Column-based Extraction Method Research Articles

Rapid, Portable, and Electricity-free Sample Extraction Method for Enhanced Molecular Diagnostics in Resource-Limited Settings.

The COVID-19 pandemic has highlighted the need for rapid and reliable diagnostics that are accessible in resource-limited settings. To address this pressing issue, we have developed a rapid, portable, and electricity-free method for extracting nucleic acids from respiratory swabs (i.e. nasal, nasopharyngeal and buccal swabs), successfully demonstrating its effectiveness for the detection of SARS-CoV-2 in residual clinical specimens. Unlike traditional approaches, our solution eliminates the need for micropipettes or electrical equipment, making it user-friendly and requiring little to no training. Our method builds upon the principles of magnetic bead extraction and revolves around a low-cost plastic magnetic lid, called SmartLid, in combination with a simple disposable kit containing all required reagents conveniently prealiquoted. Here, we clinically validated the SmartLid sample preparation method in comparison to the gold standard QIAamp Viral RNA Mini Kit from QIAGEN, using 406 clinical isolates, including 161 SARS-CoV-2 positives, using the SARS-CoV-2 RT-qPCR assays developed by the US Centers for Disease Control and Prevention (CDC). The SmartLid method showed an overall sensitivity of 95.03% (95% CI: 90.44-97.83%) and a specificity of 99.59% (95% CI: 97.76-99.99%), with a positive agreement of 97.79% (95% CI: 95.84-98.98%) when compared to QIAGEN's column-based extraction method. There are clear benefits to using the SmartLid sample preparation kit: it enables swift extraction of viral nucleic acids, taking less than 5 min, without sacrificing significant accuracy when compared to more expensive and time-consuming alternatives currently available on the market. Moreover, its simplicity makes it particularly well-suited for the point-of-care where rapid results and portability are crucial. By providing an efficient and accessible means of nucleic acid extraction, our approach aims to introduce a step-change in diagnostic capabilities for resource-limited settings.

DNA Extraction Methods

KATIE LONG and MARK FLOOD, Forensic Science Program, Fairmont State University, Fairmont, WV 26554. Evaluation of DNA Extraction Methods. Three forms of DNA extraction types include solution-based, column-based, and magnetic bead-based methodologies, with the latter being the accepted methodology used in forensic laboratories. Magnetic bead-based DNA extraction uses the negative charge of DNA and the positive charge of the magnetic beads to isolate the DNA strands and produce a purified sample. However, solution and column-based extraction methods use the same techniques of washing to purify DNA and typically have comparable DNA yields. Cheek swabs were obtained from ten individuals and the three extraction methods were performed to determine the quantitation of double-stranded DNA and the quality of the gel electrophoresis profile. Preliminary results indicate that solution-based extraction produces a slightly higher yield of double-stranded DNA having a 3.92% difference between the average quantitation of 26.29 ng/µL from solution-based and 25.28 ng/µL from magnetic bead-based methods. However, it is expected that the magnetic bead-based extraction will produce higher quality gel electrophoresis results. Overall, the methods examined produced similar DNA extraction efficiencies. This research was made possible by NASA West Virginia Space Grant Consortium, Training Grant #80NSSC20M0055 and the STaR Sure Grant.

Comparing analytical methods to detect SARS-CoV-2 in wastewater

Wastewater based epidemiology (WBE) has emerged as a reliable strategy to assess the coronavirus disease 2019 (COVID-19) pandemic. Recent publications suggest that SARS-CoV-2 detection in wastewater is technically feasible; however, many different protocols are available and most of the methods applied have not been properly validated. To this end, different procedures to concentrate and extract inactivated SARS-CoV-2 and surrogates were initially evaluated. Urban wastewater seeded with gamma-irradiated SARS-CoV-2, porcine epidemic diarrhea virus (PEDV), and mengovirus (MgV) was used to test the concentration efficiency of an aluminum-based adsorption-precipitation method and a polyethylene glycol (PEG) precipitation protocol. Moreover, two different RNA extraction methods were compared in this study: a commercial manual spin column centrifugation kit and an automated protocol based on magnetic silica beads. Overall, the evaluated concentration methods did not impact the recovery of gamma-irradiated SARS-CoV-2 nor MgV, while extraction methods showed significant differences for PEDV. Mean recovery rates of 42.9 ± 9.5%, 27.5 ± 14.3% and 9.0 ± 2.2% were obtained for gamma-irradiated SARS-CoV-2, PEDV and MgV, respectively. Limits of detection (LoD95%) for five genomic SARS-CoV-2 targets (N1, N2, gene E, IP2 and IP4) ranged from 1.56 log genome equivalents (ge)/mL (N1) to 2.22 log ge/mL (IP4) when automated system was used; while values ranging between 2.08 (N1) and 2.34 (E) log ge/mL were observed when using column-based extraction method. Different targets were also evaluated in naturally contaminated wastewater samples with 91.2%, 85.3%, 70.6%, 79.4% and 73.5% positivity, for N1, N2, E, IP2 and IP4, respectively. Our benchmarked comparison study suggests that the aluminum precipitation method coupled with the automated nucleic extraction represents a method of acceptable sensitivity to provide readily results of interest for SARS-CoV-2 WBE surveillance.

Refined detection and phasing of structural aberrations in pediatric acute lymphoblastic leukemia by linked-read whole-genome sequencing

Structural chromosomal rearrangements that can lead to in-frame gene-fusions are a leading source of information for diagnosis, risk stratification, and prognosis in pediatric acute lymphoblastic leukemia (ALL). Traditional methods such as karyotyping and FISH struggle to accurately identify and phase such large-scale chromosomal aberrations in ALL genomes. We therefore evaluated linked-read WGS for detecting chromosomal rearrangements in primary samples of from 12 patients diagnosed with ALL. We assessed the effect of input DNA quality on phased haplotype block size and the detectability of copy number aberrations and structural variants in the ALL genomes. We found that biobanked DNA isolated by standard column-based extraction methods was sufficient to detect chromosomal rearrangements even at low 10x sequencing coverage. Linked-read WGS enabled precise, allele-specific, digital karyotyping at a base-pair resolution for a wide range of structural variants including complex rearrangements and aneuploidy assessment. With use of haplotype information from the linked-reads, we also identified previously unknown structural variants, such as a compound heterozygous deletion of ERG in a patient with the DUX4-IGH fusion gene. We conclude that linked-read WGS allows detection of important pathogenic variants in ALL genomes at a resolution beyond that of traditional karyotyping and FISH.

MRNA Extraction from Gill Tissue for RNA-sequencing.

Adaptation is thought to proceed in part through spatial and temporal changes in gene expression. Fish species such as the threespine stickleback are powerful vertebrate models to study the genetic architecture of adaptive changes in gene expression since divergent adaptation to different environments is common, they are abundant and easy to study in the wild and lab, and have well-established genetic and genomic resources. Fish gills, due to their respiratory and osmoregulatory roles, show many physiological adaptations to local water chemistry, including differences in gene expression. However, obtaining high-quality RNA using popular column-based extraction methods can be challenging from small tissue samples high in cartilage and bone such as fish gills. Here, we describe a bead-based mRNA extraction and transcriptome RNA-seq protocol that does not use purification columns. The protocol can be readily scaled according to sample size for the purposes of diverse gene expression experiments using animal or plant tissue.

P1.03-11 Molecular Testing of Small Bronchoscopy Specimens Using NanoString Technology

Molecular testing for driver variants in oncogenes is crucial for NSCLC management to predict response to targeted therapy. In the majority of cases, NSCLC is diagnosed by trans-thoracic needle aspiration or more commonly bronchoscopy techniques resulting in small diagnostic tissue biopsies or cytological samples. As such specimens may be inadequate for molecular testing, we tested the accuracy of a novel digital molecular barcoding assay to detect actionable mutations in a single-centre cohort. A consecutive cohort of 46 specimens (19 endobronchial biopsy, EnBx; 18 transbronchial biopsy, TBBx; seven bronchoalveolar lavage, BAL; and two transbronchial needle aspirate, TBNA) were obtained ancillary to primary diagnostic specimens from 36 patients undergoing EBUS-guided bronchoscopy at The Prince Charles Hospital. Specimens containing at least 5ng DNA after standard column-based extraction methods were analysed using the NanoString SNV Solid Tumour Panel for testing of 104 somatic variants across 25 genes of clinical significance. NanoString variants calls were compared with routine clinical testing results from the primary diagnostic sample. Agreement analyses for variants common to both methods revealed the positive, negative and overall percentage agreement (PPA, NPA, OPA). One discordant case was validated using droplet digital PCR. Using NanoString, molecular analysis was feasible for 60.1% (28/46) of specimens. At least one variant was identified in 8/28 (28.6%) cases (Table 1). Two (7.1%) cases harboured dual mutations. KRAS mutations were detected in six (21.4%) cases, and EGFR in two (7.1%). Two patients would be eligible for targeted therapy. Agreement analysis for the two methods revealed PPA, NPA and OPA of 100%, 88.9% and 92.3%. In one discordant case, NanoString identified a KRAS G12C mutation and was confirmed by ddPCR with a mutant allele frequency of 5.5%. The mean time for reporting clinical mutation test results was 19.6 days. Of the 18 excluded cases with insufficient DNA, five had routine testing results for comparison however 3/5 cases cited insufficient DNA for reliable EGFR testing.Table 1Clinical and molecular characteristics of bronchoscopy samples used for molecular testingClinical molecular testingNanoStringConcordanceCase no.Sample typeHistological classificationDNA yield (μg)Mutation testing result (MAF)Mutation testing methodTAT to result (days)SNV panel resultAgreement (Yes/No)1BALNo evidence of malignancy2.81Not performedN/AN/AWTNCA2TBNAAC4.39Not performedN/AN/AWTNCA3TBBxSCC0.39Not performedN/AN/AKRAS G12RNCA4TBBxAC1.82EGFR exon 19 (L747_P753>S) del (24%)NGS TruSight21EGFR exon 19 (L747_P753>S) delYes5TBBxNo evidence of malignancy1.24Not performedN/AN/AWTNCA6TBBxNo evidence of malignancy0.62Not performedN/AN/AWTNCA7EnBxAC1.41WT for EGFR, KRAS, NRAS, BRAFNGS TruSight13WTYes8EnBxSCC0.57Not performedN/AN/AWTNCA9TBBxSCC0.53Not performedN/AN/AWTNCA10TBBxAC0.51WT for EGFR, KRAS, NRAS, BRAFNGS TruSight20KRAS G12CNo*11TBBxAC0.31EGFR L858R (12%), EGFR T790M (6%)NGS TruSight13EGFR L858R, EGFR T790MYes12TBBxNo evidence of malignancy0.69Not performedN/AN/AWTNCA13TBBxNSCLC4.26WT for EGFRcastPCR48WTYes14EnBxAC1.37KRAS G12C (10%)NGS TruSight23KRAS G12CYes15EnBxNSCLC19.6KRAS G12A (25%)NGS TruSight20KRAS G12AYes16EnBxCarcinoid1.16Not performedN/AN/AWTNCA17TBBxSCC5.4Not performedN/AN/AWTNCA18EnBxAC4.03WT for EGFR, KRAS, NRAS, BRAFNGS TruSight19WTYes19EnBxSCC2.12Not performedN/AN/AWTNCA20TBBxAC3.85WT for EGFRcastPCR14KRAS G12C, NRAS Q61KNCA21EnBxSCC6.4Not performedN/AN/AWTNCA22TBBxAC1.63WT for EGFRcastPCR20KRAS G12CNCA23EnBxAC0.234WT for EGFR, KRAS, NRAS, BRAFNGS TruSight16WTYes24BALAC0.871BRAF G466V (12%)NGS TruSight24WTYes25TBBxAC0.0918BRAF G466V (12%)NGS TruSightN/AWTYes26TBBxAC0.893WT for EGFRcastPCR13WTYes27TBBxSCC0.66Not performedN/AN/AWTNCA28EnBxAC4.4WT for EGFRcastPCR10WTYesMean2.5819.6 Open table in a new tab The performance of the NanoString platform for SNV characterisation was highly concordant with alternate clinical testing methods for those with sufficient DNA. Advantages of NanoString include its multiplex capacity, high sensitivity, low nucleic acid input, reduced turn-around time (<24hr) compared to alternate testing methods. The NanoString platform is a robust method for identification of actionable variants in NSCLC where at least 5ng of DNA is available.

Multicenter Evaluation of Circulating Plasma MicroRNA Extraction Technologies for the Development of Clinically Feasible Reverse Transcription Quantitative PCR and Next-Generation Sequencing Analytical Work Flows.

In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters. By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals. There were significant differences between the tested methods. Although column-based extraction methods were highly effective for the isolation of endogenous miRNA, phenol extraction combined with column-based miRNA purification and ultracentrifugation resulted in lower quality and quantity of isolated miRNA. Among all extraction methods, the ubiquitously expressed miR-16 was represented with high abundance when compared with other targeted miRNAs. In addition, the use of miR-16 as an endogenous control for normalization of quantification cycle values resulted in a decreased variability of column-based cfmiRNA extraction methods. Cluster analysis of normalized NGS counts clearly indicated a method-dependent bias. The choice of plasma miRNA extraction methods affects the selection of potential miRNA marker candidates and mechanistic interpretation of results, which should be done with caution, particularly across studies using different protocols.

Improved yield and accuracy for DNA extraction in microbiome studies with variation in microbial biomass.

A major challenge for microbiome studies is maintaining an even and accurate DNA extraction in the presence of samples with a wide range of bacterial content. Here we compare five DNA extraction methods using replicate stool samples that were diluted to create high and low biomass samples. Our results indicate greater variation in microbiome composition between high and low biomass samples than variation between methods. Many of the extraction methods had reduced yield from low biomass samples; however, our adapted plate column-based extraction method was evenly efficient and captured the largest number of high-quality reads. Based on these results, we have identified a DNA extraction method that ensures adequate yield in metagenomic microbiome studies that have samples with a broad range ofbacterial content.

An optimised direct lysis method for gene expression studies on low cell numbers.

There is increasing interest in gene expression analysis of either single cells or limited numbers of cells. One such application is the analysis of harvested circulating tumour cells (CTCs), which are often present in very low numbers. A highly efficient protocol for RNA extraction, which involves a minimal number of steps to avoid RNA loss, is essential for low input cell numbers. We compared several lysis solutions that enable reverse transcription (RT) to be performed directly on the cell lysate, offering a simple rapid approach to minimise RNA loss for RT. The lysis solutions were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in low cell numbers isolated from four breast cancer cell lines. We found that a lysis solution containing both the non-ionic detergent (IGEPAL CA-630, chemically equivalent to Nonidet P-40 or NP-40) and bovine serum albumin (BSA) gave the best RT-qPCR yield. This direct lysis to reverse transcription protocol outperformed a column-based extraction method using a commercial kit. This study demonstrates a simple, reliable, time- and cost-effective method that can be widely used in any situation where RNA needs to be prepared from low to very low cell numbers.

DNA Extraction Methods for Panbacterial and Panfungal PCR Detection in Intraocular Fluids

Purpose: Three different methods of DNA extraction from intraocular fluids were compared with subsequent detection for bacterial and fungal DNA by universal PCR amplification.Material and Methods: Three DNA extraction methods, from aqueous and vitreous humors, were evaluated to compare their relative efficiency. Bacterial (Gram positive and negative) and fungal strains were used in this study: Escherichia coli, Staphylococcus epidermidis and Candida albicans. The quality, quantification, and detection limit for DNA extraction and PCR amplification were analyzed. Validation procedures for 13 aqueous humor and 14 vitreous samples, from 20 patients with clinically suspected endophthalmitis were carried out.Results: The column-based extraction method was the most time-effective, achieving DNA detection limits ≥102 and 103 CFU/100 µL for bacteria and fungi, respectively. PCR amplification detected 100 fg, 1 pg and 10 pg of genomic DNA of E. coli, S. epidermidis and C. albicans respectively. PCR detected 90.0% of the causative agents from 27 intraocular samples collected from 20 patients with clinically suspected endophthalmitis, while standard microbiological techniques could detect only 60.0%. The most frequently found organisms were Streptococcus spp. in 38.9% (n = 7) of patients and Staphylococcus spp. found in 22.2% (n = 4).Conclusions: The column-based extraction method for very small inocula in small volume samples (50–100 µL) of aqueous and/or vitreous humors allowed PCR amplification in all samples with sufficient quality for subsequent sequencing and identification of the microorganism in the majority of them.

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.

A two-step method for the extraction of high-quality RNA from endoscopic biopsies.

The use of molecular techniques such as quantitative RT-PCR depends on the quality of cellular RNA. In particular, RNA extraction from endoscopic biopsies is difficult with respect to yield, and especially integrity. Endoscopic biopsies taken from the gastric antrum, corpus and duodenum were subjected to various RNA extraction protocols, and the RNA was used for quantitative RT-PCR. The subsequent use of two methods, (i) a phenol/chloroform extraction and (ii) a column-based extraction method, resulted in a yield of 4.5 microg total RNA per biopsy with reliable quality in 80% of samples. The quantitative RT-PCR analysis revealed that only RNA samples that clearly show both 18S- and 28S-RNA bands in agarose gel electrophoresis were suitable for quantitative RT-PCR as shown by expression of corpus-specific pepsinogen C-mRNA and the duodenum-specific multi-drug resistance protein-1 (mdr-1)-mRNA. In partially degraded RNA, pepsinogen C, mdr-1, or beta-actin mRNAs were still detectable, but the quantitative determination gave inconsistent data. The two-step method described here is a suitable option for extracting high-quality RNA from endoscopic biopsies when other standard protocols fail.