RNA Extraction Methods Research Articles

B-242 Fast and Cost-effective RNA Extraction in Onco-hematology Laboratories

Abstract Background Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of a BCR-ABL1 rearrangement; its detection and quantification by real-time quantitative PCR (RQ-PCR) plays a central role in CML diagnosis, therapy monitoring, and sequencing to identify resistance mutations in case of therapy failure. To evaluate the molecular response of CML, BCR-ABL1 quantification must be calculated on the international scale (IS%) for which RQ-PCR has the necessary sensitivity (up to 10−5). Currently, BCR-ABL1 quantifications are ubiquitous in onco-hematology laboratories (OHLs; ∼70% of tests performed in our OHL), whereby quality, cost, and turn-around-time are of critical importance. Today, the majority of OHLs in Brazil rely on laborious manual RNA-extraction methods (e.g., Trizol) that usually require 8 mL of peripheral blood (PB) to monitor CML. Here, we report a strategy that requires only 2 mL of PB in combination with the automated magnetic DNA/RNA-extraction kit Extracta-MPTA (Loccus do Brasil) for CML diagnosis and monitoring. Methods: BCR:ABL1 was quantified and its IS% determined using our developed kit, which was calibrated using ERM-AD263 and UK-NEQAS (external quality assessment). Total RNA was extracted from 40 PB samples, after erythrocyte lysis using two different methods: 1) Leukocytes from 8 mL of PB were extracted manually using Trizol; 2) leukocytes from 2 mL of PB were automatically extracted using Extracta-MPTA (Loccus do Brasil). BCR-ABL1 quantification, BCR-ABL1 IS%, and BCR quantification (internal control) were compared statistically (paired t-test). The reproducibility was evaluated by analyzing 20 replicates; the low limit of the leucocyte number to BCR:ABL1 quantification using Extracta-MPTA were also evaluated. Median and moving means of 135 unpaired samples were used to monitor BCR-ABL1 IS% between Trizol, Extracta-MPTA, while EQA from UK-NEQAS was also evaluated using both methods. In parallel, RNA was extracted by both methods from 20 samples for the diagnosis of acute lymphoblastic leukemia (ALL) (BCR-ABL p190 transcript) and acute promyelocytic leukemia (APL) (PML-RARA transcript). Results A comparison of BRC-ABL1, BCR, and %IS in 40 paired samples showed that the results obtained from Trizol or Extracta-MPTA are comparable (paired t-test: P = 0.4222, P = 0.9263, and P = 0.071, respectively). The reproducibility was evaluated by BCR quantification (24%). A comparison of the medians from 135 unpaired samples extracted using Trizol and Extracta-MPTA revealed that BCR-ABL1 IS% distribution among our patients is stable over time (P = 0.7704; Mann Whitney: r2 = 0.0012). EQA was extracted using both methods and the results were identical. A comparison between Trizol and EXTRACTA_MPTA for ALL and APL was 100% identical (Fischer; P = 1.000). Conclusion Both RNA extraction methods are suitable for OHLs for PML-RARA and BCR-ABL (p190 and p210) detection and CML diagnosis/monitoring by BCR-ABL1 detection/quantification. By using Extracta-MPTA the amount of PB used to obtain RNA can be reduced by 75%, test results can be obtained within 4 h, and direct reagent costs are reduced by 25% with a minimum of hands-on time.

Testing of worn face mask and saliva for SARS-CoV-2.

Exhaled SARS-CoV-2 can be detected on face masks. We compared tests for SARS-CoV-2 RNA on worn face masks and matched saliva samples. We conducted this prospective, observational, case-control study between December 2021 and March 2022. Cases comprised 30 in-center hemodialysis patients with recent COVID-19 diagnosis. Controls comprised 13 hemodialysis patients and 25 clinic staff without COVID-19 during the study period and the past 2 months. Disposable 3-layer masks were collected after being worn for 4 hours together with concurrent saliva samples. ThermoFisher COVID-19 Combo Kit (A47814) was used for RT-PCR testing. Mask and saliva testing specificities were 99% and 100%, respectively. Test sensitivity was 62% for masks, and 81% for saliva (p = 0.16). Median viral RNA shedding duration was 11 days and longer in immunocompromised versus non-immunocompromised patients (22 vs. 11 days, p = 0.06, log-rank test). While SARS-CoV-2 testing on worn masks appears to be less sensitive compared to saliva, it may be a preferred screening method for individuals who are mandated to wear masks yet averse to more invasive sampling. However, optimized RNA extraction methods and automated procedures are warranted to increase test sensitivity and scalability. We corroborated longer viral RNA shedding in immunocompromised patients.

Comparison and improvement of RNA extraction methods in Sargassum (Phaeophyta).

Sargassum (Sargassaceae) is widely distributed globally and plays an important role in regulating climate change, but the landscape of genomes and transcripts is less known. High-quality nucleic acids are the basis for molecular biology experiments such as high-throughput sequencing. Although extensive studies have documented methods of RNA extraction, these methods are not very applicable to Sargassum, which contains high levels of polysaccharides and polyphenols. To find a suitable method to improve the quality of RNA extracted, we compared and modified several popular RNA extraction methods and screened one practical method with three specific Sargassum spp. The results showed that three CTAB methods (denoted as Methods 1, 2, and 3) and the RNAprep Pure Plant Kit (denoted as Method 4) could, with slight modifications, effectively isolate RNA from Sargassum species, except for Method 4 used with S. fusiforme. By performing further screening, we determined Method 4 was the best choice for S. hemiphyllum and S. henslowianum, as revealed by RNA yields, RNA Integrity Number (RIN), extraction time, and unigene mapped ratio. For S. fusiforme, Methods 1, 2, and 3 showed no obvious differences among the yields, quality, or time to perform. In addition, one other method was tested, but we found the quality of the RNA extracted by TRIzol reagent methods (denoted as Method 5) performed the worst when compared with the above four methods. Therefore, our study provides four suitable methods for RNA extraction in Sargassum and is essential for future genetic exploration of Sargassum.

Detection of SARS-CoV-2 RNA in wastewater from an enclosed college campus serves as an early warning surveillance system.

SARS-CoV-2, the causative agent of Covid-19, is shed from infected persons in respiratory droplets, feces, and urine. Using quantitative PCR (qPCR), our group hypothesized that we could detect SARS-CoV-2 in wastewater samples collected on a university campus prior to the detection of the virus in individuals on campus. Wastewater samples were collected 3 times a week from 5 locations on the main campus of the University of North Carolina Wilmington (UNCW) from July 24, 2020 to December 21, 2020. Post-collection, total RNA was extracted and SARS-CoV-2 RNA in the samples was detected by qPCR. SARS-CoV-2 signal was detected on campus beginning on August 19 as classes began and the signal increased in both intensity and breadth as the Fall semester progressed. A comparison of two RNA extraction methods from wastewater showed that SARS-CoV-2 was detected more frequently on filter samples versus the direct extracts. Aligning our wastewater data with the reported SARS-CoV-2 cases on the campus Covid-19 dashboard showed the virus signal was routinely detected in the wastewater prior to clusters of individual cases being reported. These data support the testing of wastewater for the presence of SARS-CoV-2 and may be used as part of a surveillance program for detecting the virus in a community prior to an outbreak occurring and could ultimately be incorporated with other SARS-CoV-2 metrics to better inform public health enabling a quick response to contain or mitigating spread of the virus.

Landscape of semi-extractable RNAs across five human cell lines.

Phase-separated membraneless organelles often contain RNAs that exhibit unusual semi-extractability using the conventional RNA extraction method, and can be efficiently retrieved by needle shearing or heating during RNA extraction. Semi-extractable RNAs are promising resources for understanding RNA-centric phase separation. However, limited assessments have been performed to systematically identify and characterize semi-extractable RNAs. In this study, 1074 semi-extractable RNAs, including ASAP1, DANT2, EXT1, FTX, IGF1R, LIMS1, NEAT1, PHF21A, PVT1, SCMH1, STRG.3024.1, TBL1X, TCF7L2, TVP23C-CDRT4, UBE2E2, ZCCHC7, ZFAND3 and ZSWIM6, which exhibited consistent semi-extractability were identified across five human cell lines. By integrating publicly available datasets, we found that semi-extractable RNAs tend to be distributed in the nuclear compartments but are dissociated from the chromatin. Long and repeat-containing semi-extractable RNAs act as hubs to provide global RNA-RNA interactions. Semi-extractable RNAs were divided into four groups based on their k-mer content. The NEAT1 group preferred to interact with paraspeckle proteins, such as FUS and NONO, implying that RNAs in this group are potential candidates of architectural RNAs that constitute nuclear bodies.

High quality RNA extraction from Pseudomonas aeruginosa and other bacteria with a novel rapid and cost-effective method

High-quality mRNA extraction is essential for gene expression assays. In this study, we developed a rapid method (20 min) named FACTS for the extraction of intact RNA from Pseudomonas aeruginosa and compared its performance to established rapid techniques like RNAsnap and CiAR. The RNA integrity, yield, purity and presence of residual genomic DNA were adopted as assessment criteria. Multiple assays for RNA integrity were applied, including the Agilent 2200 TapeStation, QIAxcel capillary electrophoresis, and the newly available Qubit RNA Integrity and Quality (IQ) Assay Kit. The RNA purity and DNA/RNA yield were assessed by spectrophotometry and fluorimetry, respectively. Following Dnase treatment, two-step RT-qPCR for the expression of the rpoD reference gene was performed to evaluate the performance of each method. In terms of RNA integrity, FACTS showed the highest RNA integrity, while in terms of purity, CiAR scored best. RNAsnap resulted in a substantial amount of residual DNA. Pilot experiments for RNA extraction with FACTS from other Gram-negative and Gram-positive bacteria revealed promising results. FACTS is a novel RNA extraction method for rapid highly effective extraction of high-quality RNA from P. aeruginosa and can be used as a cost-efficient alternative to other methods in gene expression studies.

A Novel Cell Membrane-Associated RNA Extraction Method and Its Application in the Discovery of Breast Cancer Markers.

Cell membrane-associated RNA (mem-RNA) has been demonstrated to be cell-specific and disease-related and are considered as potential biomarkers for disease diagnostics, drug delivery, and cell screening. However, there is still a lack of methods specifically designed to extract mem-RNA from cells, limiting the discovery and applications of mem-RNA. In this study, we propose the first all-in-one solution for high-purity mem-RNA isolation based on two types of magnetic nanoparticles, named MREMB (Membrane-associated RNA Extraction based on Magnetic Beads), which achieved ten times enrichment of cell membrane components and over 90% recovery rate of RNA extraction. To demonstrate MREMB's potential in clinical research, we extracted and sequenced mem-RNA of typical breast cancer MCF-7, MDA-MB-231, and SKBR-3 cell lines and non-neoplastic breast epithelial cell MCF-10A. Compared to total RNA, sequencing results revealed that membrane/secreted protein-encoding mRNAs and long noncoding RNAs (lncRNAs) were enriched in the mem-RNA, some of which were significantly overexpressed in the three cancer cell lines, including extracellular matrix-related genes COL5A1 and lncRNA TALAM1. The results indicated that MREMB could enrich membrane/secreted protein-coding RNA and amplify the expression differences of related RNAs between cancer and non-neoplastic cells, promising for cancer biomarker discovery.

Comparison of Four Methods of RNA Extraction and cDNA Synthesis from The Venom of Peruvian Snakes of the Genus Bothrops of Clinical Importance.

RNA purification and cDNA synthesis represents the starting point for molecular analyses of snake venom proteins-enzymes. Usually, the sacrifice of snakes is necessary for venom gland extraction to identify protein-coding transcripts; however, the venom can be used as a source of transcripts. Although there are methods for obtaining RNA from venom, no comparative analysis has been conducted in the Bothrops genus. In the present study, we compared four commercial methods for RNA purification and cDNA synthesis from venom (liquid, lyophilized, or long-term storage) of four clinically relevant species of Peruvian Bothrops. Our results show that the TRIzol method presents the highest yield of RNA purified from venom (59 ± 11 ng/100 µL or 10 mg). The SuperScript First-Strand Synthesis System kit produced high amounts of cDNA (3.2 ± 1.2 ng cDNA/ng RNA), and the highest value was from combination with the Dynabeads mRNA DIRECT kit (4.8 ± 2.0 ng cDNA/ng RNA). The utility of cDNA was demonstrated with the amplification of six relevant toxins: thrombin-like enzymes, P-I and P-III metalloproteinases, acid and basic phospholipases A2, and disintegrins. To our knowledge, this is the first comparative study of RNA purification and cDNA synthesis methodologies from Bothrops genus venom.

Comparison of Modified Manual Acid-Phenol Chloroform Method and Commercial RNA Extraction Kits for Resource Limited Laboratories.

In a comparative experimental cross-sectional study, RNA was extracted from oral swabs and blood samples from 25 healthy individuals at the Department of Molecular Medicine, KNUST. RNA was extracted by the manual AGPC extraction method and commercial RNA extraction kits. The quantity (ng/μl) and purities (260/280 nm) of the extracted RNA were measured spectrophotometrically using the IMPLEN NanoPhotometer® N60. The presence of RNA in the extracts was confirmed using 2% agarose gel electrophoresis. Statistical analyses were conducted using R language. The yield of RNA extracted from blood and oral swab samples using modified AGPC was significantly higher compared to the commercial methods (p < 0.0001). However, the purity of RNA extracted by the manual AGPC method from blood was significantly lower than the commercial methods (p < 0.0001). Moreover, the purity from oral swabs using the manual AGPC method was significantly lower compared to QIAamp (p < 0.0001) and the OxGEn kits method (p < 0.001). The modified manual AGPC method has a very high yield of RNA extracts using blood samples, which could serve as an alternate cost-effective method for RNA extraction in resource-limited laboratories; however, its purity may not be suitable for downstream processes. Moreover, the manual AGPC method may not be suitable for extracting RNA from oral swab samples. Future investigation is needed to improve the purity of the manual AGPC RNA extraction method and also confirmation of the obtained results by PCR amplification and RNA purity verification by sequencing.

Soil Metatranscriptomics: An Improved RNA Extraction Method Toward Functional Analysis Using Nanopore Direct RNA Sequencing

Soil microbes play an undeniable role in sustainable agriculture, plant health, and soil management. A deeper understanding of soil microbial composition and function has been gained through next-generation sequencing. Although soil metagenomics has provided valuable information about microbial diversity, issues stemming from RNA extraction, low RNA abundance in some microbial populations (e.g., viruses), and messenger RNA enrichment have slowed the progress of soil metatranscriptomics. A variety of soil RNA extraction methods have been developed thus far yet none of the available protocols can obtain RNA with high quality, purity, and yield for third-generation sequencing. The latter requires RNA with high quality and large quantities (with no or low contamination such as humic acids). Also, use of commercial kits for in-batch soil RNA extraction is quite expensive, and these commercial kits lack buffer composition details, which prevents the optimization of protocols for different soil types. An improved and cost-effective method for extracting RNAs from mineral and organic soils is presented in this article. An acidic sodium acetate buffer and phosphate buffer with modifications to bead beating and nucleic acid precipitation lead to higher RNA yields and quality. Using this method, we obtained almost DNA-free RNA. By using nanopore's direct RNA sequencing, the extracted contamination-free RNAs were successfully sequenced. Finally, taxonomic groups such as bacteria, fungi, archaea, and viruses were classified and profiled, and functional annotation of the datasets was carried out using an in-house customized bioinformatics workflow.

A simplified viral RNA extraction method based on magnetic nanoparticles for fast and high-throughput detection of SARS-CoV-2.

The ongoing outbreak of the novel coronavirus disease 2019 (COVID-19) draws worldwide concerns due to its long incubation period and strong infectivity. Although RT-PCR-based methods are being widely applied for clinical diagnosis, timely and accurate diagnosis towards COVID-19 causing virus, the SARS-CoV-2, is still limited due to labor-intensive and time-consuming operations. Herein, we report a new viral RNA extraction method based on poly-(amino ester) with carboxyl group (PC)-coated magnetic nanoparticles (pcMNPs) for the sensitive detection of SARS-CoV-2. This method combines the lysis and binding steps into one step, and refines multiple washing steps into one step, giving a turnaround time of less than 9min. Furthermore, the extracted pcMNP-RNA complexes can be directly introduced into subsequent RT-PCR reactions without elution. This simplified viral RNA method could be well adapted in fast manual and automated high-throughput nucleic acids extraction protocols suitable for different scenarios. A high sensitivity down to 100 copies/mL and a linear correlation between 100 and 106 copies/mL of SARS-CoV-2 pseudovirus particles are achieved in both protocols. Benefitting from the simplicity and excellent performances, this new method can dramatically improve the efficiency and reduce operational requirements for the early clinical diagnosis and large-scale SARS-CoV-2 nucleic acid screening.

Optimised protocols for RNA extraction from a broad taxonomic range of algae

AbstractDespite advancements in RNA extraction methods, RNA extraction from sources rich in polyphenols and polysaccharides such as algae and seagrasses remains a challenge. Here we present a RNA extraction strategy using a hexadecyltrimethylammonium bromide (CTAB) extraction buffer and demonstrate its effectiveness on a broad range of red, green, and brown algae, as well as on the cyanobacterium Arthrospira platensis and the seagrass Zostera marina. For the vast majority of tested samples we achieved high yields of RNA comparable to those obtained from higher plants by commercially available kits (ranging from 3.9 to 125.9 µg RNA g−1 fresh weight). Analysis by UV/Vis spectrometry and capillary electrophoresis revealed high purity and integrity of obtained RNA extracts. For highly challenging species of brown algae like Fucus vesiculosus, Fucus serratus and Dictyosiphon foeniculaceus, we established an alternative procedure using a sodium dodecyl sulfate (SDS) extraction buffer in combination with a commercial kit. With this protocol, even higher RNA yields up to 317.0 µg g−1 fresh weight were extracted from polysaccharide-rich brown algae tissues. This study can serve as a guideline and starting point for the development of RNA extraction protocols for so far unstudied algal species from very diverse taxa.

Simplified protocol modification of TRIzol method for extraction of high-quality RNA yield from RNase-rich rat pancreas

The extraction of a sufficient quantity of intact RNA from RNase-rich rat pancreas for estimation of gene transcript levels has been a long-standing challenge. This study provided simple modifications in the steps involved in the standard TRIzol RNA extraction method to set up a reproducible, applicable, and efficient protocol to be routinely used in the laboratory. A Nanodrop spectrophotometer was used to evaluate the RNA concentration, and purity (A260/A280, and A260/A230 absorbent ratios). Gel electrophoresis was used to assess RNA integrity. Further analysis included sequential RT-qPCR of a housekeeping gene to evaluate the optimized protocol. The current study demonstrated that the modified protocol rendered significantly more RNA concentration (mean: 635.8 ± 210.5 ng/μL) compared to the standard protocol (mean: 77.2 ± 25.9 ng/μL). Moreover, the modified protocol produced an improvement in the RNA purity (mean :A260/A280 ratio = 1.89 ± 0.13; A260/A230 ratio = 1.7 ± 0.16) compared to the standard protocol (mean: A260/A280 ratio = 1.7 ± 0.26; A260/A230 ratio = 2.5 ± 1.9). Additionally, RT-qPCR analysis revealed that the RNA extracted using the modified protocol resulted in significantly decreased (P < 0.001) Ct values (decreased by 6 Ct units) compared to the standard protocol, reflecting the good quality of the RNA. Conclusion: This study succeeded in providing golden tips to extract a high yield of good-quality RNA from RNase-rich rat pancreas for the sequential gene expression assays.

An optimized RNA extraction method for diverse leaves of Hawaiian Metrosideros, a hypervariable tree species complex.

The isolation of RNA from trees is challenging due to the interference of polyphenols and polysaccharides with downstream processes. Furthermore, many RNA extraction protocols are time consuming and involve hazardous chemicals. To address these issues, we aimed to develop a safe protocol for high-quality RNA extraction from diverse Metrosideros taxa representing a broad range of leaf toughness, pubescence, and secondary metabolites. We tested popular RNA isolation kits and protocols that were effective on other recalcitrant trees, including a broad range of optimization and purification steps. We optimized a protocol involving two silica-membrane column-based kits that yielded high-quantity RNA with an RNA integrity number >7 and without DNA contamination. All RNA samples were used successfully in a follow-on RNA-Seq experiment. We present an optimized high-throughput RNA extraction protocol that yielded high-quality and high-quantity RNA from three contrasting leaf phenotypes within a hyperdiverse woody species complex.

Evaluation of the AdvanSure One-Stop COVID-19 Plus Kit for SARS-CoV-2 Detection Using a Streamlined RNA Extraction Method.

Real-time reverse transcription (rRT)-PCR, which is the reference standard for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, generally involves a time-consuming and costly RNA extraction step prior to amplification. We evaluated the performance of the AdvanSure One-Stop COVID-19 Plus Kit (LG Chem, Seoul, Korea), a novel rRT-PCR assay that can detect SARS-CoV-2 within 90 minutes using a streamlined RNA extraction method. In total, 509 nasopharyngeal swab (NPS) specimens (SARS-CoV-2 positive: N=205; SARS-CoV-2 negative: N=304) previously tested using the PowerChek SARS-CoV-2 Real-time PCR Kit (Kogene Biotech, Seoul, Korea) were tested using the AdvanSure assay. The limit of detection (LOD) of the AdvanSure assay was determined using serially diluted inactivated SARS-CoV-2. The positive and negative percent agreements between the AdvanSure and PowerChek assays were 99.5% (204/205) and 99.3% (302/304), respectively. The LODs of the AdvanSure assay for SARS-CoV-2 nucleocapsid and spike/RNA-dependent RNA polymerase genes were 672 and 846 copies/mL, respectively. The results show that the performance of the AdvanSure assay is comparable to that of the PowerChek assay used for routine SARS-CoV-2 testing, suggesting that the AdvanSure assay is a useful diagnostic tool for rapid and accurate detection of SARS-CoV-2 infection.

Abstract 1332: Extracted mRNA from frozen buffy coat samples shows promise for use in downstream sequencing analyses

Abstract Frozen blood samples are routinely collected and stored in medical biobanks across the world, often for periods of several decades, and later used for medical research such as biomarker studies. Some common biomarker targets have included DNA alterations, protein or antibody levels or metabolomics. However, despite the potential importance of gene-expression alterations in circulating immune cells, transcriptional studies on blood samples from biobanks have been limited due to preanalytical technical difficulties of extracting high-quality RNA. In the absence of an RNA preservative, RNA degrades rapidly during the thawing process. Our aim was, therefore, to investigate the possibility of using biobanked blood samples for transcriptomics analyses. First, we used a CryoXtract CXT 350 to establish a protocol capable of isolating high-quality RNA from frozen core aliquots of buffy coat without thawing the entire sample tube. Then, we performed RNA sequencing on RNA extracted from 20 pre-diagnostic buffy coat samples from colorectal cancer patients and matched controls included in the population-based, prospective Northern Sweden Health and Disease Study (NSHDS). For all samples, two different library preparation methods were used (Illumina TruSeq Stranded mRNA poly-A selection and Illumina Stranded Total RNA with Ribo-Zero Globin). We obtained RNA yields of more than 1 µg from a majority of the NSHDS samples (mean=2.57 µg), and more than 92% of samples had RIN values of 6 or higher, indicating suitability for downstream analyses. Preliminary random forest analyses showed expected discriminatory ability between men and women based on data generated from each of the two library preparation methods. In conclusion, our newly established RNA extraction method enables extraction of RNA from frozen, biobanked buffy coat samples, collected in standard tubes, that is of sufficient quantity and quality for use in downstream whole-genome transcriptomic analyses, such as RNA sequencing. Citation Format: Erik Bovinder Ylitalo, Linda Vidman, Sophia Harlid, Bethany Van Guelpen. Extracted mRNA from frozen buffy coat samples shows promise for use in downstream sequencing analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1332.

Comparison of Manual and Automated Nucleic Acid (RNA) Extraction Methods for the Detection of SARS-CoV-2 by qRT-PCR.

Objectives During the COVID-19 pandemic, several laboratories used different RNA extraction methods based on the resources available. Hence this study was done to compare the Ct values in qRT-PCR, time taken (sample processing-loading to PCR), manpower requirement, and cost of consumables between manual and automated methods. Materials and methods A cross-sectional study was done on 120 nasopharyngeal/oropharyngeal swabs received in VRDL for RT-PCR testing. Based on the results of automated RNA extraction (Genetix, HT 96 Purifier) and RT-PCR (Trivitron PCR Kit) detecting E gene (screening) and ORF gene (confirmatory), the division into Group- I (Ct 15-22), Group- II (Ct 23-29), Group-III (Ct 30-36) and Group-IV (Ct >36) was done. Manual RNA extraction was done using magnetic beads (Lab system, Trivitron). Statistical analysis Data were analyzed by SPSS 19.0 version software. Ct values obtained in the two methods were compared by paired t-test, GroupWise. Z test was used to compare the other parameters. Results The difference in Ct values for target genes was statistically significant (p<0.05) in Group-I to III; however, no variation in result interpretation. The difference in time, manpower, and cost were statistically significant (p<0.05). The manual method required twice more manpower; 40 minutes more time & automated method cost 3.5 times more for consumables. Conclusion The study showed that RNA yield was better with automated extraction in comparison to manual extraction. The samples extracted by the automated method detected the virus at a lower Ct range by PCR than the manual method. Automated method processed samples in less time and with less manpower. Considering the cost factor, manual extraction can be preferred in resource-limited settings as there was no difference in the results of the test. The manual method requires more hands-on time with potential chances of cross-contamination and technical errors.

Comparison of Eleven RNA Extraction Methods for Poliovirus Direct Molecular Detection in Stool Samples.

Direct detection by PCR of poliovirus RNA in stool samples provides a rapid diagnostic and surveillance tool that can replace virus isolation by cell culture in global polio surveillance. The sensitivity of direct detection methods is likely to depend on the choice of RNA extraction method and sample volume. We report a comparative analysis of 11 nucleic acid extraction methods (7 manual and 4 semiautomated) for poliovirus molecular detection using stool samples (n = 59) that had been previously identified as poliovirus positive by cell culture. To assess the effect of RNA recovery methods, extracted RNA using each of the 11 methods was tested with a poliovirus-specific reverse transcription-quantitative PCR (RT-qPCR), a pan-poliovirus RT-PCR (near-whole-genome amplification), a pan-enterovirus RT-PCR (entire capsid region), and a nested VP1 PCR that is the basis of a direct detection method based on nanopore sequencing. We also assessed extracted RNA integrity and quantity. The overall effect of extraction method on poliovirus PCR amplification assays tested in this study was found to be statistically significant (P < 0.001), thus indicating that the choice of RNA extraction method is an important component that needs to be carefully considered for any diagnostic based on nucleic acid amplification. Performance of the methods was generally consistent across the different assays used. Of the 11 extraction methods tested, the MagMAX viral RNA isolation kit used manually or automatically was found to be the preferable method for poliovirus molecular direct detection considering performance, cost, and processing time. IMPORTANCE Poliovirus, the causative agent of poliomyelitis, is a target of global eradication led by the World Health Organization since 1988. Direct molecular detection and genomic sequencing without virus propagation in cell culture is arguably a critical tool in the final stages of polio eradication. Efficient recovery of good-quality viral RNA from stool samples is a prerequisite for direct detection by nucleic acid amplification. We tested 11 nucleic acid extraction methods to identify those facilitating sensitive, fast, simple, and cost-effective extraction, with flexibility for manual and automated protocols considered. Several different PCR assays were used to compare the recovered viral RNA to test suitability for poliovirus direct molecular detection. Our findings highlight the importance of choosing a suitable RNA extraction protocol and provide useful information to diagnostic laboratories and researchers facing the choice of RNA extraction method for direct molecular virus detection from stool.

Fast, cheap and sensitive: Homogenizer-based RNA extraction free method for SARS-CoV-2 detection by RT-qPCR.

The SARS-CoV-2 gold standard detection method is an RT-qPCR with a previous step of viral RNA extraction from the patient sample either by using commercial automatized or manual extraction kits. This RNA extraction step is expensive and time demanding. The aim of our study was to evaluate the clinical performance of a simple SARS-CoV-2 detection protocol based on a fast and intense sample homogenization followed by direct RT-qPCR. 388 nasopharyngeal swabs were analyzed in this study. 222 of them tested positive for SARS-CoV-2 by the gold standard RNA extraction and RT-qPCR method, while 166 tested negative. 197 of those 222 positive samples were also positive for the homogenization protocol, yielding a sensitivity of 88.74% (95% IC; 83.83 - 92.58). 166 of those negative samples were also negative for the homogenization protocol, so the specificity obtained was 97% (95% IC; 93.11 - 99.01). For Ct values below 30, meaning a viral load of 103 copies/uL, only 4 SARS-CoV-2 positive samples failed for the RNA extraction free method; for that limit of detection, the homogenizer-based method had a sensitivity of 97.92% (95% CI; 96.01 - 99.83). Our results show that this fast and cheap homogenization method for the SARS-CoV-2 detection by RT-qPCR is a reliable alternative of high sensitivity for potentially infectious SARS-CoV-2 positive patients. This RNA extraction free protocol would help to reduce diagnosis time and cost, and to overcome the RNA extraction kits shortage experienced during COVID-19 pandemic.

Molecular detection of SARS-CoV-2 in Argentina: Evaluation of alternative diagnostic tools for the decentralization of the diagnosis

The rocketing number of COVID-19 cases highlighted the critical role that diagnostic tests play in medical and public health decision-making to contain and mitigate the SARS-CoV-2 pandemic. This study reports the evaluation and implementation of different tests for the molecular detection of SARS-CoV-2 in the central region of Argentina. We evaluated 3 real time RT-PCR kits (GeneFinder COVID-19 Plus RealAmp Kit, DisCoVery SARS-CoV-2 RT-PCR Detection Kit and WGene SARS-CoV-2 RT Detection), 2 nucleic acid extraction methods [MagaBio plus Virus DNA/RNA Purification Kit II (BioFlux), 35-min vs. 9-min], a pre-analytical reagent (FlashPrep®) and 2 isothermal amplification tests (Neokit Plus and ELA CHEMSTRIP®). The order according to the best performance of the 3 real-time RT-PCR kits evaluated was: DisCoVery>GeneFinderTM>WGene. The 2 RNA extraction methods showed similar good results: MagaBio plus Virus RNA Purification Kit II (BioFlux) 9-min was selected due to its faster performance. FlashPrep® reagent showed excellent results to perform direct RNA detection. Isothermal amplification assays showed acceptable sensitivity and specificity values (>80%), except in samples with Ct>30. Our data show optimal real time RT-PCR kits and alternative molecular methods for SARS-CoV-2 diagnostic. These alternative assays proved to be acceptable for their use in adverse contexts, decentralization, and different epidemiological scenarios, for rapid and accurate SARS-CoV-2 detection.