scholarly journals VariantDirect: An extraction-free screening approach to detect circulating SARS-CoV-2 virus strains from pooled specimens

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S19-S20
Author(s):  
Abdulrahman Saadalla ◽  
Brooke Stroup ◽  
Bijal Parikh
Keyword(s):  
Proteinase K ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Validation Data ◽  
Viral Receptor ◽  
Epidemiologic Surveillance ◽  
Clinical Laboratories ◽  
Time Capsule ◽  
Analytical Sensitivities ◽  
Viral Titers

Abstract Coronavirus disease (COVID-19) caused by the SARS-CoV-2 virus has exposed clinical laboratories to unprecedented challenges. With surging case numbers, clinical laboratories were forced to acquiesce and integrate multiple testing platforms with varying workflows and analytical sensitivities in order to meet testing volumes. Now a new challenge has emerged with the evolution of viral variants, both globally and locally, raising concerns for uncontrolled spread, increased disease severity, and weakened responses to vaccinations. Preliminary data suggests that these variants may be associated with higher viral titers and prolonged infections. While primarily leveraged for epidemiologic surveillance, the clinical utility of variant detection may quickly become paramount. Furthermore, laboratories must remain vigilant and nimble enough to pivot should variant identification play a role in the patient care. To prepare for the validation of clinical assays that identify important viral variants, we designed a novel method, termed VariantDirect, to screen SARS-CoV-2 positive samples for the presence of variants, focusing initially on the increasingly prevalent UK and South African (SA) variants. The detection strategy is based on primers designed to specifically target the viral receptor-binding domain mutation, N501Y, shared by the UK and SA strains. Screening for variants will be limited to nasopharyngeal swab samples of high viral titers (Ct values <25 by RT-qPCR assay, Roche Diagnostics). Pools of 9 different samples, 50 µl each, are mixed and stored at -80°C along with aliquots of the 9 original samples. These pools will then be tested, and if positive for the N501Y variant, the pooled 9 samples will be thawed and tested separately to identify the affected specimen. Most of these specimens are also being independently sequenced via a comprehensive but more resource-intensive NGS approach. Advantages of our pooled workflow are primarily in time and cost, with the capacity of screening up to 837 specimens on a single run. In addition, our collection strategy establishes a “time capsule” to document the evolution of viral strains within our geographical region. Finally, these studies serve to optimize technical parameters for the development of clinical assays. A validated nucleic acid (NA) extraction-free RT-qPCR method will be utilized for this assay. Our internal validation data showed comparable analytical sensitivities to NA extraction-based methods. Pooled samples in transport medium are diluted in normal saline at a ratio of 1:1, and then heat-inactivated in the presence of proteinase-K and ultimately analyzed on the Applied Biosystems™ 7500 Fast Dx instrument. As new variants of interest emerge, primers and probes can be quickly redesigned and validated on clinical samples within our NGS-confirmed “time capsule”. This study will provide important information needed for current or future genomic and epidemiologic studies.

scholarly journals Comparison Between a Standard and SalivaDirect RNA Extraction Protocol for Molecular Diagnosis of SARS-CoV-2 Using Nasopharyngeal Swab and Saliva Clinical Samples

2021 ◽  
Vol 9 ◽  
Author(s):  
Sofía N. Rodríguez Flores ◽  
Luis Mario Rodríguez-Martínez ◽  
Bernardita L. Reyes-Berrones ◽  
Nadia A. Fernández-Santos ◽  
Elthon J. Sierra-Moncada ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Proteinase K ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Sample Collection ◽  
Extraction Protocol ◽  
Assay Performance ◽  
Two Samples ◽  
Oropharyngeal Swab

During the COVID-19 pandemic, a certified laboratory of Tamaulipas, Mexico has processed over 100,000 samples of COVID-19 suspected patients, working a minimum of 100 tests daily. Thus, it would be beneficial for such certified laboratories nationwide to reduce the time and cost involved in performing the diagnosis of COVID-19, from sample collection, transportation to local lab, processing of samples, and data acquisition. Here, 30 nasopharyngeal swab and saliva samples from the same COVID-19 individuals were assessed by a standard nucleic acid extraction protocol, including protein lysis with proteinase K followed by binding to column, washing, and elution, and by the SalivaDirect protocol based on protein lysis, skipping the other steps to reduce processing time and costs. The genomic RNA was amplified using a SARS-CoV-2 Real-Time PCR kit. A variation (P > 0.05) in the 95% CIs = 72.6%–96.7% was noted by using the SalivaDirect protocol and saliva samples (sensitivity of 88.2%) in comparison to those of standard protocol with oropharyngeal swab samples (95% CIs = 97.5%–100%; sensitivity of 100%) as reported elsewhere. However, when using nasopharyngeal swab samples in the SalivaDirect protocol (sensitivity of 93.6%; 95% CIs = 79.2%–99.2%), it was in concordance (P < 0.05) with those of the standard one. The logical explanation to this was that two samples with Ct values of 38, and 40 cycles for gene E produced two false negatives in the SalivaDirect protocol in relation to the standard one; thus, there was a reduction of the sensitivity of 6.4% in the overall assay performance.

scholarly journals Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247792
Author(s):  
Valeria Genoud ◽  
Martin Stortz ◽  
Ariel Waisman ◽  
Bruno G. Berardino ◽  
Paula Verneri ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Standard Technique ◽  
Proteinase K ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Nasopharyngeal Swab ◽  
Reverse Transcription Pcr ◽  
Extraction Step ◽  
Commercial Kits ◽  
Inexpensive Procedure

Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.

scholarly journals Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

2020 ◽  
Author(s):  
Julien Fassy ◽  
Caroline Lacoux ◽  
Sylvie Leroy ◽  
Latifa Noussair ◽  
Sylvain Hubac ◽  
...  
Keyword(s):  
High Throughput ◽  
Cellular Response ◽  
Rna Extraction ◽  
Reference Method ◽  
Proteinase K ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Rna Purification ◽  
Large Populations ◽  
Reaction Volumes

AbstractThe emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a couple of probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction steps. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the Biomark instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring in addition to SARS-CoV-2 probes of other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). Its 10 nL range volume is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several procedures, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.

scholarly journals Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0243333
Author(s):  
Julien Fassy ◽  
Caroline Lacoux ◽  
Sylvie Leroy ◽  
Latifa Noussair ◽  
Sylvain Hubac ◽  
...  
Keyword(s):  
High Throughput ◽  
Cellular Response ◽  
Rna Extraction ◽  
Reference Method ◽  
Proteinase K ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Extraction Step ◽  
Large Populations ◽  
Reaction Volumes

The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the BiomarkTM instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of BiomarkTM reactions is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several protocols, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.

scholarly journals Urine lipoarabinomannan in HIV uninfected, smear negative, symptomatic TB patients: effective sample pretreatment for a sensitive immunoassay and mass spectrometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anita G. Amin ◽  
Prithwiraj De ◽  
Barbara Graham ◽  
Roger I. Calderon ◽  
Molly F. Franke ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Sample Pretreatment ◽  
Positive Association ◽  
Proteinase K ◽  
Clinical Samples ◽  
Sputum Smear ◽  
Smear Negative ◽  
Tuberculostearic Acid ◽  
Negative Sputum ◽  
Adult Culture

AbstractOur study sought to determine whether urine lipoarabinomannan (LAM) could be validated in a sample cohort that consisted mainly of HIV uninfected individuals that presented with tuberculosis symptoms. We evaluated two tests developed in our laboratory, and used them on clinical samples from Lima, Peru where incidence of HIV is low. ELISA analysis was performed on 160 samples (from 140 adult culture-confirmed TB cases and 20 symptomatic TB-negative child controls) using 100 μL of urine after pretreatment with Proteinase K. Two different mouse monoclonal antibodies-CS35 and CHCS9-08 were used individually for capture of urine LAM. Among cases, optical density (OD450) values had a positive association with higher bacillary loads. The 20 controls had negative values (below the limit of detection). The assay correctly identified all samples (97–100% accuracy confidence interval). For an alternate validation of the ELISA results, we analyzed all 160 urine samples using an antibody independent chemoanalytical approach. Samples were called positive only when LAM surrogates—tuberculostearic acid (TBSA) and d-arabinose (d-ara)—were found to be present in similar amounts. All TB cases, including the 40 with a negative sputum smear had LAM in detectable quantities in urine. None of the controls had detectable amounts of LAM. Our study shows that urinary LAM detection is feasible in HIV uninfected, smear negative TB patients.

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Percentage Agreement ◽  
Testing Method ◽  
Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

scholarly journals Purification and characterization of bacteriocins-like inhibitory substances from food isolated Enterococcus faecalis OS13 with activity against nosocomial enterococci

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ahmed O. El-Gendy ◽  
Dag A. Brede ◽  
Tamer M. Essam ◽  
Magdy A. Amin ◽  
Shaban H. Ahmed ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Antimicrobial Agents ◽  
Food Samples ◽  
Proteinase K ◽  
Clinical Samples ◽  
Bile Salt Hydrolase ◽  
Antibiotic Resistant ◽  
Molecular Weights ◽  
Global Threat

AbstractNosocomial infections caused by enterococci are an ongoing global threat. Thus, finding therapeutic agents for the treatment of such infections are crucial. Some Enterococcus faecalis strains are able to produce antimicrobial peptides called bacteriocins. We analyzed 65 E. faecalis isolates from 43 food samples and 22 clinical samples in Egypt for 17 common bacteriocin-encoding genes of Enterococcus spp. These genes were absent in 11 isolates that showed antimicrobial activity putatively due to bacteriocins (three from food, including isolate OS13, and eight from clinical isolates). The food-isolated E. faecalis OS13 produced bacteriocin-like inhibitory substances (BLIS) named enterocin OS13, which comprised two peptides (enterocin OS13α OS13β) that inhibited the growth of antibiotic-resistant nosocomial E. faecalis and E. faecium isolates. The molecular weights of enterocin OS13α and OS13β were determined as 8079 Da and 7859 Da, respectively, and both were heat-labile. Enterocin OS13α was sensitive to proteinase K, while enterocin OS13β was resistant. Characterization of E. faecalis OS13 isolate revealed that it belonged to sequence type 116. It was non-hemolytic, bile salt hydrolase-negative, gelatinase-positive, and sensitive to ampicillin, penicillin, vancomycin, erythromycin, kanamycin, and gentamicin. In conclusion, BLIS as enterocin OS13α and OS13β represent antimicrobial agents with activities against antibiotic-resistant enterococcal isolates.

scholarly journals Enrichment of Exosome-Like Extracellular Vesicles from Plasma Suitable for Clinical Vesicular miRNA Biomarker Research

2019 ◽  
Vol 8 (11) ◽  
pp. 1995 ◽  
Author(s):  
Moon ◽  
Shin ◽  
Kim ◽  
Lee ◽  
Mankhong ◽  
...  
Keyword(s):  
Human Plasma ◽  
Extracellular Vesicles ◽  
Proteinase K ◽  
High Yield ◽  
Clinical Samples ◽  
Diagnostic Biomarkers ◽  
Micro Rnas ◽  
Biomarker Research ◽  
Protein Rich ◽  
Yield And Purity

Exosome-like extracellular vesicles (ELVs) contain biomolecules that have potential as diagnostic biomarkers, such as proteins, micro-RNAs (miRNAs), and lipids. However, it is difficult to enrich ELVs consistently with high yield and purity from clinical samples, which hampers the development of ELV biomarkers. This is particularly true for miRNAs in protein-rich plasma. Hence, we modified ELV isolation protocols of three commercially available polymer-precipitation-based kits using proteinase K (PK) treatment to quantify ELV-associated miRNAs in human plasma. We compared the yield, purity, and characteristics of enriched plasma ELVs, and measured the relative quantity of three selected miRNAs (miR-30c, miR-126, and miR-192) in ELVs using six human plasma samples. Compared with the original protocols, we demonstrated that ELVs can be isolated with PK treatment with high purity (i.e., lack of non-exosomal proteins and homogeneous size of vesicles) and yield (i.e., abundancy of exosomal markers), which were dependent on kits. Using the kit with the highest purity and yield with PK treatment, we successfully quantified ELV miRNAs (levels of 45%–65% in total plasma) with acceptable variability. Collectively, ELV enrichment using the modified easy-to-use method appears suitable for the analysis of miRNAs, although its clinical applicability needs to be confirmed in larger clinical studies.

scholarly journals Human Respiratory Syncytial Virus-Induced Immune Signature of Infection Revealed by Transcriptome Analysis of Clinical Pediatric Nasopharyngeal Swab Samples

2020 ◽  
Author(s):  
Claire Nicolas De Lamballerie ◽  
Andrés Pizzorno ◽  
Julia Dubois ◽  
Blandine Padey ◽  
Thomas Julien ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Transcriptome Analysis ◽  
Real Life ◽  
Human Respiratory Syncytial Virus ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Immune Signature ◽  
Real Life Setting ◽  
Syncytial Virus ◽  
New Diagnosis

Abstract Human respiratory syncytial virus (HRSV) constitutes one the main causes of respiratory infection in neonates and infants worldwide. Transcriptome analysis of clinical samples using high-throughput technologies remains an important tool to better understand virus-host complex interactions in the real-life setting but also to identify new diagnosis/prognosis markers or therapeutics targets. A major challenge when exploiting clinical samples such as nasal swabs, washes, or bronchoalveolar lavages is the poor quantity and integrity of nucleic acids. In this study, we applied a tailored transcriptomics workflow to exploit nasal wash samples from children who tested positive for HRSV. Our analysis revealed a characteristic immune signature as a direct reflection of HRSV pathogenesis and highlighted putative biomarkers of interest such as IP-10, TMEM190, MCEMP1, and TIMM23.

scholarly journals Comparative evaluation of published cytomegalovirus primers for rapid real-time PCR: which are the most sensitive?

2009 ◽  
Vol 58 (7) ◽  
pp. 878-883 ◽  
Author(s):  
Wafa Habbal ◽  
Fawza Monem ◽  
Barbara C. Gärtner
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Pcr Product ◽  
Highly Sensitive ◽  
Primer Sets ◽  
Analytical Sensitivities ◽  
Strain Ad169

Standardization of human cytomegalovirus (CMV) PCR is highly recommended. As primer design is essential for PCR sensitivity, this study evaluated all published CMV primer pairs to identify the most sensitive for single-round real-time PCR. PubMed (1993–2004) was searched for original papers aimed at CMV PCR. Fifty-seven papers were identified revealing 82 different primer pairs. Of these, 17 primer sets were selected for empirical study, as they were either used in real-time PCR or were evaluated comparatively by conventional PCR. After optimizing the PCR conditions, these primer sets were evaluated by real-time PCR using a SYBR Green format. Analytical sensitivities were assessed by testing the reference standard CMV strain AD169. A blast search was performed to identify mismatches with published sequences. Additionally, 60 clinical samples were tested with the three primer sets showing highest analytical sensitivity and the best match to all CMV strains. Three primer sets located in the glycoprotein B (UL55) gene region were found to be the most sensitive using strain AD169. However, two of these showed a considerable number of mismatches with clinical isolates in a blast search. Instead, two other pairs from the lower matrix phosphoprotein (UL83) gene and DNA polymerase (UL54) gene showed reasonable sensitivity and no mismatches with clinical isolates. These three pairs were further tested with clinical samples, which indicated that the two primer sets from UL55 and UL54 were the most sensitive. Interestingly, the analytical sensitivity of the PCR was inversely correlated with the size of the PCR product. In conclusion, these two primer pairs are recommended for a standardized, highly sensitive, real-time PCR.

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