC Instrument (Cat.# AS4500)
  • BioRobot® EZ1™ Instrument (Qiagen Cat.# 9000705)
  • nuclease-free water

Methods

Nucleic acid purification

Human serum or plasma (100µl) was mixed with 100μl of CMV viral particles (100,000 copies per ml) in a plasma like-matrix. Nucleic acid purification was then performed according to manufacturer’s instructions. Total nucleic acid was eluted in 50μl for the Maxwell® RSC Kit and 60μl for the EZ1 Virus Mini Kit. Three replicates were run for plasma and two replicates were run for serum for each kit.

Five milliliters of plasma was mixed with 300µl of 1 × 105, 5 × 104 or 2.5 × 104 viral copies of armored HIV RNA (aHIV). For the EZ1 Virus Mini Kit, 400µl of plasma was dispensed into sample tubes, the tubes loaded into and the instrument was run according to manufacturer’s instructions. For the Maxwell® RSC Viral Total Nucleic Acid Kit, the three different viral copy level plasma samples were dispensed into five aliquots of 300μl in a 1.5ml tube, and 300μl of lysis buffer and 30μl of proteinase K was added to each tube aliquot and heated for 10 minutes at 56°C. Lysates were cooled for 10 minutes and pooled. A 630μl aliquot of this pool was loaded into a Maxwell® Viral Total Nucleic Acid cartridge in triplicate for each viral copy level. Nucleic acid was eluted in 50μl for the Maxwell® RSC Kit and 60μl for the EZ1 Virus Mini Kit.

Viral Quantitation by quantitative PCR analysis

qPCR was performed with isolated CMV nucleic acid using the GoTaq® Probe qPCR System. Five microliters of purified nucleic acid was amplified in total volume of 20μl qPCR using the probe and primer set from Life Technologies, Assay ID: Pa03453400_s. Standard cycling conditions were used as recommended in the GoTaq® Probe qPCR Master Mix Technical Manual #TM378 (i.e., initial incubation at 95°C for 2 minutes followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute.)

One-step RT-qPCR was performed with isolated HIV nucleic acid using the AgPAth-ID™ One-Step RT-PCR Kit according to the manufacturer’s instructions. Briefly, a 25μl reaction was used with 5μl of purified nucleic acid. A standard curve of aHIV RNA was prepared by heating a 1 × 107 copy/ml HIV stock to 75°C for 3 minutes and then serially diluting the stock seven times to 40 copies/μl, in nuclease-free water. Five microliters of each serial dilution was used for RT-qPCR in a total reaction volume of 25μl with the following HIV-specific primer and probe sequences:
Forward: 5´-TGC AGA ATG GGA TAG ATT GC
Reverse: 5´-CCC TTG GTT CTC TCA TCT GG
Probe: 5´-FAM/CCT GGT GCA ATA GGC CCT CCA/BHQ-3´
Cycling conditions were: Reverse transcription for 10 minutes at 45°C followed by denaturation at 95°C for 10 minutes and amplification for 40 cycles at 95°C for 15 seconds and 60°C for 45 seconds.

Results

Recovery of CMV Viral DNA

When qPCR was performed using the purified nucleic acid with CMV-specific primers, the samples extracted using the Maxwell® RSC System had lower Cq value, indicating a higher DNA concentration and more efficient extraction of nucleic acids compared with the Qiagen EZ1 Virus Mini Kit (Figure 1).

13054MA.epsFigure 0. Cq values from CMV viral DNA purified from human serum and plasma.

Cq values from CMV-specific qPCR with nucleic acid isolated from 200µl of plasma or serum mixed with 10,000 CMV viral particles. For each purification, n = 3 for plasma and n = 2 for serum.

Armored HIV RNA Recovery

HIV viral RNA was detected by RT-qPCR using HIV RNA-specific primers (Figure 2). Cq values from the Maxwell® RSC Kit were consistently lower than those from the Qiagen EZ1 kit, indicating higher RNA concentration and more efficient recovery in the Maxwell® RSC-purified samples. HIV RNA yield in copy number per ml input was calculated using a HIV RNA standard curve (Figure 3). Samples purified using the Maxwell® RSC Viral Total Nucleic Acid Kit recovered more viral RNA than those purified using the Qiagen EZ1 kit. Of note, even with a higher copy number of virus mixed with plasma was isolated using the EZ1 protocol, the total recovery of viral RNA compared to input copy number was less than the Maxwell® RSC Kit (Figure 3).

13053MA.epsFigure 0. Cq values from HIV viral RNA purified from human plasma.

Cq values from HIV-specific RT-qPCR with nucleic acid isolated from plasma with known amounts of Armored RNA® HIV (aHIV; Asuragen). n = 3 for each HIV input copy number.

13055MA.epsFigure 0. Total HIV viral RNA yield from human plasma.

Yield in copies per milliliters of input from HIV specific RT-qPCR with nucleic acid isolated from plasma spiked with Armored RNA® HIV (Asuragen). n = 3 for each HIV input copy number.

Conclusion

The Maxwell® RSC Viral Total Nucleic Acid Purification Kit efficiently isolated viral nucleic acid from serum and plasma in a semi-automated format. Samples purified with the Maxwell® RSC outperformed samples purified using the Qiagen EZ1 Virus Mini Kit on the BioRobot® EZ1™ in both concentration and yield for both DNA and RNA viruses. Because the Maxwell® RSC System had recovered greater viral copy numbers compared to the input amount, less plasma is needed for sample inputs compared to the EZ1 Virus Mini Kit. The greater purification efficiency of the Maxwell® RSC Kit maximizes viral nucleic acid recovery from limited samples.

How to Cite This Article

Lewis, S. Comparing Two Automated Methods for Viral Total Nucleic Acid Purification. [Internet] May 2015; tpub_163. [cited: year, month, date]. Available from: http://www.promega.com/resources/pubhub/comparing-two-automated-methods-for-viral-total-nucleic-acid-purification/

Lewis, S. Comparing Two Automated Methods for Viral Total Nucleic Acid Purification. Promega Corporation Web site. http://www.promega.com/resources/pubhub/comparing-two-automated-methods-for-viral-total-nucleic-acid-purification/ Updated May 2015; tpub_163. Accessed Month Day, Year.

GoTaq and Maxwell are registered trademarks of Promega Corporation.

AgPAth-ID is a trademark of Life Technologies. Armored RNA is a registered trademark of Asuragen, Inc. BioRobot is a registered trademark and EZ1 is a trademark of Qiagen.

Figures

13054MA.epsFigure 0. Cq values from CMV viral DNA purified from human serum and plasma.

Cq values from CMV-specific qPCR with nucleic acid isolated from 200µl of plasma or serum mixed with 10,000 CMV viral particles. For each purification, n = 3 for plasma and n = 2 for serum.

13053MA.epsFigure 0. Cq values from HIV viral RNA purified from human plasma.

Cq values from HIV-specific RT-qPCR with nucleic acid isolated from plasma with known amounts of Armored RNA® HIV (aHIV; Asuragen). n = 3 for each HIV input copy number.

13055MA.epsFigure 0. Total HIV viral RNA yield from human plasma.

Yield in copies per milliliters of input from HIV specific RT-qPCR with nucleic acid isolated from plasma spiked with Armored RNA® HIV (Asuragen). n = 3 for each HIV input copy number.