370 Comparison of MagNA Pure 96, Chemagic MSM1, and QIAamp MinElute for Hepatitis B Virus Nucleic Acid Extraction Seong-Ho Kang 1, Eun Hee Lee 2, Geon Park 1, Sook Jin Jang 1, and Dae Soo Moon 1 1 Department of Laboratory Medicine, Chosun University Medical School, Gwang-Ju, Korea; 2 Greencross Reference Laboratory, Yongin, Kyunggi-do, Korea. Abstract. This study was designed to compare two automated systems and one manual system for hepatitis B virus (HBV) nucleic acid extraction. The two automated systems were the MagNA Pure 96 system (Roche Applied Science, Manheim, Germany) and the Chemagic system (Chemagen, Baesweiler, Germany), and the manual system was the QIAamp system (Qiagen, Hilden, Germany). Sixty-eight samples that were within the detection range of the Cobas Ampliprep/Cobas TaqMan (CAP/CTM) platform (Roche Molecular Systems, Manheim, Germany) were selected. Extracted viral nucleic acids from the three systems were quantified using an AccuPower HBV Quantitative PCR kit (Bioneer, Daejon, Korea). The MagNA Pure 96 system and QIAamp system did not detect viral loads in one sample. The Chemagic system did not detect low viral loads in nine samples (range, 26 290 IU/mL by the CAP/CTM platform). Comparisons of the viral loads of the samples from the MagNA Pure 96 system, the Chemagic system, and the QIAamp system with those from the CAP/CTM platform yielded correlation coefficients of 0.977, 0.914, and 0.967, respectively. Comparisons of the MagNA Pure 96 system and the Chemagic system with the QIAamp system yielded correlation coefficients of 0.987 and 0.939, respectively. The MagNA Pure 96 system demonstrated better performance than the Chemagic system for HBV nucleic acid extraction. The MagNA Pure 96 system demonstrated comparable performance with the QIAamp system. Key words: Nucleic acid, Extraction, Hepatitis B virus, Quantification Introduction Because of their high sensitivity, a large number of polymerase chain reaction (PCR) tests are performed daily in clinical laboratories. Advances in these techniques, such as real-time PCR, have significantly improved the hands-on time and decreased contamination rates. Real-time PCR has enabled quantification of viral loads and fusion transcripts of various leukemia. Efficient nucleic acid extraction protocols are required to benefit from the advantages of nucleic acid amplification. Various techniques to extract nucleic acids have been introduced. Conventional manual extraction Address correspondence to Seong-Ho Kang, M.D., Ph.D., Department of Laboratory Medicine, Chosun University Medical School, 365 Pilmun-daero, Dong-gu, Gwangju, 501-717, South Korea; tel : +82-62-220-3272; fax : +82-62-232-2063; e-mail: seonghomed@hanmail. net of nucleic acids is labor-intensive and susceptible to handling variations. Automated nucleic acid extraction is currently being widely performed and has become an essential part of molecular diagnostics. Hepatitis B virus (HBV) infection is a major cause of chronic liver disease, liver cirrhosis, and hepatocellular carcinoma. It is estimated that nearly 2 billion people worldwide are infected with HBV and that more than 350 million have a persistent and chronic infection [1]. Recent advances in antiviral therapy for chronic HBV infection, using new or more powerful nucleoside/nucleotide analogues, have led to better management of chronic HBV infection [2]. The introduction of highly sensitive tests for monitoring HBV DNA has supported the success of antiviral therapy. Quantification of HBV 0091-7370/12/0400-370. 2012 by the Association of Clinical Scientists, Inc.
Available online at www.annclinlabsci.org Comparison of MagNA Pure 96, Chemagic MSM 1, and QIAmp MinElute 371 Table 1. Real-time quantitative PCR from extracted DNA by three systems (Number of samples) Real-time quantitative PCR MagNApure 96 Chemagic QIAamp Detected 67 59 67 Not detected 1 a 9 b 1 c a 43 IU/mL by the CAP/CTM platform b Median value of 56 IU/mL (range, 26-290 IU/mL) by the CAP/CTM platform c 26 IU/mL by the CAP/CTM platform viral load is one of the most frequently performed tests in molecular diagnostics, especially in Asia. This study was designed to compare HBV nucleic acid extraction in two automated systems and one manual system. One of the two automated systems was the MagNA Pure 96 DNA and Viral NA small volume kit (Roche Applied Science, Manheim, Germany) with MagNA pure 96 (Roche Applied Science). The second automated system was the Chemagic Viral DNA/RNA kit (Chemagen, Baesweiler, Germany) with Chemagic MSM1 (Chemagen). The manual system was the QIAamp MinElute Virus spin kit (Qiagen, Hilden, Germany). Both the MagNA Pure 96 system and the Chemagic system use magnetic bead technology for nucleic acid extraction. The QIAamp system uses a silica membrane-based method; extraction using this system is time-consuming and requires experienced personnel. The method relying on silica columns is considered the gold standard for nucleic acid extraction [3], so it served as the reference method in the present study. Materials and Methods Samples. Samples that were requested for HBV quantification were used. We first quantified HBV loads using the Cobas Ampliprep/Cobas TaqMan (CAP/CTM) platform (Roche Molecular Systems, Manheim, Germany). Sixty-eight samples that were within the detection range of CAP/CTM (20 108 IU/mL) were selected. DNA extraction. Viral nucleic acids of these serum samples were extracted using the automated MagNA Pure 96 system, the Chemagic system, and the manual QIAamp system. For viral DNA extraction using the MagNA Pure 96 system, 200 µl samples were mixed with 300 µl lysis binding buffer. Magnetic glass particles were added to a mixture of samples. After the incubation, the nucleic acids bound to the beads were separated with a magnetic separator. After washing, the pure nucleic acids were eluted in a volume of 50 μl. For viral DNA extraction using the Chemagic system, samples were incubated with protease and lysis buffer with an input volume of 200 µl, and magnetic beads were added to the mixture. After the incubation, nucleic acids bound to the beads were separated with a magnetic separator. After washing, the nucleic acids were eluted in a volume of 50 μl. For viral DNA extraction using the QIAamp system, the manufacturer s protocol was used, input volume was 200 µl, and viral DNA was eluted in a volume of 50 μl. Real-time quantitative PCR. Extracted viral nucleic acids were quantified using an AccuPower HBV Quantitative PCR kit (Bioneer, Daejon, Korea) and an Exicycler 96 real-time Quantitative Thermal Block (Bioneer). The AccuPower HBV Quantitative PCR kit is a Conformité Européene certified product. Fifty microliters of each DNA
372 sample was mixed with the TaqMan probe and primers specific to the surface antigen S gene. PCR conditions were initial denaturation at 95 C for 10 min followed by 45 cycles of denaturation at 95 C for 20 sec, and extension at 55 C for 30 sec. The lower limit of detection was 22 IU/mL. The quantified viral loads from the three systems were compared with those from the CAP/CTM platform, and the loads from two automated systems (MagNA Pure 96 system and Chemagic system) were compared with those from the manual system (QIAamp system). In order to evaluate reproducibility, two positive control DNA samples with different concentrations (1 10 6 copies/reaction and 1 10 2 copies/reaction) were extracted twenty times using the three extraction methods described above and were subsequently quantified. HBV loads were log10 transformed for analysis. SPSS version 12.0 (SPSS, Chicago, IL, USA) was used for the statistical analysis. Results Real-time PCR from DNA extracted by the MagNA Pure 96 system detected HBV in 67 of the 68 samples but did not detect a low viral load in one sample (Table 1). The HBV load of the latter sample was 43 IU/mL using the CAP/CTM platform. Real-time PCR using DNA extracted by the Chemagic system detected HBV in 59 of 68 samples but did not detect the low viral load in nine samples. The median HBV load of samples that was not detected was 56 (range, 26 290 IU/mL) in the CAP/CTM platform. Real-time PCR using DNA extracted by the QIAamp system detected HBV in 67 of 68 samples but did not detect HBV in one sample. The HBV value of the latter sample was 26 IU/mL in the CAP/CTM platform.
Comparison of MagNA Pure 96, Chemagic MSM 1, and QIAmp MinElute 373 Figure 2. Linear regression analysis of HBV load (log10 IU/mL). (A) MagNA Pure 96 sysem vs. QIAamp system (B) Chemagic system vs. QIAamp system. Comparisons of the viral loads of samples from the MagNA Pure 96 system, the Chemagic system, and the QIAamp system with those from the CAP/ CTM platform yielded correlation coefficients of 0.977, 0.914, and 0.967, respectively (Figure 1). The slopes for the MagNA Pure 96 system, the Chemagic system, and the QIAamp system compared with CAP/CTM platform were 0.953, 0.906, and 0.940, respectively. Comparisons of the MagNA Pure 96 system and the Chemagic system with the QIAamp system yielded correlation coefficients of 0.987 and 0.939, respectively (Figure 2). The slopes of the MagNA Pure 96 system and the Chemagic system compared with the QIAamp system were 0.995 and 0.964, respectively. In reproducibility testing using high positive control DNA, the means of the MagNA Pure 96 system, the Chemagic system, and the QIAamp system were 6.62 log 10 IU/mL, 6.65 log 10 IU/mL and 6.69 log 10 IU/mL, respectively. The standard deviations of the three systems were 5.51 log 10 IU/mL, 5.55 log 10 IU/mL, and 5.50 log 10 IU/mL, respectively. The coefficients of variations were 7.76%, 7.94%, and 8.08%, respectively. In reproducibility testing using low positive control DNA, the means of the MagNA Pure 96 system, the Chemagic system, and the QIAamp system were 2.80 log 10 IU/ ml, 2.74 log 10 IU/mL and 2.74 log 10 IU/mL, respectively. The standard deviations of the three systems were 1.90 log 10 IU/mL, 1.85 log 10 IU/mL, and 1.85 log 10 IU/mL, respectively. The coefficients of variations were 12.58%, 12.87%, and 12.89%, respectively. These results were not shown to be statistically different by analysis of variance (ANOVA). Discussion The present study compared the performance of three systems for HBV nucleic acid extraction. Some studies have evaluated Nuclisens easy MAG (biomerieux, Marcy l Etoile, France), QIAsymphony SP (Qiagen, Valencia, CA, USA), COBAS Ampliprep (Roche Molecular Systems), and MagNA Pure (Roche Applied Science) for nucleic acid extracts of cytomegalovirus and reported comparable performance with the manual system [3-6]. However, only one report has evaluated the performance of automated extraction systems for HBV quantification [7]. This could be due to the fact that large companies have developed HBV quantification systems for extraction and detection. HBV is a DNA virus, and quantification of HBV is the most frequent quantification test in molecular diagnostics in some countries. The extraction performance of HBV could be a barometer of viral DNA extraction in many extraction systems. In Korea, there are real-time quantification HBV kits that are commercially available and applicable to different real-time PCR machines. Thus,
374 it is important to select an efficient and sensitive extraction system when using these kits. The present study used the CAP/CTM platform as a reference method for HBV quantification. Previous studies have shown that the CAT/CTM HBV assay is sensitive, specific, and reproducible, and that it accurately quantifies HBV DNA levels [8, 9]. In the present study, the DNA viral loads detected by the MagNA Pure 96 system and the QIAamp system significantly correlated with those of the CAP/CTM platform, and the DNA viral loads of the Chemagic system also significantly correlated with those of the CAP/CTM platform. However, the correlation coefficient of the Chemagic system was lower than that of the MagNA Pure 96 system and that of the QIAamp system, and the slopes of the MagNA Pure 96 system and the QIAamp system were closer to 1 than was the slope of the Chemagic system. Compared with the QIAamp system, the reference method for viral nucleic acid extraction, the MagNA Pure 96 system correlated with a higher correlation coefficient than that of the Chemagic system. A previous report compared performance of the Chemagic system with the QIAamp system by testing qualitative rather than quantitative HBV PCR, and concluded that the Chemagic system is not distinguishable from the QIAamp [10]. Both the MagNA Pure 96 system and the Chemagic system use magnetic bead technology, but in the present study the Chemagic system did not detect low viral loads in nine samples in which viral DNA was detected by the MagNA Pure 96 system. These collective observations indicate that the nucleid acid extraction efficiency of the MagNA Pure 96 system is better than that of the Chemagic system. Both the MagNA Pure 96 system and the QIAamp system did not detect viral load in one sample with a low viral load. The viral load of the sample that the MagNA Pure 96 system did not detect (43 IU/ ml by the CAP/CTM platform) was slightly higher than that of the sample that the QIAamp system did not detect (26 IU/mL by the CAP/CTM platform). These samples might not have been detected due to random error or were perhaps close to the detection limit of the HBV quantification kit used in the present study. In the present study, specificity testing of the three systems with HBV negative samples or hepatitis C virus positive samples was not performed. The present study is a limited comparison and further study is needed to evaluate the specificity performance of the three systems. In conclusion, the MagNA Pure 96 system demonstrated better performance than the Chemagic system for HBV nucleic acid extraction. The MagNA Pure 96 system demonstrated comparable performance with the QIAamp system. Acknowledgements This study was supported by research funds from Chosun University Hospital in 2012. 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