RNA Purification Kit Research Articles

Standardisation of an efficient RNA isolation protocol from the seeds of Momordica charantia var. muricata (Willd.): A Comparative Evaluation

Extraction of high-quality total RNA from Momordica charantia var. muricata seeds is difficult due to the presence of polyphenolic and polysaccharide compounds which bind to nucleic acids and thus they co-precipitate and contaminate the RNA samples. The current study aimed to find out a reliable and reproducible RNA extraction method for obtaining good-quality RNA from the seeds of Momordica charantia var. muricata (Willd). The RNA was isolated from seed tissues with four different extraction protocols including the cetyltrimethylammonium bromide (CTAB) method, TRIzol (Invitrogen) method, GenElute™ total RNA purification kit (Sigma-Aldrich) and Plant RNeasy mini kit (Qiagen). The Qiagen RNeasy plant mini kit was the most efficient extraction method providing the highest RNA yield and good quality. The method successfully enhanced the yield of RNA to 923.27 ± 58.64 ng/μl and electrophoresis revealed two distinct bright 28S and 18S rRNA bands and spectrophotometric quantification detected A260/ A280 ratio of 2.04±0.01 within the desired range of 1.8-2. To verify that the RNA quality obtained was appropriate for downstream molecular applications, the isolated RNA samples were converted into cDNA and PCR amplification of the cDNA was performed using a housekeeping gene (Actin 7) and a dormancy-specific gene (DOG1). The current investigation reports on a method for isolating high-quality RNA from M. charantia var. muricata seeds for future dormancy gene expression studies.

The virome of the kitome: small circular virus-like genomes in laboratory reagents.

In the course of studying the virome of protozoan parasites, we identified small circular genomes resembling viruses, which turned out to be contaminants from an RNA purification kit. We report their sequences here so others can detect possible contamination in their samples by aligning them to these targets.

Examination and comparison of the RNA extraction methods using mouse serum.

Serum microRNAs (miRNAs) are considered useful as non-invasive biomarkers for different diseases. However, the optimal method for extracting RNAs from serum is currently unknown. In the present study, several RNA extraction kits were used to examine the optimal kit. RNAs were extracted from the serum of 8-week-old C57BL/6NJcl male mice following the protocol of each RNA extraction kit. The yield of the extracted RNA samples was calculated, and an Agilent Bioanalyzer was used to assess the electrophoretic patterns. An Agilent mouse miRNA microarray was utilized to confirm the expression patterns of the extracted RNA samples. The results revealed significant differences in RNA yields from the miRNeasy Serum/Plasma Advanced kit and mirVana™ PARIS™ RNA and Native Protein Purification Kit compared with almost all other samples. Further, two peaks were determined in the miRNeasy Serum/Plasma Advanced kit using a small RNAs kit of Agilent Bioanalyzer, including one at 20-40 nucleotides (nt) and another at ~40-100 nt, whereas the other reagents had a single peak. This revealed that the extracted RNAs may differ in composition based on the RNA extraction method. Some types of miRNAs were only detected with certain RNA extraction reagents. This suggested that different RNA extraction reagents may cause differences in the types of miRNAs detected. On the other hand, the miRNAs commonly expressed by the three RNA extraction reagents are highly correlated in expression levels.

Fine-tuning RNA extraction protocols for polysaccharide-rich freshwater red algae (Rhodophyta).

Freshwater red algae are important primary producers, widely distributed, contributing significantly to nutrient cycling in aquatic ecosystems. Recent studies have focused on identifying the effect of different environmental conditions such as light on the gene expression in photosynthesis pathways. However, obtaining the necessary RNA quantity and quality for sequencing from these algae has been challenging. Although RNA extractions have been optimized for model organisms, RNA extraction for non-model organisms, such as gelatinous (polysaccharide-rich) red algae, requires considerable troubleshooting. The common freshwater red alga, Batrachospermum gelatinosum , was used to test protocols. The extraction efficiency of various sample disruption methods in combination with seven RNA extraction kits was compared. Using a 2-minute disruption procedure with a modification of TRIzol ™ Plus RNA Purification Kit (PureLink ™ RNA Mini Kit + Trizol ™ ) protocol resulted in significantly higher RIN scores (p < 0.05) and high RNA concentration, compared to other methods. The fine-tuned protocol yielded quality RNA (RIN>7) in high concentrations for subsequent sequencing.

First molecular detection of influenza D virus in cattle from commercial farm in Nigeria, Sub-Saharan Africa

Objective: To carrry out molecular surveillance of influenza D virus (IDV) in cattle in Lagos, Nigeria. Methods: Prospective epidemiological investigation was initiated in a large commercial farm market where animals in open pens were reared, sold, and butchered under poor hygienic conditions without adequate biosecurity measures. A total of 80 nasopharyngeal swabs were collected from cattle between October and November 2021. The samples were extracted using an RNA purification kit (NIMR). RNA extracts were amplified following a two-step PCR using FIREScript RT cDNA synthesis kit (Solis Biodyne, Estonia), followed by PCR OneTaq Quick-load 2X master-mix (NEB, UK) in a Rotor-Gene thermocycler (Qiagen, Germany). Amplicons were detected using a 1.5% Gel electrophoresis. Results: In total, 32.5% (26/80) IDV was detected in cattle. Sick animals showed higher burden of IDV with 65.4% (17/26) than 34.6% (9/26) in a healthy population, including 88.2% (15/17) cattle with diarrhoea and 11.8% (2/17) with nausea having IDV. An incidence of 69.2% (18/26) by sex was recorded in bull, which was more than twice compared to 30.7% (8/26) in cow. Age prevalence showed 62.2% (18/26), the highest detection in cattle of four years old, followed by 23.1% (6/26) in five years old, while the lowest 7.7% (2/26) was recorded in three years old. Conclusions: To the best of our knowledge, this study presents the first molecular detection of IDV in Nigeria and West Africa sub-region. It underscores the need for continuous surveillance of IDV at the animal-human interface.

First report of cucumber mosaic virus infecting Azorina vidalii in Azores Islands.

Virus diseases that occur in crops pose a major threat not only to global food security but also to wild plant communities growing in natural ecosystems (Jones, 2020, and references therein). In Azores (Portugal) little is known about viruses present on native flora and therefore, they have not been taken into consideration in conservation programs. Considering this, we selected Azorina vidalii (Campanulaceae), an endangered (IUCN) plant, endemic to Azores (Bilz, 2011), to survey for plant viruses. A. vidalii, the sole species of its genus, is often found in crevices with no soil accumulation on coastal cliffs, exposed to storms and sea spray, and is used as an ornamental. Leaves from 53 plants of A. vidalii from three populations from Terceira Island and three populations from Flores Island were randomly collected without obvious symptoms of virus infection, between the summer of 2021 and fall of 2022. RNA extraction was performed using the Plant/Fungi Total RNA Purification Kit (Norgen Biotek, Canada). RNA extracts from each population were pooled into six distinct composite samples (AvT1, AvT2, AvT3, AvF1, AvF4 and AvF5) and sent to Lexogen (Austria) for small RNA library preparation and High-Throughput Sequencing. Single-end RNA sequencing using Illumina NextSeq2000 system yielded between 10.1 M and 33.8 M raw reads. Adaptors and low-quality reads were removed with Trim Galore! and PRINSEQ. Trimmed reads were mapped to the genome phylogenetically nearest to A. vidalii available at the NCBI database (Adenophora triphylla). The resulting 2.5 M - 13.5 M unmapped reads were analysed with VirusDetect online version (database v248) (Zheng et al., 2017) for virus detection and identification. Sequences of cucumber mosaic virus (CMV) (contigs of up to 3045 nt for RNA1, 2917 nt for RNA2 and 2086 nt for RNA3) were identified in five (AvT1, AvT2, AvT3, AvF1 and AvF5) of the six composite samples and CMV satellite sequences (two contigs with 145 and 197 nt) were identified in only one (AvT1) of the composite samples. To confirm the presence of CMV, all samples were tested by two-step RT-PCR using primers targeting CMV-specific RdRp gene (513 bp) (Grieco et al., 2000), which retrieved 18 positive samples (34%). Nine samples were selected for Sanger sequencing (six out of 13 from Terceira and three out of five from Flores) based on digestion profile obtained with AluI and MboI. The resulting sequences (OQ176229-OQ176233, OQ732757-OQ732760) share an identity of 97.2-100% and BLASTn showed them to have 98.3-99.6% identity to CMV strain TN (AB176848). A Neighbour-Joining tree (Supplementary material) inferred in MEGA11 (Tamura et al., 2021) with 237 additional CMV-RdRp sequences, showed that A. vidalii CMV-derived isolates clustered together with reference strains of subgroup II, as those used by Roossinck (2002) for phylogenetic analysis of the 2a ORF. Besides CMV, tomato spotted wilt virus and polerovirus-associated RNAs sequences were found in one of the A. vidalii populations, but with lower coverage, and need to be further investigated. To the best of our knowledge, this is the first report of CMV infecting A. vidalli. CMV, genus Cucumovirus, is an agriculturally important virus and one of the most successful viruses known, infecting over 1,200 species of plants (Palukaitis & García-Arenal, 2003). In addition to A. vidalii being a CMV reservoir, which may have implications on adjacent crop fields, further research is needed to investigate the impact of CMV on A. vidalli fitness.

Evaluation of a novel real-time polymerase chain reaction assay for identifying H3 equine influenza virus in Kazakhstan.

Equine influenza (EI) is a highly contagious disease that causes fever and upper respiratory tract inflammation. It is caused by influenza virus A, belonging to the Orthomyxoviridae family, with subtypes H3N8 and H7N7. This study presents data on the development of a real-time polymerase chain reaction (RT-PCR) assay using TaqMan probes to detect the H3 subtype of EI virus (EIV). The evaluation of the developed RT-PCR assay involved five strains of EIV as positive controls and ten nasopharyngeal swab samples collected from horses. RNA was isolated using the GeneJet Viral DNA and RNA Purification Kit, and primers and probes were designed using the Integrated DNA Technology PrimerQuest Tool. The assay was optimized by investigating the annealing temperature, primer and probes concentrations, sensitivity, and specificity. Sequencing was performed using the Thermo Fisher 3130 Genetic Analyzer, and the evolutionary history was inferred using the Neighbor-Joining method. The designed primers and probes, targeting the H3 gene, were found to be specific to the EIV. The RT-PCR assay was capable of detecting as low as 50 femtogram (f) or 3 × 103 copies of genomic RNA. No cross-reactions were observed with other respiratory viral and bacterial pathogens, indicating the high specificity of the assay. To evaluate its effectiveness, ten nasopharyngeal swab samples collected from farms in North Kazakhstan regions during disease monitoring were analyzed. The accuracy of the analysis was confirmed by comparing the results with those obtained from a commercial RT-PCR assay for EI identification. The developed RT-PCR assay exhibited high sensitivity and specificity for detecting the EIV. The results demonstrate that the developed RT-PCR assay is suitable for diagnosing EI. This simple, highly sensitive, and specific assay for detecting H3 EIV can be a reliable tool for diagnosing and surveilling EI. Implementing this RT-PCR assay in veterinary practice will enhance and expedite the timely response to potential outbreaks of EI, thus positively impacting the overall epizootic well-being of EI in Kazakhstan.

Development and Validation of a Duplex RT-qPCR for Detection of Peach Latent Mosaic Viroid and Comparison of Different Nucleic-Acid-Extraction Protocols.

Peach latent mosaic viroid (PLMVd) is an important pathogen that causes disease in peaches. Control of this viroid remains problematic because most PLMVd variants are symptomless, and although there are many detection tests in use, the reliability of PCR-based methods is compromised by the complex, branched secondary RNA structure of the viroid and its genetic diversity. In this study, a duplex RT-qPCR method was developed and validated against two previously published single RT-qPCRs, which were potentially able to detect all known PLMVd variants when used in tandem. In addition, in order to simplify the sample preparation, rapid-extraction protocols based on the use of crude sap or tissue printing were compared with commercially available RNA purification kits. The performance of the new procedure was evaluated in a test performance study involving five participant laboratories. The new method, in combination with rapid-sample-preparation approaches, was demonstrated to be feasible and reliable, with the advantage of detecting all different PLMVd isolates/variants assayed in a single reaction, reducing costs for routine diagnosis.

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.

Obtaining miRNA from Saliva-Comparison of Sampling and Purification Methods.

The use of saliva as a biomarker source has advantages over other biofluids and imaging techniques, and miRNAs are ideal biomarker candidates. They are involved in numerous cellular processes, and their altered expression suggests that miRNAs play a crucial regulatory role in disease development. We wanted to find an easily reproducible and executable miRNA-obtaining methodology suitable for quantification. Three commercial miRNA extraction kits (mirVana, Nucleospin and miRNeasy) and three saliva collectors (50 mL tubes, Salimetrics and Oragene) were tested. Several features, including RNA quality and technical parameters, were evaluated. The expression of five synthetic spike-in controls and seven saliva-miRNAs was analyzed independently and grouped by the collectors and the extraction kits. The combination of Oragene and miRNeasy assured the most sensitive detection of all seven saliva miRNAs. Testing different combinations of saliva collectors and RNA purification kits permitted the establishment of combinations for different uses. The results of our study highlight that optimization of resources for biomarker studies is possible after careful planning of each study.

Microparticles as Viral RNA Carriers from Stool for Stable and Sensitive Surveillance.

Since its discovery, polymerase chain reaction (PCR) has emerged as an important technology for the diagnosis and identification of infectious diseases. It is a highly sensitive and reliable nucleic acids (NA) detection tool for various sample types. However, stool, which carries the most abundant micro-organisms and physiological byproducts, remains to be the trickiest clinical specimen for molecular detection of pathogens. Herein, we demonstrate the novel application of hydrogel microparticles as carriers of viral RNA from stool samples without prior RNA purification for real-time polymerase chain reaction (qPCR). In each microparticle of primer-incorporated network (PIN) as a self-sufficient reaction compartment, immobilized reverse transcription (RT) primers capture the viral RNA by hybridization and directly initiate RT of RNA to generate a pool of complementary DNA (PIN-cDNA pool). Through a simple operation with a portable thermostat device, a PIN-cDNA pool for influenza A virus (IAV) was obtained in 20 min. The PIN-cDNA pools can be stored at room temperature, or directly used to deliver cDNA templates for qPCR. The viral cDNA templates were freely released in the subsequent qPCR to allow amplification efficiency of over 91%. The assay displayed good linearity, repeatability, and comparable limit of detection (LoD) with a commercialized viral RNA purification kit. As a proof of concept, this technology carries a huge potential for onsite application to improve human and animal infectious disease surveillance activities using stool samples without the need for a laboratory or centrifuge for sample preparation.

Shotgun metagenomic sequencing in culture negative microbial keratitis.

To evaluate the microbiota of culture negative Corneal Impression Membrane (CIM) microbial keratitis samples with the use of shotgun metagenomics analysis. DNA of microbial keratitis samples were collected with CIM and extracted using the MasterPure™ Complete DNA and RNA Purification Kit (Epicentre). DNA was fragmented by sonication into fragments of 300 to 400 base pairs (bp) using Bioruptor® (Diagenode, Belgium) and then used as a template for library preparation. DNA libraries were sequenced on Illumina® HiSeq2500. The resulting reads were quality controlled, trimmed and mapped against the human reference genome. The unmapped reads were taxonomically classified using the Kraken software. 18 microbial keratitis samples were included in the study. Brevundimonas diminuta was found in 5 samples while 6 samples showed the presence of viral infections. Cutibacterium acnes, Staphylococcus aureus, Moraxella lacunata and Pseudomonas alcaligenes were also identified as the presumed putative cause of the infection in 7 samples. Shotgun sequencing can be used as a diagnostic tool in microbial keratitis samples. This diagnostic method expands the available tests to diagnose eye infections and could be clinically significant in culture negative samples.

MicroRNA and Hemostasis Profile of Carotid Atherosclerosis.

Carotid atherosclerosis (CA) is an important risk factor for ischemic stroke. We described the miRNA and hemostasis profile of patients with moderate and advanced stages of carotid atherosclerosis and elucidated potential correlations with hemostatic activation. A prospective case-control study included 61 patients with evidence of carotid atherosclerosis (via ultrasound). The study population was divided into groups depending on the degree of carotid artery stenosis: 60% or more (advanced) and <60% (moderate). All patients underwent the following blood tests: general blood test, hemostatic parameters and microRNA. Extraction of microRNA was performed using Leukocyte RNA Purification Kit (NORGEN Biotec Corp., Thorold, ON, Canada); miRNA quantification was performed via RT-PCR. Statistical analysis was performed in R programming language (v. 4.1.0) using RSudio. MicroRNA expression profile was different depending on CA degree. MiR-33a-5p/3p levels were higher in patients with ≥60% carotid stenosis (42.70 and 42.45 versus 38.50 and 38.50, respectively, p < 0.05). Almost complete separation can be visualized with the levels of miR-126-5p: 9.50 in the moderate CA group versus 5.25 in the advanced CA (p < 0.001). MiR-29-5p was higher in the moderate CA group: 28.60 [25.50;33.05] than in advanced CA group: 25.75 [24.38;29.50] (p = 0.086); miR-29-3p was also higher in the moderate CA group: 10.36 [8.60;14.99] than in advanced CA group: 8.46 [7.47;10.3] (p = 0.001). By-group pairwise correlation analyses revealed at least three clusters with significant positive correlations in the moderate CA group: miR-29-3p with factors V and XII (r = 0.53 and r = 0.37, respectively, p < 0.05); miR-21-5p with ADAMTS13, erythrocyte sedimentation rate and D-dimer (r = 0.42, r = 0.36 and r = 0.44, respectively, p < 0.05); stenosis degree with miR-33a-5p/3p and factor VIII levels (r = 0.43 (both) and r = 0.62, respectively, p < 0.05). Hemostasis parameters did not reveal significant changes in CA patients: the only statistically significant differences concerned factor VIII, plasminogen and (marginally significant) ADAMTS-13 and protein C. Down-regulation of miR-126-5p expression has been identified as a promising biomarker of advanced carotid atherosclerosis with high specificity and sensitivity. Correlation cluster analysis showed potential interplay between miRNAs and hemostatic activation in the setting of carotid atherosclerosis.

Calystegia hederacea: a new natural host of Sweet potato latent virus in China.

Sweet potato is a global root crop, with a worldwide production of 91.5 million tons in 2019 (FAOSTAT, 2019). However, virus diseases cause significant yield losses and quality decline in sweet potato. Up to now, over 30 different viruses have been identified in sweet potato (Clark et al. 2012). Expanding knowledge of the host range of sweet potato viruses will provide a benefit for the understanding of virus occurrence and designing appropriate virus control measures. In August 2019, ten Calystegia hederacea and two Convolvulus arvensis (Convolvulaceae) weed plants with or without symptoms of leaf yellowing symptoms were collected from various virus disease-affected sweet potato fields in four cities (Jiaozuo, Xinxiang, Zhengzhou and Kaifeng) of Henan Province for virus detection. The leaves of these plants were harvested and pooled for total RNA extraction using a Plant Total RNA Purification Kit (GMbiolab, Taichung, Taiwan). A library for high-throughput sequencing (HTS) was constructed and sequenced using the Illumina HiSeq 2000 platform by BGI Tech (Shenzhen, China). Clean reads (n = 100,570,346), each 150 bp in length, were de novo assembled using CLC Genomics Workbench 9.5 (Qiagen, USA). The assembled contigs were analyzed against the viral reference genome database in GenBank using the BLASTN and BLASTX searches. Three contigs related to sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus, family Closteroviridae) were identified (Liu et al. 2021). In addition, a total of 20 contigs, ranging from 1,019 to 9,859 bp in length with an average depth of coverage of 1439.26, showed 74.80-87.59% nucleotide (nt) sequence identities with corresponding sequences of sweet potato latent virus (SPLV, genus Potyvirus, family Potyviridae). The sequence of the 9,859-bp contig covering nearly complete genome sequence for SPLV, was deposited in GenBank (accession no.OL625609). These results demonstrated the presence of genetically diverse isolates of SPLV in the pooled samples. To further confirm the HTS result, each of the 12 samples were tested by RT-PCR using SPLV primers (SPLV-F1: 5'-AATGCCAAGGCTACAAGGAGT-3' and SPLV-R1: 5'-CAAGTAGTGTGTGTATGTTCC-3') that targets a partial conserved region of the coat protein gene in SPLV and SPCSV primers designed based on three contigs (ctg1-F1/R1, ctg2-F1/R1, and ctg3-F1/R1) (Liu et al. 2021), respectively. As a result, four symptomless C. hederacea samples tested positive for SPLV, yielding the expected approximately 500 bp PCR fragment, and one leaf yellowing C. hederacea sample tested positive for SPCSV (Liu et al. 2021). The sequences obtained from two of the four amplicons of SPLV (MZ089700 and OM056706) showed 90.2 and 89.8% nt (100 and 99.4% amino acid) identities with the corresponding sequences of the SPLV isolate Shaanxi1 from sweet potato (HQ844148). In 2021, a further 45 C. hederacea plants collected from Shangqiu (n = 6), Xinxiang (n =30) and Pingdingshan (n = 9) cities in Henan Province, were screened by RT-PCR with SPLV-F1/R1 primers, giving an incidence of 33.33%. SPLV is an important potyvirus infecting sweet potato. SPLV is asymptomatic in most sweet potato cultivars in single infection but is able to mediate synergistic viral disease in co-infection with SPCSV (Untiveros et al. 2007). To the best of our knowledge, this is the first report of SPLV in C. hederacea. The finding reported here indicated that C. hederacea may act as a reservoir of SPLV and possible infection source for the sweet potato crop.

Authentication of Rusa timorensis DNA Using Sequencing Assay with Modified Extraction Method

A technique to isolate DNA from deer meat was established to authenticate the deer species and was successfully extracted from deer meat. The method was based on the Epicentre MasterPureTM Complete DNA and RNA Purification Kit extraction kit with slight modification by increasing the volume of Proteinase K and RNase from 5 µL to 20 µL each. The recovery of deer DNA was then analysed by Polymerase Chain Reaction using cytochrome b gene oligonucleotide primers for the deer family, which targeted the mitochondrial DNA. The increasing volume of Proteinase K and RNase resulted in clean DNA and no smearing after performing Polymerase Chain Reaction. Furthermore, the amplified DNA product was extended to sequence assay to authenticate deer species above 95% maximum identification. The result showed that the increasing volume of Proteinase K and RNase offered a high yield of DNA recovery, which provided more than 100 ng per μL and was useful in validating animal-based sources, especially in deer products.

Nanomolecular Magnetic Probe for Colorimetric RT-LAMP of Viral RNA

Amplification of the RNA from the Covid-19 virus is considered the main objective of the molecular diagnosis of SARS-Cov-2. However, the use of target-based amplification methods such as polymerase chain reaction requires a step to convert the RNA of the Covid-19 virus into a DNA template to lead to amplification. In addition, isolating the RNA of the Covid-19 virus requires RNA purification kits, which will increase the time and costs of molecular detection of this virus. In this study, the magnetic nanoprobe is introduced that it could capture and amplify Covid-19 RNA through an isothermal amplification process called loop-mediated isothermal amplification without requiring a step to convert the viral RNA into a DNA template. By using the engineered sequences corresponding to the target nucleic acid attached to magnetic nanoparticles, it becomes possible to identify the target RNA of this virus through color changes due to pH changes that can be seen with the naked eye due to the presence of pH indicators in the reaction mix. According to the isothermal amplification of the viral RNA via LAMP assisted with the magnetic nanoprobe, the nanomolecular method eliminated the need for special equipment and the time for detecting Covid-19 in specimens.

P-196 Assessing human embryo implantation competence through analysis of extracellular vesicles and their contents

Abstract Study question Could extracellular vesicles (EVs) derived from spent media serve as a non-invasive quality marker for embryo implantation and pregnancy? Summary answer EVs isolated from spent media derived from human blastocysts that either did or didn’t establish a pregnancy after transfer differ in size, concentration, miRNA expression. What is known already EVs are important for communication from one cell to another, influencing the translation of genes to proteins. They play a regulating role during biological processes, including pregnancy. We hypothesized that EVs released by a high-quality human embryo, which has the potential to create a pregnancy after the transfer, differ from EVs released by poor quality non-implanting embryos. Study design, size, duration Patients scheduled for a fresh single blastoycst transfer in IVF/ICSI cycles using own gametes consented to sampling of spent culture media (n = 102). Spent media from the transferred blastocyst (25 μl) were collected from implanted (hCG+/ET) (n = 45) and non-implanted (hCG-/ET) (n = 51). Spent media from degenerated embryos (n = 21) were collected from 6 patients. Media samples were pooled randomly into three replicates for each group (implanted (I), non-implanted (NI), degenerated (DG)). Participants/materials, setting, methods Extracellular vesicles were isolated by qEV single size exclusion column(SEC):a maximum of 200 µl of implanted or non-implanted or degenerated spent media was loaded onto the qEV SEC. Identification and characterization of EVs was performed by nanoparticle tracking analysis, transmission electron microscopy, and western blotting (CD9, CD63). Next, EV-RNA isolation was performed on these three groups (three replicates from each group) by Norgen Biotek Exosomal RNA Purification Kit, followed by RT-qPCR, and further data analysis. Main results and the role of chance Extracellular vesicles isolated from the SEC column are referred to as I-EVs, NI –EVs, and DG-EVs (implanted, non-implanted, and degenerated, respectively). Western blot confirmed the presence of EV-specific markers (CD63, CD9) in all EV samples (I-EVs, NI-EVs, and DG-EVs). By NTA, a higher particle concentration was observed in I-EVs (2.69±1.9 × 1010 particles/mL) compared to NI-EVs (6.13±0.2 × 1010 particles/mL) (p &amp;lt; 0.05), whereas DG-EVs particle concentration (4.97±0. 2 × 109 particles/mL) was significantly (P &amp;lt; 0.01). I-EVs were also characterized by a larger particle size with an average mean diameter of 180.3±5.4 nm, whereas NI-EVs displayed a smaller particle size with a mean diameter of 141.5±2.5 nm (p &amp;lt; 0.05). TEM confirmed the presence of larger-sized vesicles in I-EVs (≥ 200 nm) compared with NI and DG samples. During earlier work in EVs from bovine blastocysts we found miR-378a-3p to be typically present in blastocyst EVs, with a prominent role in the hatching process of the blastocyst. Here, we compared miR-378a-3p expression levels by RT-qPCR in I-EVs vs. NI-EVs vs DG-EVs. Higher expression of miR-378a-3p was identified in NI-EVs and I-EVs, compared to DG-EVs (log2fc= 30.10, log2fc = 8.94 p &amp;lt; 0.001 respectively), but was not correlated with implantation rate. Limitations, reasons for caution Currently, the methodology is unable to study EVs from a single droplet of spent media, hence pooling of the samples is necessary. As a consequence, data on EV-miRNA from individual blastocyst is lost. Wider implications of the findings Our data demonstrated that EVs and their cargo (miRNAs) have potential to be used as a non-invasive method to predict embryo implantation:miR-378a-3p has been associated with hatching in bovine embryos, but was not related with human implantion. Other embryo-derived miRNAs may be tested as a possible biomarker for human pregnancy. Trial registration number EC 2018/0266 – B670201835577

Comparison of Two RNA Extraction Methods for the Molecular Detection of SARS-CoV-2 from Nasopharyngeal Swab Samples.

Background: Rapid diagnosis of COVID-19 is essential in order to restrict the spread of the pandemic, and different approaches for SARS-CoV-2 testing have been proposed as cost-effective and less time-consuming alternatives. For virus detection, the real-time reverse transcriptase–polymerase chain reaction (RT-PCR) technique is still the “gold standard” for accuracy and reliability, but its performance is affected by the efficiency of nucleic acid extraction methods. Objective: In order to improve the SARS-CoV-2 diagnostic workflow, we compared a “standard” commercially available kit, based on viral RNA extraction from human swab samples by magnetic beads, with its technological evolution. The two methods differ mainly in their time consumption (9 vs. 35 min). Methods: We adopted the MAGABIO PLUS VIRUS DNA/RNA PURIFICATION KIT II (BIOER), defined as “standard”, with the automatic extractor BIOER (GenePure Pro fully automatic nucleic acid purification system) to isolate RNA from nasopharyngeal swabs for the detection of SARS-CoV-2 by RT-PCR. We tested this kit with a new faster version of the first one, defined as “rapid” (MAGABIO PLUS VIRUS RNA PURIFICATION KIT II). Results and Conclusion: The two evaluated procedures provided similar analytical results, but the faster method proved to be a more suitable tool for the detection of SARS-CoV-2 from nasopharyngeal swabs, due to a more rapid availability of results, which may contribute to improving both clinical decision making and patient safety.

Centrifugal Microfluidic Method for Enrichment and Enzymatic Extraction of Severe Acute Respiratory Syndrome Coronavirus 2 RNA.

The diversification of analytical tools for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is imperative for effective virus surveillance and transmission control worldwide. Development of robust methods for rapid, simple isolation of viral RNA permits more expedient pathogen detection by downstream real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) to minimize stalled containment and enhance treatment efforts. Here, we describe an automatable rotationally driven microfluidic platform for enrichment and enzymatic extraction of SARS-CoV-2 RNA from multiple sample types. The multiplexed, enclosed microfluidic centrifugal device (μCD) is capable of preparing amplification-ready RNA from up to six samples in under 15 min, minimizing user intervention and limiting analyst exposure to pathogens. Sample enrichment leverages Nanotrap Magnetic Virus Particles to isolate intact SARS-CoV-2 virions from nasopharyngeal and/or saliva samples, enabling the removal of complex matrices that inhibit downstream RNA amplification and detection. Subsequently, viral capsids are lysed using an enzymatic lysis cocktail for release of pathogenic nucleic acids into a PCR-compatible buffer, obviating the need for downstream purification. Early in-tube assay characterization demonstrated comparable performance between our technique and a “gold-standard” commercial RNA extraction and purification kit. RNA obtained using the fully integrated μCDs permitted reliable SARS-CoV-2 detection by real-time RT-PCR. Notably, we successfully analyzed full-process controls, positive clinical nasopharyngeal swabs suspended in viral transport media, and spiked saliva samples, showcasing the method’s broad applicability with multiple sample matrices commonly encountered in clinical diagnostics.

The effect of ENEAS application in patients with endometriosis and its influence on the level of IL-8 and MCP-1.

Endometriosis is a gynecological disease that endometrial cells develop outside the uterus. This event happens when the endometrial glands grow outside the endometrium and uterine muscles, especially in the pelvis. Although endometriosis is widespread, the clinical manifestations of the disease are very different, and it is challenging to adapt to the conventional classification system to divide patients into homogeneous groups. Given the importance of endometriosis, a correct, accurate, and timely diagnosis of this disease can significantly prevent its complications. Using health-related software is one of these ways. Enhanced Endometriosis Archiving Software (ENEAS) is a web-based application based on one of the most widely used open-source database management systems (MySQL), allowing the direct link to other open-source software for data management and storage. In the current study, the effect of ENEAS application was considered in patients with endometriosis, and its influence on IL-8 and MCP-1 gene expression was evaluated. For this purpose, 100 women with endometriosis were divided into two groups of 50 patients. The first group (control group) was examined by a gynecologist and received medication and treatment. In the case group, their demographic and clinical information were entered into ENEAS software. To study the expression of the IL-8 gene and MCP-1 gene, after collecting 5 ml of blood samples in tubes containing anticoagulant, RNA extraction was performed by Total RNA Purification Kit (Cat. 17200, 37500, 17250). Then cDNA synthesis was performed for this purpose, and a Bioneer DNA synthesis kit (South Korea) was used. The results showed that the expression level of the IL-8 gene in the case group was significantly reduced compared to the control group (P = 0.035). MCP-1 gene expression was also decreased compared to the control group, but this decrease was not significant. Therefore, those who used this application for treatment had reduced expression of IL-8 and MCP-1 genes. This event indicates that this application has reduced the amount of inflammation caused by endometriosis with proper analysis.