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Fully Automated Nucleic Acid Extraction: MagNA Pure LC

¹ Institute of Hygiene, Karl-Franzens-University Graz, A-8010 Graz, Austria, and
² Roche Diagnostics GmbH, A-1211 Wien, Austria

^aaddress correspondence to this author at: Molecular Diagnostics Laboratory, Institute of Hygiene, KF-University Graz, Universitaetsplatz 4, A-8010 Graz, Austria; fax 43-316-380-9649, e-mail harald.kessler{at}uni-graz.at

Kinetic PCR analysis by real-time monitoring of DNA amplification was first described 8 years ago (1). Recently, the LightCycler^TM instrument (Roche Molecular Biochemicals, Mannheim, Germany), which allows high-speed thermal cycling by use of air instead of thermal blocks and on-line real-time fluorescence monitoring, was introduced (2). Several reports have been published with regard to utilization of the LightCycler technology for detection of pathogens (3)(4)(5)(6)(7)(8)(9)(10)(11).

Real-time PCR has greatly decreased the amount of hands-on time needed to generate and detect amplification products. Before amplification, pathogen-specific DNA or RNA must be extracted from the specimen. This procedure, also called sample preparation, remains the most labor-intensive and time-consuming part of widely automated molecular assays and may be considered the major weakness in most molecular assays today (12)(13). Therefore, a fully automated sample preparation system is urgently needed for the routine diagnostic laboratory.

In this study, a fully automated specimen preparation instrument, the MagNA Pure LC^TM (Roche) was evaluated. The new instrument was used for extraction of herpes simplex virus (HSV) DNA in combination with real-time PCR on the LightCycler instrument.

In the first experiment, the interassay variation and the detection limit were tested. The Second European Union Concerted Action HSV Proficiency Panel, which consists of 12 vials with different concentrations of HSV type 1 (HSV-1), strain MacIntyre (American Type Culture Collection), HSV type 2 (HSV-2), strain MS (American Type Culture Collection), varicella-zoster virus, and negative samples, was used. Samples were analyzed three times on different days. Results were compared with a molecular assay, which consisted of an in-house DNA extraction protocol and real-time PCR.

In the second experiment, intraassay variation of the new molecular assay was tested. Plasma was collected from a patient without clinical presentations compatible with HSV infection. Aliquots were supplemented with dilutions of a culture supernatant of commercially available HSV-1-infected Vero cells (VR-260; American Type Culture Collection). Each dilution was analyzed eight times. Each assay contained five positive and three negative controls (water).

In the third experiment, precision and the influence of different sample materials were tested. Whole blood (3-mL EDTA tubes), serum, and plasma were collected from a patient without clinical presentations compatible with HSV infection. Aliquots were supplemented with a culture supernatant of HSV-1 as described above. Different sample volumes were analyzed three times with the new molecular assay. Each assay contained three negative controls (blank reagent and water). For experiments 2 and 3, each sample was processed independently through both the MagNA Pure LC and LightCycler stages.

The MagNA Pure LC is a benchtop instrument that can extract 32 samples in parallel. The extraction protocol "Total NA Serum, Plasma, Blood" was used. We used a 200-µL sample volume for experiments 1 and 2 and sample volumes of 50, 100, 150, or 200 µL for the third experiment. An elution volume of 100 µL and a dilution volume of 0 µL were chosen for each analytical run. Other details, such as reagent volumes and number of reaction tips needed for the run, were automatically calculated by the software. The MagNA Pure LC automatically performed all steps of the procedure.

After DNA extraction was completed, the MagNA Pure LC Cooling Block, which included a sample carousel with the correct number of LightCycler capillaries, and the reaction vessel, including the master mixture, were placed into the postelution area. After the start of the postelution protocol, which had been programmed before the start of the first run, the MagNA Pure LC automatically pipetted 15 µL of the master mixture and 5 µL of the processed sample into each of the LightCycler capillaries.

For the in-house DNA extraction method, a rapid DNA extraction protocol, which has recently been described in detail, was used (6). After completion, 15 µL of the master mixture and 5 µL of the processed sample were manually pipetted into each of the LightCycler capillaries.

For the real-time PCR assay, oligonucleotides deduced from the published sequence of the DNA polymerase gene-coding region of HSV were used as described previously (6). The LightCycler FastStart DNA Master Hybridization Probes reagent set (Roche) with the TaqMan^TM probe described previously was used (6).

After completion of the postelution protocol, the LightCycler capillaries were sealed. The sample carousel with the capillaries was then centrifuged in the LightCycler Carousel Centrifuge and placed into the LightCycler. After denaturation for 10 min at 95 °C, a total of 55 PCR cycles were run. Each cycle consisted of 10 s at 95 °C and 20 s at 60 °C. Fluorescence was measured once every cycle immediately after the end of the 60 °C incubation. After the final cycle, the capillaries were cooled for 2 s at 40 °C.

Fluorescence curves were analyzed with the LightCycler software, Ver. 3.5. Cycles 15 to 55 were selected for calculation of crossing points, which were defined as the maximum of the second derivative from the fluorescence curves. Automated calculation was done by the second derivative maximum method.

When samples of the Second European Union Concerted Action HSV Proficiency Panel were tested with the new molecular assay, 0.7–2 x 10³ estimated HSV-1 genomes/mL, i.e., 7–20 estimated HSV-1 genomes/LightCycler capillary, could be detected consistently. With the dilutions containing 2–6 x 10² estimated HSV-1 genomes/mL, i.e., 2–6 estimated HSV-1 genomes/LightCycler capillary, no signal was detected. When HSV-2 samples from the same panel were tested, even the lowest concentrations, 3–7 x 10² estimated HSV-2 genomes/mL, i.e., 3–7 estimated HSV-2 genomes/LightCycler capillary, were detected consistently. With the real-time PCR assay combined with the in-house extraction protocol, 2–6 x 10³ HSV-1 genomes/mL, i.e., 20–60 estimated HSV-1 genomes/LightCycler capillary, were detected consistently.

Testing of the HSV-2 samples revealed identical results with the assay that included DNA extraction on the MagNA Pure LC, i.e., all positive samples of the Second European Union Concerted Action HSV Proficiency Panel produced signals. Negative samples and the varicella-zoster virus sample always gave negative results. When the intraassay variation of the new molecular assay was tested, crossing points were found to be mostly within one cycle. Standard deviations were between 0.22 and 0.30 and showed a tendency to increase (up to 0.82) when less-concentrated samples were tested. When different sample volumes supplemented with the same amount of HSV-1 were analyzed with the new molecular assay, it was possible to distinguish among sample volumes of 50, 100, 150, and 200 µL by use of the mean value of the triplicates (Table 1 and Fig. 1 ). As shown by the increase of fluorescence, the quality of the different DNA extracts was independent of the input sample volume. When different sample materials were compared, EDTA blood, serum, and plasma showed similar crossing points.

View larger version (81K): [in this window] [in a new window]   Figure 1. Reproducibility of signals produced by different sample volumes. Different volumes (50, 100, 150, and 200 µL) of serum 10-3 were supplemented with HSV-1 and subjected to MagNA Pure LC extraction followed by real-time PCR on the LightCycler instrument.

Both the in-house extraction procedure and the automated DNA extraction with the MagNA Pure LC could be completed within 105 min for extraction of 32 samples. This included a 15-min set-up of the MagNA Pure LC. The time required for the postelution protocol was 15 min. After centrifugation, the combined cycling and detection procedure took another 60 min. No contamination was observed during the study.

The detection limit for HSV-1 genomes was slightly higher with the in-house protocol than with the new molecular assay. There may be two reasons for this difference: for DNA extraction on the MagNA Pure LC, a higher sample volume (200 µL compared with 100 µL for the in-house protocol) is used and samples are concentrated during the procedure to a volume of 100 µL, in contrast to the Chelex protocol, in which samples are diluted to a total volume of 400 µL.

Variations in real-time PCR results are caused mainly by extraction procedures (14). In this study, the new molecular assay that included automated nucleic acid extraction showed high reproducibility. Furthermore, no contamination occurred during the study. The probability of false-positive results because of contamination increases in relation to the number of hands-on manipulations involved in sample processing (15)(16). Although no contamination occurred with the in-house extraction protocol in our study, the study was done in an ISO 9002-certified laboratory under strict safety precautions. In a routine diagnostic laboratory, exclusion of contamination may be one of the major advantages of a fully automated nucleic acids extraction instrument. It may replace a clean room for specimen preparation.

The MagNA Pure LC extraction protocol used in this study was suitable for recovery of HSV-1 DNA from serum, plasma, and whole blood. To extend the future use of this instrument, it will be important to have nucleic acid isolation protocols for other materials, such as cerebrospinal fluid, respiratory samples, and swabs.

Time for extraction of 32 samples was similar with both extraction methods. If fewer than eight samples are extracted, in-house extraction methods may be quicker. It must, however, be taken into consideration that our hands-on time for extraction was only 15 min when we used the MagNA Pure LC.

This example of a largely automated molecular assay may provide a way for converting significant amounts of conventional microbiological testing to a chemistry-based assay technique (17)(18). Judging from the history of clinical chemistry, new and better instrumentation will emerge and the testing menu will be expanded (19). This development will lead to widespread use of molecular assays in the routine diagnostic laboratory and to replacement of conventional microbiological techniques.

In summary, the MagNA Pure LC instrument is a reliable tool for isolation of nucleic acids. Because of full automation, the probability of false-positive results attributable to contamination by hands-on manipulation may be eliminated. The MagNA Pure LC instrument helps to avoid human error, thus increasing the precision and reproducibility of results and making better standardization of molecular testing possible.

Acknowledgments

This project was supported in part by a grant from Roche Molecular Biochemicals. We gratefully acknowledge Jutta Fischer for technical assistance and stimulating discussions.

References

1. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology 1992;10:413-417.[Medline] [Order article via Infotrieve]
2. Wittwer CT, Ririe KM, Andrew RV, David DA, Gundry RA, Balis UJ. The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques 1997;22:176-181.[ISI][Medline] [Order article via Infotrieve]
3. Espy MJ, Uhl JR, Mitchell PS, Thorvilson JN, Svien KA, Wold AD, Smith TF. Diagnosis of herpes simplex virus infections in the clinical laboratory by LightCycler PCR. J Clin Microbiol 2000;38:795-799.[Abstract/Free Full Text]
4. Espy MJ, Teo R, Ross TK, Svien KA, Wold AD, Uhl JR, Smith TF. Diagnosis of varicella-zoster virus infections in the clinical laboratory by LightCycler PCR. J Clin Microbiol 2000;38:3187-3189.[Abstract/Free Full Text]
5. Ke D, Menard C, Picard FJ, Boissinot M, Ouellette M, Roy PH, Bergeron MG. Development of conventional and real-time PCR assays for the rapid detection of group B streptococci. Clin Chem 2000;46:324-331.[Abstract/Free Full Text]
6. Kessler HH, Mühlbauer G, Rinner B, Stelzl E, Berger A, Dörr HW, et al. Detection of herpes simplex virus DNA by real-time PCR. J Clin Microbiol 2000;38:2638-2642.[Abstract/Free Full Text]
7. Loeffler J, Hagmeyer L, Hebart H, Henke N, Schumacher U, Einsele H. Rapid detection of point mutations by fluorescence resonance energy transfer and probe melting curves in Candida species. Clin Chem 2000;46:631-635.[Abstract/Free Full Text]
8. Nitsche A, Steuer N, Schmidt CA, Landt O, Siegert W. Different real-time PCR formats compared for the quantitative detection of human cytomegalovirus DNA. Clin Chem 1999;45:1932-1937.[Abstract/Free Full Text]
9. Pietila J, He Q, Oksi J, Viljanen MK. Rapid differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto by LightCycler fluorescence melting curve analysis of a PCR product of the recA gene. J Clin Microbiol 2000;38:2756-2759.[Abstract/Free Full Text]
10. Ratge D, Scheiblhuber B, Nitsche M, Knabbe C. High-speed detection of blood-born hepatitis C virus RNA by single-tube real-time fluorescence reverse transcription-PCR with the LightCycler. Clin Chem 2000;46:1987-1989.[Free Full Text]
11. Reischl U, Linde HK, Metz M, Leppmeier B, Lehn N. Rapid identification of methicillin-resistant Staphylococcus aureus and simultaneous species confirmation using real-time fluorescence PCR. J Clin Microbiol 2000;38:2429-2433.[Abstract/Free Full Text]
12. Deggerdal A, Larsen F. Rapid isolation of PCR-ready DNA from blood, bone marrow, and cultured cells, based on paramagnetic beads. Biotechniques 1997;22:554-557.[ISI][Medline] [Order article via Infotrieve]
13. Verhofstede C, Fransen K, Marissens D, Verhelst R, van der Groen G, Lauwers S, et al. Isolation of HIV-1 RNA from plasma: evaluation of eight different extraction methods. J Virol Methods 1996;60:155-159.[Medline] [Order article via Infotrieve]
14. Loeffler J, Henke N, Hebart H, Schmidt D, Hagmeyer L, Schumacher U, Einsele H. Quantification of fungal DNA by using fluorescence resonance energy transfer and the LightCycler system. J Clin Microbiol 2000;38:586-590.[Abstract/Free Full Text]
15. Clewley JP. The polymerase chain reaction, a review of the practical limitations for human immunodeficiency virus diagnosis. J Virol Methods 1989;25:179-188.[Medline] [Order article via Infotrieve]
16. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989;339:237-238.[Medline] [Order article via Infotrieve]
17. Lisby G. Application of nucleic acid amplification in clinical microbiology. Mol Biotechnol 1999;12:75-99.[Medline] [Order article via Infotrieve]
18. Tang YW, Procop GW, Persing DH. Molecular diagnostics of infectious diseases. Clin Chem 1997;43:2021-2038.[Abstract/Free Full Text]
19. Jungkind D. Automation of laboratory testing for infectious diseases using the polymerase chain reaction—our past, our present, our future. J Clin Virol 2001;20:1-6.[Medline] [Order article via Infotrieve]

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