Nucleic Acid Extraction Platforms Research Articles

O-279 The lower reproductive tract microbiota composition alters upon ovarian stimulation and is associated with, but not accurately predictive of reproductive outcomes following ART

Abstract Study question Are ovarian stimulation (OS) and ART success associated with the microbiota of the lower reproductive tract (LRT), i.e. vagina and cervix? Summary answer Estrogen rise during OS has a pivotal role in the LRT microbiota variation. The LRT microbiota pre-OS, although associated, may not accurately predict ART success. What is known already OS is a key component to enhance success rates in ART. OS triggers a significant rise in serum estrogen levels, which are known to influence the vaginal microbiota. The role of the female LRT microbiota in ART success is increasingly recognized, yet the impact of OS, particularly the estrogen rise, on these microbial communities and how the change may influence ART success remains largely unknown. Additionally, finding putative biomarkers within LRT microbiota could aid in predicting ART success. Understanding this relationship could refine and personalize fertility treatments, but robust evidence is needed to guide microbiota-based clinical decisions in ART. Study design, size, duration Vaginal and cervical swabs were collected longitudinally from patients undergoing ART and this at baseline (pre-OS) and at oocyte retrieval (post-OS). We gathered a final dataset comprising 275 vaginal samples at baseline and 240 samples at oocyte retrieval. Additionally, we defined a prospective cohort of n = 228x2 samples corresponding to patients for which both pre- and post-OS samples were available. The main results as detected within the vaginal samples were confirmed in the cervical samples. Participants/materials, setting, methods Bacterial DNA from −80 °C preserved swabs was extracted in a Tecan Nucleic Acid Extraction Platform, using the QIAamp DNA-Stool-Mini-Kit. Samples for paired-end Illumina MiSeq were constructed using a two-step PCR amplicon approach targeting the V4 region of the 16S rRNA. Then, LotuS, DADA2, the RDP classifier trainset 16 and variance stabilisation (poscounts, DESeq2) were used for read de-multiplexing, pre-processing, taxonomy assignment and transformation, respectively, as recommended for zero-inflated compositional data. Main results and the role of chance First, a confounder analysis revealed that both the menstrual phase and intercourse correlated with the LRT’s baseline microbiota composition (multivariate distance-based redundancy analysis [dbRDA], AdjP<0.05, N = 275). Next, in the paired cohort, we observed that the OS-associated rise in estrogen was correlated with confounder-independent compositional (confounder-adjusted dbRDA, AdjP=0.002, R2=1.14%, N = 228x2) and microbial-feature changes (Wilcoxon test, AdjP<0.05, Ndelta=228) in the LRT. Interestingly, post-OS we noted a decrease in Lactobacillus proportions compared to baseline (in participants sampled at either the follicular or luteal phase of the menstrual cycle). Additionally, post-OS microbiota composition was not associated with the live birth rate (LBR, following the first embryo transfer of the cycle) or the cumulative LBR (CLBR, following all embryo transfers of the cycle), nor with the total number of high-quality (HQ) embryos available (dbRDA, AdjP>0.05, N = 240). Notably, pre-OS microbiota communities and LRT Anaerococcus and Peptoniphilus proportions were associated with (C)LBR (confounder-adjusted dbRDA, AdjP<0.05; Logistic regression, odds-ratio<1, AdjP<0.05, N = 275). Machine learning models determined that vaginal Peptoniphilus and Anaerococcusproportions alone did not have good accuracy/ROC for predicting (C)LBR (test-set;<70%). Notably, the number of HQ embryos showed good accuracy/ROC for CLBR (test-set; 80%/84.5%). Including vaginal Anaerococcus proportions only slightly improved the model’s accuracy/ROC (test-set; 82.5%/85.6%). Limitations, reasons for caution The data’s proportional nature restricted assessing whether the estrogen rise decreased Lactobacillus proportions or increased other bacteria. Luteal phase sampling was avoided given the intravaginal progesterone support. Larger datasets may further improve predictive models. Wider implications of the findings Our study highlights the confounding impact of baseline variables and the influence of the OS-associated estrogen rise on LRT microbiota. It questions the clinical relevance of the LRT microbiota’s impact on reproductive outcomes following ART, warning for caution in microbiota-based decisions in ART. Trial registration number NCT03105453

Evaluation of the Roche MagNA Pure 96 nucleic acid extraction platform for the SPF10 line probe assay system.

To evaluate the MagNA Pure 96 (MP96) nucleic acid extraction system as an alternative for cervical specimen processing for human papillomavirus (HPV) genotyping detection by reverse hybridization line probe assay (LiPA-25), compared to the well-established extraction system MagNA Pure LC 2.0 (MPLC). A total of 200 cervical samples preserved in ThinPrepCyt® solution were extracted by MP96 and MPLC, respectively, and then purified nucleic acids were amplified using the SPF10 PCR primer set. Amplification products were subjected to SPF10-DNA enzyme immunoassay (DEIA) and LiPA-25. The concordance between different extraction methods in this study was reflected in the comparison of the results of the DEIA and LiPA-25. Agreement of HPV-positive results (DEIA) was 97.5% (Kappa=0.932). Pair-wise analyses of either HPV grouping (any HPV genotypes, any high-risk HPV genotypes, any low-risk HPV genotypes, any nonavalent vaccine-targeted HPV, and any non-vaccine-targeted oncogenic HPV genotypes) identified >95% agreement (all Kappa>0.900). For the two extraction methods, there was no statistical difference (chi-square test: P=0.690) for single versus multiple genotypes, and concordant, compatible, and discordant genotypes were observed in 87.0%, 9.5%, and 3.5% of 200 samples, respectively. HPV genotyping results of the MPLC system and the MP96 system showed a high degree of concordance. Combined with the advantages of high-throughput and anti-contamination of MP96, the MP96 extraction system could be more suitable for the testing of samples in future studies.

A wash-free, elution-free and low protein adsorption paper-based material for nucleic acid extraction.

Nucleic acid detection technologies have been widely utilized for various diseases. Conventional laboratory tests are less suitable for use in resource-limited settings as they are time-consuming, high-cost, complex, and heavily dependent on benchtop equipment. Rapid nucleic acid detection methods that consist of rapid nucleic acid extraction steps could overcome these challenges. A paper-based platform has been utilized to develop various rapid nucleic acid extraction methods owing to its cost-effectiveness, portability, and easy-modification. However, the existing paper-based nucleic acid extraction technologies mainly focus on improving the adsorption capacity of nucleic acids without reducing the non-specific adsorption capacity of proteins. In this study, paper-based nucleic acid extraction technology with wash-free, elution-free, and low protein adsorption was developed. The fabrication of paper involves the mixing of polyethylene glycol (PEG)-modified cotton fiber, chitosan (COS)-modified cotton fiber, and cotton fiber to form PEG-modified cotton fiber/chitosan-modified cotton fiber/cotton fiber (PEG-CF/COS-CF/CF) paper by the wet molding method. The result showed that PEG-CF/COS-CF/CF paper has a desirable pore size (23.9 ± 4.03 μm), good mechanical strength (dry: 9.37 Mpa and wet: 0.28 Mpa), and hydrophilicity (contact angle: 42.6° ± 0.36°). NH3+ groups of COS and OH- groups of PEG were observed on its surface and the adsorption efficiency of nucleic acid in TE buffer was 42.48% ± 0.30%. The limit of detection of pure DNA with this PEG-CF/COS-CF/CF paper by qPCR was as low as 25 ng. Additionally, this platform could successfully extract nucleic acid from 30 μL of a saliva sample, highlighting its potential use for clinical sample testing. The proposed paper-based nucleic acid extraction platform shows tremendous potential for disease diagnosis in resource-limited settings.

Assay System for Simultaneous Detection of SARS-CoV-2 and Other Respiratory Viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggers disease with nonspecific symptoms that overlap those of infections caused by other seasonal respiratory viruses (RVs), such as the influenza virus (Flu) or respiratory syncytial virus (RSV). A molecular assay for accurate and rapid detection of RV and SARS-CoV-2 is crucial to manage these infections. Here, we compared the analytical performance and clinical reliability of Allplex™ SARS-CoV-2/FluA/FluB/RSV (SC2FabR; Seegene Inc., Seoul, South Korea) kit with those of four commercially available RV detection kits. Upon testing five target viral strains (SARS-CoV-2, FluA, FluB, RSV A, and RSV B), the analytical performance of SC2FabR was similar to that of the other kits, with no significant difference (p ≥ 0.78) in z-scores. The efficiency of SC2FabR (E-value, 81–104%) enabled reliable SARS-CoV-2 and seasonal RV detection in 888 nasopharyngeal swab specimens processed using a fully automated nucleic acid extraction platform. Bland–Altman analyses revealed an agreement value of 95.4% (SD ± 1.96) for the kits, indicating statistically similar results for all five. In conclusion, SC2FabR is a rapid and accurate diagnostic tool for both SARS-CoV-2 and seasonal RV detection, allowing for high-throughput RV analysis with efficiency comparable to that of commercially available kits. This can be used to help manage respiratory infections in patients during and after the coronavirus disease 2019 pandemic.

Evaluation of the Roche MagNA Pure 96 nucleic acid extraction platform for the Seegene Anyplex II HPV28 detection assay.

Validate the Roche, MagNAPure96 (MP96) nucleic acid extraction platform for Seegene Anyplex II HPV28 (Anyplex28) detection of Human Papillomavirus. Comparisons were made for Anyplex28 genotyping from 115 cervical samples extracted on the Hamilton, STARlet and the MP96. Two DNA concentrations were used for the MP96, one matched for sample input to the STARlet and another 5× concentration (laboratory standard). Agreement of HPV detection was 89·8% (κ=0·798; P=0·007), with HPV detected in 10 more samples for the MP96. There was a high concordance of detection for any oncogenic HPV genotype (κ=0·77; P=0·007) and for any low-risk HPV genotype (κ=0·85; P=0·008). DNA extracted at laboratory standard had a lower overall agreement 85·2% (κ=0·708; P<0·001), with 17/115 discordant positive samples that tested negative after STARlet extraction. Of the discordant genotypes, 72·7% were detected in the lowest signal range for Anyplex28 ('+'). MP96 performed with high concordance to STARlet, although produced DNA with a higher analytical sensitivity on the Anyplex28. This analysis supports the use of samples extracted on the MP96 for HPV genotyping using the Anyplex28. Furthermore, an increase in DNA concentration increased analytical sensitivity of the Anyplex28, particularly appropriate for prevalence studies.

Impact of nucleic acid extraction platforms on hepatitis virus genome detection

Detection and quantification of viral nucleic acids are important for diagnosing current viral infections and monitoring response to antiviral therapy. Automated nucleic acid extraction and purification platforms are routinely used during the first step in these processes in clinical and research laboratories. Here, we compare the extraction efficiencies of four MagNA Pure magnetic bead-based nucleic acid extraction platforms and associated kits using samples positive for nucleic acids from HAV, HBV, HCV, HDV, and HEV. These five hepatitis viruses are diverse in their virion structures and type of nucleic acid that compose their genomes. We found that the most efficient nucleic acid extraction platform and corresponding kit, when averaged across all tested viruses, was the MagNA Pure 96, which yielded twice as much detectable nucleic acid as the other platforms. However, the relative efficiencies of the different platforms varied by virus type, suggesting that an extraction platform that is more efficient for one virus type will not necessarily function better with a different virus type. Our results show that the choice of a nucleic acid extraction platform influences the sensitivity of the methodology and has the potential to generate false-negative results especially in samples with low levels of viral nucleic acids.

Automated Extraction of Microbial DNA From Formalin-Fixed, Paraffin-Embedded Tissue Using the Siemens Tissue Preparation System

We describe the adoption of an automated nucleic acid extraction platform, Siemens Tissue Preparation System (TPS), for use with formalin-fixed, paraffin-embedded (FFPE) tissues intended for PCR microbial testing. The TPS uses a liquid handling robot to automate deparaffinization, followed by extraction and purification of nucleic acids using magnetic particle-based isolation. While it has been used previously for human DNA extraction, this is the first incorporation of the TPS into clinical microbiology practice for microorganism detection to our knowledge. Our laboratory transitioned to use of the TPS from manual deparaffinization of FFPE tissue with subsequent DNA extraction using Roche MagNA Pure (MP) instruments (manual/MP method). Unpublished data from our group demonstrated that the TPS performed better than the manual/MP method for FFPE tissue extraction (27% higher positivity in 86 FFPE specimens tested by both methods). We tracked total testing volumes, laboratory efficiency, TPS errors, and workflow. Additionally, we added a microtome instrument to our clinical microbiology laboratory to section FFPE blocks in order to increase efficiency by avoiding reliance on the histopathology laboratory. Since implementation, 710 FFPE specimens were processed on the TPS, with 724 downstream PCR tests run on extracts for eight laboratory-developed assays: Mycobacterium tuberculosis (MTB), Bartonella spp, Tropheryma whipplei, Coxiella burnetii, Kingella kingae, Coccidioides spp, Acanthamoeba spp, and Balamuthia mandrillaris/Naegleria fowleri. The most common tests run after TPS extraction included 468 (64.6%) MTB, 156 (21.5%) Bartonella, and 70 (9.7%) T whipplei; positivity rates were 4.9%, 6.4%, and 4.3%, respectively. Regarding workflow, use of the TPS decreased total processing time by approximately 70%. The TPS workflow from tissue block sectioning to DNA extraction averaged a total of 3.2 hours, of which 0.7 hours (21.8%) were hands-on time (microtome sectioning and TPS specimen loading/unloading). Comparatively, our manual/MP method averaged 10.6 hours, of which 1.1 hours (10.5%) involved hands-on time (microtome sectioning, manual deparaffinization using xylene and 95% ethanol, and MP loading/unloading). Approximately 7 hours of the manual/MP method were devoted to proteinase K lysis. The TPS accommodates 48 specimens per run, with each run lasting 2.5 hours. Our laboratory currently performs one run daily with an average of four specimens per run. Eleven (6.25%) failure events were noted in 176 runs over the past 9 months. Nine (82% of the errors) were attributed to robotic mechanical problems and two (18%) to human errors (reagent misplacement). The system software alerts the user when errors occur so the problem can be fixed and/or the run repeated. In conclusion, the automated TPS extraction system for FFPE is rapid and limits exposure to hazardous chemicals with minimal run failures. Transition of FFPE sectioning to the microbiology laboratory from the histology laboratory also increased efficiency.

Evaluation of a new extraction platform in combination with molecular assays useful for monitoring immunosuppressed patients

BackgroundIn the immunosuppressed, detection of viral reactivation at the earliest convenience and molecular monitoring are of paramount importance. Nucleic acid extraction has a major impact on the reliability of results obtained from molecular assays. ObjectivesThe aim of this study was to investigate the accuracy of the new EMAG® nucleic acid extraction platform and to compare the performance of the new platform to that of the standard NucliSENS® easyMAG® instrument in the routine clinical laboratory. Study designFor accuracy testing, reference material and for comparison studies, clinical specimens were used. In addition, a lab-flow analysis including estimation of hands-on time and that for automated extraction was performed. ResultsWhen accuracy was tested, all 89 results obtained were found to be concordant with the results expected. When 648 clinical results were compared, 85.7% were found to be within ±0.5 log10 unit, 9.5% between ±0.5 and ±1.0 log10 unit, and 4.8% more than ±1.0 log10 unit. The overall time required for nucleic acid extraction of 8 samples in parallel was 94 min for the fully automated extraction mode and 82 min for the partly automated mode with the new platform, and 73 min with the standard instrument. Hands-on time was found to be shorter with the new platform. ConclusionsThe extraction performance of both platforms was found to be similar for EDTA whole blood, BAL, and urine specimens. The total turn-around time for nucleic acid extraction was found to be longer with the EMAG® platform, whereas hands-on time was reduced.

Comparison of eMAG™ versus NucliSENS® EasyMAG® performance on clinical specimens

BackgroundeMAG™ (bioMerieux) is a new nucleic acid extraction platform based on magnetic silica technology, like its predecessor, NucliSENS® easyMAG® (bioMerieux). Using the same reagents and disposables, eMAG™ adds further automation, allowing simultaneous extraction of 48 samples directly from primary tubes, and distribution of nucleic acid extracts on PCR strips or in tubes at the end of the extraction process. ObjectiveTo compare the performance of eMAG™ and easyMAG® on various clinical specimens. Study designRespiratory (n=199), whole blood (n=50), plasma (n=25) and urine (n=25) specimens were extracted in parallel on both platforms. Both qualitative (respiratory virus, cell control, CMV, EBV, HHV6 and BKV detection) and quantitative (respiratory virus and cell control cycle thresolds, and CMV, EBV, HHV6 and BKV viral loads) results were compared. ResultsDetection of qualitative targets showed good agreement, ranging from 84.6% for whole blood to 95.9% for respiratory specimens. Correlations between quantitative results were good, with R2 ranging from 0.802 to 0.995. Quantitative results showed average overall differences below 0.10 log10 copies/mL between eMAG™ and easyMAG®. ConclusionsThe two platforms showed comparable performance on the types of clinical specimen tested. With higher automation and throughput than easyMAG®, the eMAG™ platform is likely to be advantageous for laboratories performing a large number of molecular analyses.

The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing

Extraction of viral RNA and the storage of sample material are extremely important factors in the detection and whole genome sequencing (WGS) of viral pathogens. Although PCR-based detection methods focus on small amplicons, viral WGS applications require RNA of high quality and integrity for adequate sequence coverage and depth. This study examined the fitness of one manual and four automated RNA extraction platforms commonly used in diagnostic laboratories for use in metagenomic sequencing, how the practice of storing sample material in Qiagen buffer AVL before extraction affected the integrity of viral RNA and its suitability for use in amplicon-based WGS methods, and how the addition of Triton X-100 to buffer AVL affected the capability of the extraction platforms and the integrity of viral RNA in stored samples. This study found that the EZ1 platform gave the best performance of the automated platforms and gave comparable results to the frequently used manual Qiagen extraction protocol when extracted viral RNA was used in metagenomics sequencing. To maintain high levels of viral RNA integrity suitable for amplicon-based WGS, nucleic acid should be extracted from samples immediately, because even short storage periods in buffer AVL have a severe effect on integrity, and the addition of Triton X-100 had little effect on the quality of viral material for WGS.

Integrated sample-to-detection chip for nucleic acid test assays.

Nucleic acid based diagnostic techniques are routinely used for the detection of infectious agents. Most of these assays rely on nucleic acid extraction platforms for the extraction and purification of nucleic acids and a separate real-time PCR platform for quantitative nucleic acid amplification tests (NATs). Several microfluidic lab on chip (LOC) technologies have been developed, where mechanical and chemical methods are used for the extraction and purification of nucleic acids. Microfluidic technologies have also been effectively utilized for chip based real-time PCR assays. However, there are few examples of microfluidic systems which have successfully integrated these two key processes. In this study, we have implemented an electro-actuation based LOC micro-device that leverages multi-frequency actuation of samples and reagents droplets for chip based nucleic acid extraction and real-time, reverse transcription (RT) PCR (qRT-PCR) amplification from clinical samples. Our prototype micro-device combines chemical lysis with electric field assisted isolation of nucleic acid in a four channel parallel processing scheme. Furthermore, a four channel parallel qRT-PCR amplification and detection assay is integrated to deliver the sample-to-detection NAT chip. The NAT chip combines dielectrophoresis and electrostatic/electrowetting actuation methods with resistive micro-heaters and temperature sensors to perform chip based integrated NATs. The two chip modules have been validated using different panels of clinical samples and their performance compared with standard platforms. This study has established that our integrated NAT chip system has a sensitivity and specificity comparable to that of the standard platforms while providing up to 10 fold reduction in sample/reagent volumes.

Comparison of BKV quantification using a single automated nucleic acid extraction platform and 3 real-time PCR assays

Monitoring peripheral blood for evidence of BK viremia through quantitative real-time PCR testing is an important management tool that allows for interventions that prevent nephropathy in renal allograft patients. This study compared the performance of 3 different real-time PCR assays for BKV quantification including 2 noncommercial tests (a historical assay “PEP” and 1 with improved genotypic inclusivity “V3T3”) and 1 using commercial reagents (Qiagen/artus, “artus”) after nucleic acid extraction of plasma with a single automated instrument (QIAsymphony). The measurable ranges (log10 copies/mL) were 2.7 to at least 8.0 for PEP and 2.0 to at least 8.0 for artus and V3T3 assays. Limits of detection (copies/mL) were 189, 56, and 28 for PEP, V3T3, and artus, respectively. Correlation experiments demonstrated linearity with original quantification results, although values obtained for the PEP assay were generally lower than those obtained for the V3T3 or artus assay across the measuring range. V3T3 and artus values were more closely related, although artus values were generally lower. The 3 assays returned different values from clinical plasma samples, likely due in part to variances in calibration. Low BKV concentrations were quantifiable by V3T3 and artus assays in plasmas that were previously deemed negative by PEP. These data underscore the need for monitoring with a single test to enable appropriate management decisions, and they suggest that an international preparation would be useful in harmonizing quantification of BKV viremia.

Comparison of nucleic acid extraction platforms for detection of select biothreat agents for use in clinical resource limited settings

High-quality nucleic acids are critical for optimal PCR-based diagnostics and pathogen detection. Rapid sample processing time is important for the earliest administration of therapeutic and containment measures, especially in the case of biothreat agents. In this context, we compared the Fujifilm QuickGene-Mini80 to Qiagen's QIAamp Mini Purification kits for extraction of DNA and RNA for potential use in austere settings. Qiagen (QIAamp) column-based extraction is the currently recommended purification platform by United States Army Medical Research Institute for Infectious Diseases for both DNA and RNA extraction. However, this sample processing system requires dedicated laboratory equipment including a centrifuge. In this study, we investigated the QuickGene-Mini80, which does not require centrifugation, as a suitable platform for nucleic acid extraction for use in resource-limited locations. Quality of the sample extraction was evaluated using pathogen-specific, real-time PCR assays for nucleic acids extracted from viable and γ-irradiated Bacillus anthracis, Yersinia pestis, vaccinia virus, Venezuelan equine encephalitis virus, or B. anthracis spores in buffer or human whole blood. QuickGene-Mini80 and QIAamp performed similarly for DNA extraction regardless of organism viability. It was noteworthy that γ-irradiation did not have a significant impact on real-time PCR for organism detection. Comparison with QIAamp showed a less than adequate performance of the Fujifilm instrument for RNA extraction. However, QuickGene-Mini80 remains a viable alternative to QIAamp for DNA extraction for use in remote settings due to extraction quality, time efficiency, reduced instrument requirements, and ease of use.

Extraction of viral nucleic acids: Comparison of five automated nucleic acid extraction platforms

BackgroundNucleic acid extraction has a major impact on the reliability of results in routine molecular diagnostics. Optimal isolation of nucleic acids and removal of inhibitors are essential. ObjectivesThis study compares five different automated extraction platforms for the extraction of norovirus RNA from stool and cytomegalovirus (CMV) DNA from plasma samples. Study designNorovirus positive stool samples and CMV positive plasma samples were aliquoted and analyzed using five different automated platforms (easyMAG, bioMerieux; m2000sp, Abbott; MagNA Pure LC 2.0, Roche; QiaSymphony, Qiagen; sample preparation module of the VERSANT kPCR Molecular System, Siemens). Similar sample input and output volumes, the identical real-time PCR cycler, and the identical assays for amplification and detection for norovirus RNA and CMV DNA, respectively, were chosen. ResultsOf 39 stool samples, 36 tested positive for norovirus RNA with all extraction platforms. The three discrepant samples showed inhibition after extraction with at least one platform. Only with the VERSANT platform all samples tested positive for both the target RNA and the internal controls. Of 42 plasma samples, 27 gave quantifiable results for CMV DNA with all extraction platforms. There was significant variance between viral concentrations when different extraction platforms were compared. The majority of the 15 discrepant samples showed low viral concentrations. The internal control of the CMV assay gave positive results for all samples tested below the limit of quantification. ConclusionsThe five automated extraction platforms yielded comparable results. However, the extraction performance was found to be impaired by inhibitory substances in stool samples.

Comparative procedures for sample processing and quantitative PCR detection of grapevine viruses

In this study different instruments and methods used for tissue homogenization, RNA extraction and quantitative PCR (qPCR) based detection of grapevine RNA viruses were evaluated. Semi-automated and automated homogenization techniques were compared to process samples from grapevine petioles and cambial tissue. Four different high throughput automated nucleic acid extraction platforms were compared with the RNeasy plant extraction kit for their capacity and efficiency of extracting viral RNA from grapevine infected tissues. The RNA prepared from each extraction platform was then used as template for a comparative analysis of qPCR by One Step RT-qPCR, Two Step RT-qPCR and low density array (LDA) detection. This study showed that a thorough homogenization of grapevine tissues using the Tissue Lyser as well as DNase digestion of the purified RNA prior to cDNA synthesis improved the virus detection and yielded the lowest quantitation cycle (Cq) values in RT-qPCR. Comparison of different RNA extraction methods showed that methods implementing the magnetic bead-based technology were superior to other methods used. Comparing different qPCR detection methods, One Step RT-qPCR showed the lowest Cq values for the same sample tested compared to Two Step RT-qPCR and LDA.

Performance evaluation of the automated NucliSens easyMAG nucleic acid extraction platform in comparison with QIAamp Mini kit from clinical specimens

The performance of the NucliSens easyMAG platform for the extraction of nucleic acid from different clinical specimens was compared with a manual procedure. A total of 308 specimens were analyzed: 209 plasma samples collected for virus detection and quantification of cytomegalovirus (CMV) and Epstein–Barr virus (EBV) ( n = 70), and 29 for HIV genotyping for drug resistance. Linearity of extraction was tested on dilution series of CMV and EBV; the correlation coefficient ( R 2) for standard curves based on repeated extraction runs was 0.99 for CMV and EBV. Inter- and intrarun variability was in accordance with previous studies, and the correlation between automated and manual extraction was very high. The concordant results were 95.7% for CMV and 100% for EBV. The results of sequence analysis for HIV drug resistance showed a concordance in 24 of 29 specimens. The NucliSens easyMAG is extremely easy to perform, is automated, and resulted in a strong reduction of hands-on time compared with manual protocol.

Clinical Evaluation of NucliSENS Magnetic Extraction and NucliSENS Analyte-Specific Reagents for Real-Time Detection of Human Metapneumovirus in Pediatric Respiratory Specimens

In this study, we evaluated the NucliSENS miniMAG (MM) and easyMAG (EM) nucleic acid extraction platforms (bioMérieux, Durham, NC) in combination with the NucliSENS EasyQ basic kit and analyte-specific reagents (ASRs) (bioMérieux) for the detection of human metapneumovirus (hMPV) in respiratory samples. Total nucleic acids from pediatric clinical samples (n = 653) and an hMPV-specific inhibition control (h-IC) were coextracted using the MM and/or the EM. Nucleic acid sequence-based amplification and real-time molecular beacon detection of hMPV were performed using a NucliSENS EasyQ analyzer (bioMérieux). Positive results were confirmed using an in-house-validated reverse transcriptase PCR ASR-based assay. The inclusion of the h-IC monitored the entire process, including the efficiency of nucleic acid extraction, amplification, and detection. The percentages of samples with inhibited amplification of the h-IC after initial NA extraction by EM and MM were 1.88% and 3.17%, respectively. After reprocessing of a new aliquot, the final h-IC inhibition rates were 0% (EM) and 1.06% (MM). The limit of detection of the assay was between 2 (EM extraction) and 10 (MM extraction) RNA copies/reaction, and specificity was 100% when testing viral respiratory isolates and clinical samples. hMPV was detected in 5.6% of pediatric samples tested and was also detected in three coinfections with respiratory syncytial virus (RSV). hMPV was the second most frequently detected respiratory virus in children of 0 to 2 years of age, after RSV. In summary, NucliSENS extraction and ASRs provided a sensitive and specific method for the detection of hMPV in respiratory samples.

Automated extraction protocol for quantification of SARS-Coronavirus RNA in serum: an evaluation study

BackgroundWe have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (SARS) based on the detection of the SARS-coronavirus RNA in serum by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). In this study, we evaluated the feasibility of automating the serum RNA extraction procedure in order to increase the throughput of the assay.MethodsAn automated nucleic acid extraction platform using the MagNA Pure LC instrument (Roche Diagnostics) was evaluated. We developed a modified protocol in compliance with the recommended biosafety guidelines from the World Health Organization based on the use of the MagNA Pure total nucleic acid large volume isolation kit for the extraction of SARS-coronavirus RNA. The modified protocol was compared with a column-based extraction kit (QIAamp viral RNA mini kit, Qiagen) for quantitative performance, analytical sensitivity and precision.ResultsThe newly developed automated protocol was shown to be free from carry-over contamination and have comparable performance with other standard protocols and kits designed for the MagNA Pure LC instrument. However, the automated method was found to be less sensitive, less precise and led to consistently lower serum SARS-coronavirus concentrations when compared with the column-based extraction method.ConclusionAs the diagnostic efficiency and prognostic value of the serum SARS-CoV RNA RT-PCR test is critically associated with the analytical sensitivity and quantitative performance contributed both by the RNA extraction and RT-PCR components of the test, we recommend the use of the column-based manual RNA extraction method.

Early detection of HCV using MONOLISA HCV Ag-Ab ULTRA

eMAG™ (bioMerieux) is a new nucleic acid extraction platform based on magnetic silica technology, like its predecessor, NucliSENS® easyMAG® (bioMerieux). Using the same reagents and disposables, eMAG™ adds further automation, allowing simultaneous extraction of 48 samples directly from primary tubes, and distribution of nucleic acid extracts on PCR strips or in tubes at the end of the extraction process.To compare the performance of eMAG™ and easyMAG® on various clinical specimens.Respiratory (n = 199), whole blood (n = 50), plasma (n = 25) and urine (n = 25) specimens were extracted in parallel on both platforms. Both qualitative (respiratory virus, cell control, CMV, EBV, HHV6 and BKV detection) and quantitative (respiratory virus and cell control cycle thresolds, and CMV, EBV, HHV6 and BKV viral loads) results were compared.Detection of qualitative targets showed good agreement, ranging from 84.6% for whole blood to 95.9% for respiratory specimens. Correlations between quantitative results were good, with R2 ranging from 0.802 to 0.995. Quantitative results showed average overall differences below 0.10 log10 copies/mL between eMAG™ and easyMAG®.The two platforms showed comparable performance on the types of clinical specimen tested. With higher automation and throughput than easyMAG®, the eMAG™ platform is likely to be advantageous for laboratories performing a large number of molecular analyses.