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High-throughput robotic workstation for fully automated purification of nucleic acids from up to 96 samples.
For in vitro diagnostic use.
Ready-to-use reagents in two prefilled sealed trays for purifying nucleic acids from up to 1,000 μL sample volume using the MagNA Pure 96 Instrument.
For in vitro diagnostic use.
Ready-to-use reagents in two prefilled sealed trays for purifying nucleic acids from up to 200 μL sample volumes using the MagNA Pure 96 Instrument.
For in vitro diagnostic use.
Background: Peripheral blood based molecular diagnostics requires automated sample processing and evaluation of RNA for RT-PCR analysis. Our aims were to compare the automated and manual RNA isolation and to determine the purity, recovery, RNA integrity (RIN) and sensitivity of downstream RT-PCR applications.
Methods: Total RNA was isolated from blood samples of 25 individuals with colorectal cancer (CRC) and 19 healthy individuals, either manually using PAXgene Blood RNA Kit and in parallel, using the MagNA Pure 96 Instrument. The expression of seven CRC and one adenoma markers was analyzed using real-time RT-PCR.
Results: OD260/280 of manually isolated samples (2.09) was equal to that of MagNA Pure 96 Instrument isolated samples (2.01), while OD230/260 of MagNA Pure 96 Instrument samples (1.85) was signifi cantly better than that of manually isolated samples (1.52). The RIN is acceptable in case of both isolation methods. The manual protocol produced slightly more intact RNA (RIN-manual: 9.19 +/- 0.26; RIN-MagNA Pure 96 Instrument: 8.25). The MagNA Pure 96 System resulted in slightly higher yields of isolated RNA (manual: 5.65 μg/tube; MagNA Pure 96 System: 5.99 μg/tube). Marker correlation between the two batches isolated using the MagNA Pure 96 Instrument and between two PAXgene batches was high. In overall qPCR reproducibility and mRNA yield/ratio were higher with the MagNA Pure 96 System. The automated method showed less standard deviation. Genomic DNA contamination could not be detected in any of the MagNA Pure 96 System isolated RNA samples.
Conclusion: Automated RNA isolation from peripheral blood samples using the MagNA Pure 96 Instrument produces high quality, high purity and high yield total RNA. MagNA Pure 96 Instrument extraction is a fully automated, fast and reliable method with 96 extractions in 85 min, compared to the equally precise, but time consuming manual extraction.
The objective of this study was to compare the MagNA Pure 96 Pathogen Universal protocol to the MagNA Pure LC DNA Isolation Kit III that is well established in our microbiological laboratory. For this we isolated nucleic acids from ten different sample materials: body fluids (Fluids, urine, sputum), feces, swabs, as well as whole blood, EDTA-, Citrate-Plasma and serum. This spectrum is typical for a microbiology laboratory and includes easy and difficult-to-process samples, as sputum and stool are. Into these sample types we spiked classified pathogen stocks from in-house cultures. Subsequently Real-time PCR analysis of fungal, bacterial and viral targets was done using the LightCycler® 480 Instrument.The 200 μl protocol performed effi cient and sensitive nucleic acid extraction from up to 96 samples requiring less than 60 minutes runtime. It yielded superior or similar results compared to the respective MagNA Pure LC protocol. Higher input volumes, 500 or 1000 μl, available only for the MagNA Pure 96 System, generally increased sensitivity of detection. Direct use of some routine microbiological samples without dilution by pretreatment reagents, resulted in improved Crossing Point (CP) values. For normal sample types including whole blood this made pretreatment dispensable. A pretreatment step did however improve pathogen detection in the more diffi cult-to-process samples, such as sputum, swabs and stool. Our results demonstrate that both methods allow efficient and convenient isolation of high quality nucleic acid. Compared to the Viral NA Universal protocol, the Pathogen Universal protocol is further optimized for diverse sample materials, including difficult samples and for the detection of various pathogen types. The MagNA Pure 96 Instrument thus enables high throughput sample preparation applications in microbiology laboratories.
In May and June 2011, there was a significant increase in the number of cases of bloody diarrhea associated with hemolyticuremic syndrome (HUS) caused by enterhemorrhagic Escherichia coli (EHEC). The majority of EHEC infected individuals lived in Germany, but significant numbers of infections have also been reported in France, Sweden, Russia and other European countries.To streamline the workflow for detecting the EHEC bacterium, the automated MagNA Pure Sample Preparation System for nucleic acid isolation and LightCycler® Real-time PCR Instrument (Roche Diagnostics) were used for high throughput examination of EHEC directly in stool samplesHuman stool samples were processed using the high throughput MagNA Pure 96 System to isolate high quality total genomic DNA. Targeting the Shiga toxin genes of enterohemorrhagic E. coli by a LightCycler® Real-time PCR test demonstrated that fecal specimens can be used as starting material to detect EHEC infection in epidemic situations reducing the turnaround from days to hours.The fully automated robotic MagNA Pure 96 Instrument used in this study isolates nucleic acids from 96 samples in approximately one hour. We have used an analysis assay containing two pairs of LightCycler® HybProbe probes for the LightCycler® 480 Instrument, detecting and distinguishing the Shiga toxin genes stx1 and stx2. The absence of E. coli intimin and enterohemolysin genes as well as a negative result for stx1 and a positive result for stx2 with a specific Tm of +71°C was characteristic for the epidemic HUSEC 41 strain. The parC gene was used as both an extraction control and inhibition control for these tests The MagNA Pure 96 and LightCycler® workflow used here to detect EHEC-infections performed accurate and sensitive tests during the EHEC outbreak. In the 181 samples examined, a sensitivity of 100% and a specificity of 98% were achieved. In contrast to routine microbiological procedures, PCR testing produced reliable data within hours. Although confirmatory tests are needed, the protocol described here using DNA extracted directly from stool specimens is tailored to EHEC outbreak situations enabling rapid screening with the required controls for managing treatment options.
E. coli (EHEC) Extraction and Detection in Stool Samples through Use of MagNA Pure 96 System and LightCycler® Instrument
To detect the EHEC bacterium, the automated MagNA Pure 96 System for nucleic acid isolation and LightCycler® * Real-Time PCR Instrument (Roche Diagnostics) were used for high throughput examination of EHEC directly in stool samples.
Utility of the MagNA Pure 96 and LightCycler® 480 Systems for Testing of Stool Samples for Toxin-producing C. Difficile
Clostridium difficile infection (CDI) is an important cause of hospital diarrhea. Conventional CDI testing by enzyme immunoassays is fast yet shows poor sensitivity and specificity, whereas the "gold standard" toxigenic culture is laborious and time consuming. Molecular tests for CDI are fast and highly sensitive and specific. Our laboratory has developed a molecular procedure that allows for rapid detection of CDI directly in stool samples. It is based on the isolation of highly pure DNA extracts from feces using the MagNA Pure 96 System, followed by detection of the C. difficile toxins tcdA and tcdB using real-time PCR on the LightCycler® 480 Instrument.
Utility of the MagNA Pure 96 and LightCycler® 480 System for the Detection of Respiratory Pathogens
Acute respiratory disease (ARD) is a major cause of morbidity in developed countries and accounts for a large number of hospitalizations of especially young children. In addition, in developing countries, it constitutes an important cause of death. Viruses were shown to be a major cause of ARD. The last decade has seen the growing importance of molecular techniques in not only the discovery of novel viral respiratory pathogens, but also in routine testing of viral respiratory disease, replacing traditional virus detection methods in many laboratories. Also in our laboratory, a multiplex real-time PCR respiratory panel was introduced a number of years ago, leading to faster analysis and detection. In 2010, the MagNA Pure 96 System was introduced into the laboratory, where it is combined with the LightCycler® 480 Instrument to yield an easy-touse, efficient set-up for molecular research. This paper describes our results obtained with this setup in analysis of viral respiratory samples in 2010 and 2011.
Detection of bacterial, fungal and viral NA in routine microbiological sample types on the MagNA Pure 96 System
The objective of this study was to compare the MagNA Pure 96 Pathogen Universal protocol to the MagNA Pure LC DNA Isolation Kit III that is well established in our microbiological laboratory. For this we isolated nucleic acids from ten different sample materials: body fluids (CSF, urine, sputum), feces, swabs, as well as whole blood, EDTA-, Citrate-plasma and serum. This spectrum is typical for a microbiology laboratory and includes easy and difficult-to-process samples, as sputum and stool are. Into these sample types we spiked classifi ed pathogen stocks from in-house cultures. Subsequently real-time PCR analysis of fungal, bacterial and viral targets was done using the LightCycler® 480 Instrument.
Comparison of Automated MagNA Pure 96 and Manual PAXgene RNA Isolation from Stabilized Peripheral Blood Samples
Experiments in this paper investigate the quality and capacity of RNA isolation from PAXgene tubes on the MagNA Pure 96 System compared to conventional manual extraction methods. Researchers used a modified RNA isolation protocol (described in the MagNA Pure 96 Cellular RNA Large Volume Kit product insert) that extracts up to 48 stabilized blood samples (or 96 aliquots) simultaneously. Attributes such as purity, recovery, RNA integrity (RIN) and sensitivity in downstream RT-PCR applications were compared and analyzed. The goal for these studies is to develop an optimized and more efficient workflow for the detection of CRC biomarkers in peripheral blood samples.