Manual Extraction Methods Research Articles

Evaluation of a manual DNA extraction protocol and an isothermal amplification assay for detecting HIV-1 DNA from dried blood spots for use in resource-limited settings

BackgroundIn resource-limited settings (RLS) dried blood spots (DBS) are collected on infants and transported through provincial laboratories to a central facility where HIV-1 DNA PCR testing is performed using specialized equipment. Implementing a simpler approach not requiring such equipment or skilled personnel could allow the more numerous provincial laboratories to offer testing, improving turn-around-time to identify and treat infected infants sooner. ObjectivesAssess performances of a manual DNA extraction method and helicase-dependent amplification (HDA) assay for detecting HIV-1 DNA from DBS. Study design60 HIV-1 infected adults were enrolled, blood samples taken and DBS made. DBS extracts were assessed for DNA concentration and beta globin amplification using PCR and melt-curve analysis. These same extracts were then tested for HIV-1 DNA using HDA and compared to results generated by PCR and pyrosequencing. Finally, HDA limit of detection (LOD) studies were performed using DBS extracts prepared with known numbers of 8E5 cells. ResultsThe manual extraction protocol consistently yielded high concentrations of amplifiable DNA from DBS. LOD assessment demonstrated HDA detected ∼470copies/ml of HIV-1 DNA extracts in 4/4 replicates. No statistical difference was found using the McNemar's test when comparing HDA to PCR for detecting HIV-1 DNA from DBS. ConclusionsUsing just a magnet, heat block and pipettes, the manual extraction protocol and HDA assay detected HIV-1 DNA from DBS at levels that would be useful for early infant diagnosis. Next steps will include assessing HDA for non-B HIV-1 subtypes recognition and comparison to Roche HIV-1 DNA v1.5 PCR assay.

Use of Conventional Bioanalytical Devices to Automate DBS Extractions in Liquid-Handling Dispensing Tips

Conventional liquid-handling devices were employed, along with an improved punching device, to semi-automate dried blood spot (DBS) extraction of alprazolam, α-hydroxyalprazolam and midazolam from human whole blood. Liquid-handling devices were used to add internal standard to the DBS cards and to extract the analytes from the DBS, in order to be analyzed by HPLC-MS/MS. The technique was shown to be accurate (±12.0%) and precise (10.3%) across the dynamic range of the assay. The semi-automated extraction reduced sample preparation time by more than 50% when compared with more conventional DBS manual extraction methods.

Methods for isolation of cell-free plasma DNA strongly affect DNA yield

Extracellular nucleic acids are present in plasma, serum, and other body fluids and their analysis has gained increasing attention during recent years. Because of the small quantity and highly fragmented nature of cell-free DNA in plasma and serum, a fast, efficient, and reliable isolation method is still a problem and so far there is no agreement on a standardized method. We used spin columns from commercial suppliers (QIAamp DNA Blood Midi Kit from Qiagen; NucleoSpin Kit from Macherey-Nagel; MagNA Pure isolation system from Roche Diagnostics) to isolate DNA from 44 plasma samples in parallel at laboratories in Berlin and Munich. DNA in all samples was quantified by real-time PCR on a LightCycler 480 using three different targets (GAPDH, ß-globin, ERV). The quantities of cell-free DNA for the different isolation methods and genes varied between medians of 1.6ng/mL and 28.1ng/mL. This considerable variation of absolute DNA values was mainly caused by the use of different isolation methods (p<0.0001). Comparable results were achieved by the use of the genes GAPDH and ERV while higher values were obtained by use of ß-globin. The laboratory site had only minor influence on DNA yield when manual extraction methods were used.

Technical note: Comparative analyses of the quality and yield of genomic DNA from invasive and noninvasive, automated and manual extraction methods

Several new automated methods have recently become available for high-throughput DNA extraction, including the Maxwell 16 System (Promega UK, Southampton, UK). The purpose of this report is to compare automated with manual DNA extraction methods, and invasive with noninvasive sample collection methods, in terms of DNA yield and quality. Milk, blood, and nasal swab samples were taken from 10 cows for DNA extraction. Nasal swabs were also taken from 10 calves and semen samples from 15 bulls for comparative purposes. The Performagene Livestock (DNA Genotek, Kanata, Ontario, Canada) method was compared with similar samples taken from the same animal using manual extraction methods. All samples were analyzed using both the Qubit Quantification Platform (Invitrogen Ltd., Paisley, UK) and NanoDrop spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE) to accurately assess DNA quality and quantity. In general, the automated Maxwell 16 System performed best, consistently yielding high quantity and quality DNA across the sample range tested. Average yields of 28.7, 10.3, and 19.2μg of DNA were obtained from 450μL of blood, 400μL of milk, and a single straw of semen, respectively. The quality of DNA obtained from buffy coat and from semen was significantly higher with the automated method than with the manual methods (260/280 ratio of 1.9 and 1.8, respectively). Centrifugation of whole blood facilitated the concentration of leukocytes in the buffy coat, which significantly increased DNA yield after manual extraction. The Performagene method also yielded 18.4 and 49.8μg of high quality (260/280 ratio of 1.8) DNA from the cow and calf nasal samples, respectively. These results show the advantages of noninvasive sample collection and automated methods for high-throughput extraction and biobanking of high quality DNA.

Comparative evaluation of automated and manual commercial DNA extraction methods for detection of Francisella tularensis DNA from suspensions and spiked swabs by real-time polymerase chain reaction

This study evaluated commercial automated and manual DNA extraction methods for the isolation of Francisella tularensis DNA suitable for real-time polymerase chain reaction (PCR) analysis from cell suspensions and spiked cotton, foam, and polyester swabs. Two automated methods, the MagNA Pure Compact and the QIAcube, were compared to 4 manual methods, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean Microbial DNA isolation kit. The methods were compared using 6 F. tularensis strains representing the 2 subspecies which cause the majority of reported cases of tularemia in humans. Cell viability testing of the DNA extracts showed that all 6 extraction methods efficiently inactivated F. tularensis at concentrations of ≤106 CFU/mL. Real-time PCR analysis using a multitarget 5′ nuclease assay for F. tularensis revealed that the PCR sensitivity was equivalent using DNA extracted by the 2 automated methods and the manual MasterPure and QIAamp methods. These 4 methods resulted in significantly better levels of detection from bacterial suspensions and performed equivalently for spiked swab samples than the remaining 2. This study identifies optimal DNA extraction methods for processing swab specimens for the subsequent detection of F. tularensis DNA using real-time PCR assays. Furthermore, the results provide diagnostic laboratories with the option to select from 2 automated DNA extraction methods as suitable alternatives to manual methods for the isolation of DNA from F. tularensis.

Robust Automatic Rodent Brain Extraction Using 3-D Pulse-Coupled Neural Networks (PCNN)

Brain extraction is an important preprocessing step for further processing (e.g., registration and morphometric analysis) of brain MRI data. Due to the operator-dependent and time-consuming nature of manual extraction, automated or semi-automated methods are essential for large-scale studies. Automatic methods are widely available for human brain imaging, but they are not optimized for rodent brains and hence may not perform well. To date, little work has been done on rodent brain extraction. We present an extended pulse-coupled neural network algorithm that operates in 3-D on the entire image volume. We evaluated its performance under varying SNR and resolution and tested this method against the brain-surface extractor (BSE) and a level-set algorithm proposed for mouse brain. The results show that this method outperforms existing methods and is robust under low SNR and with partial volume effects at lower resolutions. Together with the advantage of minimal user intervention, this method will facilitate automatic processing of large-scale rodent brain studies.

Performance Evaluation of Manual and Automated (MagNA Pure) Nucleic Acid Isolation in HPV Detection and Genotyping Using Roche Linear Array HPV Test

Nucleic acids of human papillomavirus (HPV) isolated by manual extraction method (AmpliLute) and automated MagNA pure system were compared and evaluated with cytohistological findings in 253 women. The concordance level between AmpliLute and MagNA was very good 93.3% (κ = 0.864, P < .0001). Overall HPVpositivity detected by AmpliLute was 57.3% (30.4% as single and 27% as multiple infections) in contrast to MagNA 54.5% (32% and 23%, resp.). Discrepant results observed in 25 cases: 11 MagNA(−)/AmpliLute(+), 10 of which had positive histology; 5 MagNA(+)/AmpliLute(−) with negative histology; 8 MagNA(+)/AmpliLute(+): in 7 of which AmpliLute detected extra HPV genotypes and 1 MagNA(invalid)/AmpliLute(+) with positive histology. Both methods performed well when compared against cytological (area under curve (AUC) of AmpliLute 0.712 versus 0.672 of MagNA) and histological diagnoses (AUC of AmpliLute 0.935 versus 0.877 of MagNA), with AmpliLute showing a slightly predominance over MagNA. However, higher sensitivities, specificities, and positive/negative predictive values were obtained by AmpliLute.

GIS and remote-sensing-based urban-information system design and development: A case study for Kozlu, Zonguldak

Urban growth is inevitable over the next two decades. The bulk of this growth will take place in less developed countries. This growth presents a formidable challenge for urban planners and managers. In this context, we consider some of the ways urban planners can make use of the recent developments in Geographic Information Systems (GIS) and Remote-Sensing (RS) technology to respond to this challenge. GIS projects were initiated in late 1980s in Turkey by both the public and private sectors. Currently, GIS and RS technology play very important roles for urban managers. At the present time, RS technologies are being continually improved; new satellites have been launched with imaging abilities enhanced by new techniques, allowing images to be obtained faster and at higher quality. Current applications use these imagery data for many purposes, among them automatic feature extraction from high-resolution satellite imagery. In this context, automatic and manual object extraction and GIS are the most important issues. In this study, the primary issue was the design and development of Urban Information Systems (UISs); another, more specific, goal was to evaluate the advantages and disadvantages of OrbView-3 and IKONOS imagery for their use in UIS. A test case, consisting of a UIS design, development and application, was executed for the Kozlu, Turkey area. Automatic and manual extraction methods were employed using pan-sharpened IKONOS and OrbView-3 images of the Kozlu urban test area. The NetCAD v5.0, eCognition v4.0.6 and MapInfo v9.0 software packages were used for automatic and manual extraction analyses. Digital reference-vector data were obtained from large-scale digital maps. The extracted results were then compared with a database of reference digital building and road information created from large-scale digital maps in a GIS software package. Thus, utilizing GIS-based analyses, the availability of UIS updating was tested with high-resolution imagery. Key words: Urban information systems, Geographic Information Systems (GIS), Remote-Sensing (RS), high-resolution imagery.

Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic microarray analysis

The human gastrointestinal tract (GI-tract) harbors a complex microbial ecosystem, largely composed of so far uncultured species, which can be detected only by using techniques such as PCR and by different hybridization techniques including phylogenetic microarrays. Manual DNA extraction from feces is laborious and is one of the bottlenecks holding up the application of microarray and other DNA-based techniques in large cohort studies. In order to enhance the DNA extraction step we combined mechanical disruption of microbial cells by repeated bead-beating (RBB) with two automated DNA extraction methods, KingFisher with InviMag Stool DNA kit (KF) and NucliSENS easyMAG (NeM). The semi-automated DNA extraction methods, RBB combined with either KF or NeM, were compared to the manual extraction method currently considered the most suited method for fecal DNA extraction by assessing the yield of 16S rRNA gene copies by qPCR and total microbiota composition by the HITChip, a phylogenetic microarray. Parallel DNA extractions from infant fecal samples by using the three methods showed that the KF and manual methods gave comparable yields of 16S rRNA gene copies as assessed by qPCR, whereas NeM showed a significantly lower yield. All three methods showed highly similar microbiota profiles in HITChip. Both KF and NeM were found to be suitable methods for DNA extraction from fecal samples after the mechanical disruption of microbial cells by bead-beating. The semi-automated methods could be performed in half of the time required for the manual protocol, while being comparable to the manual method in terms of reagent costs.

Comparison of five RNA extraction methods from rabbit’s blood

Extraction of RNA from eukaryotic cells is a crucial stage in many molecular biology studies. Thus it is essential to use the most appropriate method that maximizes the yield and maintains the integrity of extracted RNA. The present work compared five manual RNA extraction methods of different dilutions of rabbit blood. The assessment of the concentration and purity of the extracted RNA were used as parameters to evaluate the efficiency of each method. Statistical analysis showed that the most effective method for RNA extraction from rabbit blood is by following the protocol of the RNeasy Lipid Tissue kit, but it requires the use of toxic reagents. The most efficient kit using no toxic reagents is the RNeasy protect animal blood.

Influence of different extraction methods and PCR techniques on the sensitivity of HCMV-DNA detection in dried blood spot (DBS) filter cards

Background Infection with human cytomegalovirus (HCMV) is the most common congenital virus infection, affecting about 0.5–2% of newborns. Using DBS on Guthrie cards, it is possible to discriminate congenital from postnatal HCMV-infection. However, a recent European trial revealed serious problems in detection of low HCMV-DNA levels from DBS-filter-cards (Barbi et al., 2008). 7 Objectives Evaluation of the most sensitive combination of sample size, DNA extraction method and PCR system for the detection of low copy numbers of HCMV-DNA from DBS-filter-cards. Study design We compared three different manual extraction methods for the detection of HCMV-DNA out of DBS: the QIAmp-blood-Mini-Kit, a heat-extraction-method and traditional phenol–chloroform extraction. Additionally, we tested an automated nucleic acid extraction system (NucliSense EasyMag/Biomerieux). Different punch-sizes of DBS spiked with defined HCMV AD169-DNA copy numbers were analyzed. For detection, we used a quantitative in-house-LightCycler-PCR targeting the gB-region using the hybridisation-probe-format. We compared the sensitivity of the real-time-PCR with IE1Ex4-targeted nested-PCR. Results The highest sensitivity with 200 copies HCMV DNA/ml was achieved using the phenol–chloroform method in combination with the nested-PCR and 6 mm, 3 × 3 mm punches or the whole DBS. The QIAmp-blood-Mini-Kit also showed a very high sensitivity by using the whole DBS and the nested-PCR. Conclusion These results may have strong implications for retrospective diagnosis of congenital HCMV (cHCMV) infection, since a defined combination of the area of punch, the extraction method, and PCR method determine the probability of detection of viral DNA from DBS according to a logistic model.

Implementation and Validation of the Teleshake Unit for DNA IQ™ Robotic Extraction and Development of a Large Volume DNA IQ™ Method

Automated platforms used for forensic casework sample DNA extraction need to be versatile to accommodate a wide variety of sample types, thus protocols frequently need modification. In this study, DNA IQ methods previously developed for the Biomek 2000 Automation Workstation were adapted for the Teleshake Unit using normal volumes and all deepwell extraction, and a large volume DNA IQ method developed. DNA purification without detectable contamination of adjacent reagent blanks is reported in the extraction of tissue samples containing several micrograms of DNA. Sensitivity and contamination studies demonstrated similar performance with the manual organic extraction method for bloodstain dilution samples. Mock casework samples demonstrated the effectiveness of the Teleshake and Teleshake large volume methods. Because of the performance and increased versatility of the DNA IQ extraction with these modifications, the Teleshake Unit has been implemented in both normal and large volume automated DNA extractions at the Virginia Department of Forensic Science.

Evaluation of Automated and Manual Commercial DNA Extraction Methods for Recovery of Brucella DNA from Suspensions and Spiked Swabs

This study evaluated automated and manual commercial DNA extraction methods for their ability to recover DNA from Brucella species in phosphate-buffered saline (PBS) suspension and from spiked swab specimens. Six extraction methods, representing several of the methodologies which are commercially available for DNA extraction, as well as representing various throughput capacities, were evaluated: the MagNA Pure Compact and the MagNA Pure LC instruments, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean microbial DNA isolation kit. These six extraction methods were performed upon three pathogenic Brucella species: B. abortus, B. melitensis, and B. suis. Viability testing of the DNA extracts indicated that all six extraction methods were efficient at inactivating virulent Brucella spp. Real-time PCR analysis using Brucella genus- and species-specific TaqMan assays revealed that use of the MasterPure kit resulted in superior levels of detection from bacterial suspensions, while the MasterPure kit and MagNA Pure Compact performed equally well for extraction of spiked swab samples. This study demonstrated that DNA extraction methodologies differ in their ability to recover Brucella DNA from PBS bacterial suspensions and from swab specimens and, thus, that the extraction method used for a given type of sample matrix can influence the sensitivity of real-time PCR assays for Brucella.

Multicenter Comparative Evaluation of Five Commercial Methods for Toxoplasma DNA Extraction from Amniotic Fluid

Over the past few years, a number of new nucleic acid extraction methods and extraction platforms using chemistry combined with magnetic or silica particles have been developed, in combination with instruments to facilitate the extraction procedure. The objective of the present study was to investigate the suitability of these automated methods for the isolation of Toxoplasma gondii DNA from amniotic fluid (AF). Therefore, three automated procedures were compared to two commercialized manual extraction methods. The MagNA Pure Compact (Roche), BioRobot EZ1 (Qiagen), and easyMAG (bioMérieux) automated procedures were compared to two manual DNA extraction kits, the QIAamp DNA minikit (Qiagen) and the High Pure PCR template preparation kit (Roche). Evaluation was carried out with two specific Toxoplasma PCRs (targeting the 529-bp repeat element), inhibitor search PCRs, and human beta-globin PCRs. The samples each consisted of 4 ml of AF with or without a calibrated Toxoplasma gondii RH strain suspension (0, 1, 2.5, 5, and 25 tachyzoites/ml). All PCR assays were laboratory-developed real-time PCR assays, using either TaqMan or fluorescent resonance energy transfer probes. A total of 1,178 PCRs were performed, including 978 Toxoplasma PCRs. The automated and manual methods were similar in sensitivity for DNA extraction from T. gondii at the highest concentration (25 Toxoplasma gondii cells/ml). However, our results showed that the DNA extraction procedures led to variable efficacy in isolating low concentrations of tachyzoites in AF samples (<5 Toxoplasma gondii cells/ml), a difference that might have repercussions since low parasite concentrations in AF exist and can lead to congenital toxoplasmosis.

A dedicated automated system for extraction, quantification and STR amplification of forensic evidence samples

Forensic DNA analysis is a multi-step process involving extraction of DNA, quantification of human DNA in the extract, amplification using multiplex STR systems, separation of products, and data analysis. The backlog of forensic casework is increasing worldwide. Automation is one significant way to alleviate the bottleneck of sample processing in forensic labs. The HID EVOlution™ Combination System described here is a robust, reliable sample processing platform, easily adapted to forensic laboratory workflows. Using a variety of forensic sample types including: blood stained FTA paper, cotton fabric and denim, dried blood spiked with known PCR inhibitors, saliva on cotton swabs, and semen stains, we found that yields of human DNA and STR profiles obtained with AmpF lSTR ® Idenitfiler ® kits were complete, highly reproducible, and equivalent to results obtained using the manual PrepFiler™ reagent extraction method. Automated operation was clean, and no cross-contamination was detected between extraction blanks and interspersed high DNA content samples.

Optimization of an Oil Extraction Process for Algae from the Treatment of Manure Effluent

AbstractIncreasing interest in the coupling of biological wastewater treatment processes with the generation of value‐added products (such as oil containing ω‐3 fatty acids (FA)) has stimulated efforts in adapting extraction methods for treatment byproducts. This study’s objective was to compare a high temperature/pressure extraction method (accelerated solvent extraction) (ASE) and a manual extraction method (modified Folch extraction) with regard to their ability to extract total oil from three algae samples from the treatment of dairy manure effluent. The efficiency of total oil and FA extraction with three solvents (chloroform/methanol, isopropanol/hexane, and hexane) was also evaluated using the ASE method. Results showed that the ASE method yielded higher values for total oil content compared to the Folch method but similar values for FA content and composition after four extraction cycles with chloroform/methanol. However, the ASE method yielded much higher amounts of FA in the first cycle (85–95% of total extracted) compared to the Folch method (44–55% of total extracted in the first cycle). As expected, the extraction efficiency of the ASE method for FA was dependent on the extraction solvent. FA content values using ASE with chloroform/methanol &gt; isopropanol/hexane &gt; hexane. FA content values using the Folch method or ASE with chloroform/methanol were not significantly influenced by sample particle size within the size range of 0.1–1 mm.

Comparison of 3 nucleic acid isolation methods for the quantification of HIV-1 RNA by Cobas Taqman real-time polymerase chain reaction system

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA viral load in blood is a clinically validated tool for the management and follow-up of patients infected with this virus. HIV-1 RNA quantification can be performed by several kinds of commercially available polymerase chain reaction (PCR) assays. The sensitivity of these assays is affected by the nucleic acid isolation procedures that are performed before PCR. Especially for the samples containing low numbers of HIV-1 RNA, an effective nucleic acid isolation method is very important to obtain a high nucleic acid output. The purpose of this study was to compare and evaluate the conventional manual extraction method, automated MagNA Pure system, and automated AmpliPrep system for the nucleic acid isolation before TaqMan real-time PCR assay for the quantification of HIV-1 RNA in plasma samples. Plasma samples of 40 patients in which anti-HIV antibodies were found as positive serologically were included in this study. Nucleic acid isolation from these samples was performed by manual High Pure Viral Nucleic Acid Isolation kit (Roche Diagnostics, Mannheim, Germany), automated MagNA Pure LC Total Nucleic Acid Isolation kit (Roche Diagnostics), and automated Cobas AmpliPrep kit (Roche Diagnostics). The nucleic acids obtained by these 3 methods were amplified and detected in the Cobas Taqman 48 (Roche Diagnostics) instrument. Viral RNA copies detected by AmpliPrep system, MagNA Pure system, and manual isolation method were found to be between 40 and 420 000 (average, 100 135), 53 and 1 200 000 (average, 220 554), and 183 and 590 000 (average, 104 761) copies/mL, respectively. The results of isolation methods were evaluated for each sample, and it was found out that the number of HIV-1 RNA obtained by MagNA Pure system was higher than the other 2 methods. All of the 3 methods were found to be reliable in detecting very small amounts of HIV-1 RNA in samples. Manual isolation method is a cheaper alternative, but because the nucleic acid isolation can be performed in a shorter time by automated systems when compared with manual isolation method, these systems can be preferred especially in the laboratories that have high number of samples.

Evaluation of Different RNA Extraction Methods and Storage Conditions of Dried Plasma or Blood Spots for Human Immunodeficiency Virus Type 1 RNA Quantification and PCR Amplification for Drug Resistance Testing

The development and validation of dried sample spots as a method of specimen collection are urgently needed in developing countries for monitoring of human immunodeficiency virus (HIV) infection. Our aim was to test some crucial steps in the use of dried spots, i.e., viral recovery and storage over time. Moreover, we investigated whether dried plasma and blood spots (DPS and DBS, respectively) give comparable viral load (VL) results. Four manual RNA extraction methods from commercial HIV type 1 (HIV-1) VL assays--a QIAamp minikit (Qiagen), the Abbott Molecular sample preparation system, the Nuclisens assay (bioMarieux), and High Pure viral nucleic acid kit (Roche Applied Science)--were compared for VL quantification and PCR amplification for genotypic drug resistance testing on dried spots from spiked plasma and residual samples from HIV-1 patients (n = 47; median VL, 4.13 log(10) copies/ml). RNA recovery from DPS was efficient using Nuclisens extraction (median difference, 0.03 log(10) copies/ml) and slightly underestimated using the Abbott Molecular sample preparation system (median difference, 0.35 log(10) copies/ml). PCR amplification results were in concordance. Measurements from DBS overestimated VL for plasma, with VL results showing <3.7 log(10) copies/ml. VL was stable for up to 3 months in spiked DPS stored at 20 degrees C but for only 1 month at 37 degrees C. A faster decline was observed in PCR efficiency: DPS could be stored for 1 week at 37 degrees C and for 1 month at 20 degrees C. In conclusion, the RNA extraction method is an important factor in obtaining reliable RNA quantification and PCR amplification of HIV-1 on DPS/DBS. DBS could be used as an alternative for DPS depending on HIV RNA cutoffs for virological failure. VL measurements remain stable over a longer period than do PCR amplification results.

Evaluation of an Automated Nucleic Acid Extractor for Hepatitis C Virus Load Quantification

The increasing use of molecular methods strongly motivates clinical laboratories to introduce automated nucleic acid extractors. We compared the easyMAG (bioMérieux) with a manual extraction method for hepatitis C virus (HCV) load quantification (RealTime HCV; Abbott). Both methods were comparable, and, therefore, the easyMAG is suitable to be implemented in our laboratory for the management of HCV-infected patients.

Evaluation of automated nucleic acid extraction methods for virus detection in a multicenter comparative trial

Five European veterinary laboratories participated in an exercise to compare the performance of nucleic acid extraction robots. Identical sets of coded samples were prepared using serial dilutions of bovine viral diarrhoea virus (BVDV) from serum and cell culture propagated material. Each laboratory extracted nucleic acid from this panel using available robotic equipment (12 separate instruments, comprising 8 different models), after which the processed samples were frozen and sent to a single laboratory for subsequent testing by real-time RT-PCR. In general, there was good concordance between the results obtained for the different automated extraction platforms. In particular, the limit of detection was identical for 9/12 and 8/12 best performing robots (using dilutions of BVDV infected-serum and cell culture material, respectively), which was similar to a manual extraction method used for comparison. The remaining equipment and protocols used were less sensitive, in an extreme case for serum, by a factor of 1000. There was no evidence for cross-contamination of RNA template in any of the negative samples included in these panels. These results are not intended to replace local optimisation and validation, but provide reassurance to laboratories to indicate that the best performing optimised nucleic acid extraction systems can have similar performance.