scholarly journals An international, inter-laboratory ring trial confirms the feasibility of an open source, extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples

2021 ◽  
Author(s):  
Margaret G. Mills ◽  
Emily A Bruce ◽  
Meei-Li Huang ◽  
Jessica W. Crothers ◽  
Ollivier Hyrien ◽  
...  
Keyword(s):  
Open Access ◽  
Qualitative Method ◽  
Clinical Samples ◽  
Correct Identification ◽  
Rt Pcr ◽  
Viral Transport ◽  
Ring Trial ◽  
Qpcr Method ◽  
Feasible Option ◽  
Source Extraction

RT-qPCR is used world-wide to test and trace the spread of SARS-CoV-2. Extraction-less or direct RT-PCR is an open-access qualitative method for SARS-CoV-2 detection from nasopharyngeal (NP) or oral pharyngeal (OP) samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged ten global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international inter-laboratory ring trial. Participating labs were provided a common protocol, common reagents, aliquots of identical pooled clinical samples and purified nucleic acids, and used their existing in-house equipment. We observed 100% concordance across labs in the correct identification of all positive and negative samples, with highly similar Ct values observed. The test also performed well when applied to locally collected patient NP samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that open access, direct RT-PCR assays are a feasible option for more efficient COVID-19 testing as demanded by the continuing pandemic.

scholarly journals An international, interlaboratory ring trial confirms the feasibility of an extraction-less “direct” RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0261853
Author(s):  
Margaret G. Mills ◽  
Emily Bruce ◽  
Meei-Li Huang ◽  
Jessica W. Crothers ◽  
Ollivier Hyrien ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Correct Identification ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Ring Trial ◽  
Feasible Option ◽  
Polymerase Chain

Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). “Extraction-less” or “direct” real time–reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

scholarly journals Rapid Screening for SARS-CoV-2 Variants of Concern in Clinical and Environmental Samples Using Nested RT-PCR Assays Targeting Key Mutations of the Spike Protein

2021 ◽  
Author(s):  
G. La Rosa ◽  
P. Mancini ◽  
G. Bonanno Ferraro ◽  
C. Veneri ◽  
M. Iaconelli ◽  
...  
Keyword(s):  
Spike Protein ◽  
Rapid Screening ◽  
Clinical Samples ◽  
Correct Identification ◽  
Potential Contribution ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Field Samples ◽  
Pcr Assays ◽  
Viral Isolates

SUMMARYNew SARS-CoV-2 mutations are constantly emerging, raising concerns of increased transmissibility, virulence or escape from host immune response.We describe a nested RT-PCR assay (∼1500 bps) to detect multiple key spike protein mutations distinctive of the major known circulating SARS-CoV-2 variants, including the three Variants of Concern (VOCs) 20I/501Y.V1 (United Kingdom), 20H/501Y.V2 (South Africa), and 20J/501Y.V3 (Brazil), as well as the 20E.EU1 variant (Spain), the CAL.20C recently identified in California, and the mink-associated variant (GR, lineage B.1.1.298). Prior to application to field samples, the discriminatory potential of this PCR assay was explored using GISAID and Nextclade. To extend variant detection to challenging matrices such as sewage, where the amplification of long fragments is problematic, two short nested RT-PCR assays (∼300 bps) were also designed, targeting portions of the region spanned by the long nested assay.The three newly-designed assays were then tested on field samples, including 7 fully-sequenced viral isolates from swab samples and 34 urban wastewater samples, some of which collected in areas where circulation of VOCs had been reported.The long assay successfully amplified all the previously characterized viral isolates, allowing the correct identification of variants 20I/501Y.V1 and 20E.EU1 present in the panel. The sequences obtained using the short assays were consistent with those obtained with the long assay. Mutations characteristic of VOCs (UK and Brazilian variant) and of other variant (Spanish) were detected in sewage samples. To our knowledge, this is the first evidence of the presence of sequences harboring key mutations of 20I/501Y.V1 and 20J/501Y.V3 in urban wastewaters, highlighting the potential contribution of wastewater surveillance to explore SARS-CoV-2 diversity.The developed nested RT-PCR assays can be used as an initial rapid screening test to select clinical samples containing mutations of interest. This can speed up diagnosis and optimize resources since it allows full genome sequencing to be done only on clinically relevant specimens. The assays can be also employed for a rapid and cost-effective detection of VOCs or other variants in sewage for the purposes of wastewater-based epidemiology. The approach proposed here can be used to better understand SARS-CoV-2 variant diversity, geographic distribution and impact worldwide.

scholarly journals Ultrafast RNA extraction-free SARS-CoV-2 detection by direct RT-PCR using a rapid thermal cycling approach

2021 ◽  
Author(s):  
Robin Struijk ◽  
Anton van den Ouden ◽  
Brian McNally ◽  
Theun de Groot ◽  
Bert Mulder ◽  
...  
Keyword(s):  
Large Scale ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Clinical Specimens ◽  
Percent Agreement ◽  
Qpcr Method ◽  
Agreement Score ◽  
Pcr Method

The surging COVID19 pandemic has underlined the need for quick, sensitive, and high-throughput SARS-CoV-2 detection assays. Although many different methods to detect SARS-CoV-2 particles in clinical material have been developed, none of these assays are successful in combining all three of the above characteristics into a single, easy-to-use method that is suitable for large-scale use. Here we report the development of a direct RT-PCR SARS-CoV-2 detection method that can reliably detect minute quantities of SARS-CoV-2 gRNA in nasopharyngeal swab samples as well as the presence of human genomic DNA. An extraction-less validation protocol was carried out to determine performance characteristics of the assay in both synthetic SARS-CoV-2 RNA as well as clinical specimens. Feasibility of the assay and analytical sensitivity was first determined by testing a dilution series of synthetic SARS-CoV-2 RNA in two different solvents (water and AMIES VTM), revealing a high degree of linearity and robustness in fluorescence readouts. Following analytical performance using synthetic RNA, the limit of detection was determined at equal to or less than 1 SARS-CoV-2 copy/ul of sample in a commercially available sample panel that contains surrogate clinical samples with varying SARS-CoV-2 viral load. Lastly, we benchmarked our method against a reference qPCR method by testing 87 nasopharyngeal swab samples. The direct endpoint ultra-fast RT-PCR method exhibited a positive percent agreement score of 98.5% and a negative percent agreement score of 100% as compared to the reference method, while RT-PCR cycling was completed in 27 minutes/sample as opposed to 60 minutes/sample in the reference qPCR method. In summary, we describe a rapid direct RT-PCR method to detect SARS-CoV-2 material in clinical specimens which can be completed in significantly less time as compared to conventional RT-PCR methods, making it an attractive option for large-scale SARS-CoV-2 screening applications.

Environment Approach on Optimizing Tourism Facilities at Tourism Village in Indonesia

2019 ◽  
Vol 4 (10) ◽  
pp. 109 ◽  
Author(s):  
Nurlisa Ginting ◽  
Satria Halim
Keyword(s):  
Open Access ◽  
Peer Review ◽  
Qualitative Method ◽  
Local Community ◽  
Publishing House ◽  
Tourist Attraction ◽  
Dynamic Movement ◽  
International Publishing ◽  
Tourist Village ◽  
Open Access Article

Tourism is a sector that always has a dynamic movement which will benefit the local community. Lumban Suhi-suhi is a tourist village famous for its ulos on Samosir Island that has become one of the tourist attraction. Due to the limitations of facilities arrangement make it becomes less of the attraction itself. Therefore, the environment-based mechanism becomes one of the concepts that will be applied to improve the tourist facilities in which could increase tourists in that area. The researcher uses a qualitative method in the form of direct observation and interview and analyzing data with related theory.Keywords: Tourism Village; Facilities; Sustainable Tourism; EnvironmenteISSN: 2398-4287© 2019. The Authors. Published for AMER ABRA cE-Bs by e-International Publishing House, Ltd., UK. This is an open access article under the CC BYNC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer–review under responsibility of AMER (Association of Malaysian Environment-Behaviour Researchers), ABRA (Association of Behavioural Researchers on Asians) and cE-Bs (Centre for Environment-Behaviour Studies), Faculty of Architecture, Planning & Surveying, Universiti Teknologi MARA, Malaysia.DOI: https://doi.org/10.21834/e-bpj.v4i10.1617

Effective optimization of SARS-CoV-2 laboratory testing variables in an era of supply chain constraints

2020 ◽  
Vol 15 (15) ◽  
pp. 1483-1487
Author(s):  
Nikhil S Sahajpal ◽  
Ashis K Mondal ◽  
Allan Njau ◽  
Sudha Ananth ◽  
Kimya Jones ◽  
...  
Keyword(s):  
Supply Chain ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Sample Collection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Short Supply ◽  
Viral Transport ◽  
3D Printed ◽  
Bridging Studies

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

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 Diagnostic amyloid proteomics: experience of the UK National Amyloidosis Centre

2020 ◽  
Vol 58 (6) ◽  
pp. 948-957 ◽  
Author(s):  
Diana Canetti ◽  
Nigel B. Rendell ◽  
Janet A. Gilbertson ◽  
Nicola Botcher ◽  
Paola Nocerino ◽  
...  
Keyword(s):  
Simple Algorithm ◽  
Enzymatic Digestion ◽  
The Body ◽  
Clinical Samples ◽  
Correct Identification ◽  
Pathogenic Variants ◽  
Amyloidogenic Protein ◽  
Patient Database ◽  
Serious Disease ◽  
The Uk

AbstractSystemic amyloidosis is a serious disease which is caused when normal circulating proteins misfold and aggregate extracellularly as insoluble fibrillary deposits throughout the body. This commonly results in cardiac, renal and neurological damage. The tissue target, progression and outcome of the disease depends on the type of protein forming the fibril deposit, and its correct identification is central to determining therapy. Proteomics is now used routinely in our centre to type amyloid; over the past 7 years we have examined over 2000 clinical samples. Proteomics results are linked directly to our patient database using a simple algorithm to automatically highlight the most likely amyloidogenic protein. Whilst the approach has proved very successful, we have encountered a number of challenges, including poor sample recovery, limited enzymatic digestion, the presence of multiple amyloidogenic proteins and the identification of pathogenic variants. Our proteomics procedures and approaches to resolving difficult issues are outlined.

scholarly journals Quantitative measurement of infectious virus in SARS-CoV-2 Alpha, Delta and Epsilon variants reveals higher infectivity (viral titer:RNA ratio) in clinical samples containing the Delta and Epsilon variants.

2021 ◽  
Author(s):  
Hannah W Despres ◽  
Margaret G Mills ◽  
David J Shirley ◽  
Madaline M Schmidt ◽  
Meei-Li Huang ◽  
...  
Keyword(s):  
Quantitative Measurement ◽  
Infectious Virus ◽  
Viral Rna ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Genome Copy ◽  
Live Virus ◽  
High Degree ◽  
The Relationship ◽  
Rna Levels

ABSTRACT Background Novel SARS-CoV-2 Variants of Concern (VoC) pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between viral RNA and infectious virus for individual variants is unknown. Methods We measured infectious viral titer (using a micro-focus forming assay) as well as total and subgenomic viral RNA levels (using RT-PCR) in a set of 165 clinical samples containing SARS-CoV-2 Alpha, Delta and Epsilon variants that were processed within two days of collection from the patient. Results We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite the variability we observed for individual samples the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (6 and 4 times as much, p=0.0002 and 0.009 respectively) or subgenomic E RNA (11 and 7 times as much, p<0.0001 and 0.006 respectively). Conclusion In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may also be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity of the Delta variant may further explain increased spread and suggests a need for increased measures to prevent viral transmission.

scholarly journals Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252687
Author(s):  
Sukalyani Banik ◽  
Kaheerman Saibire ◽  
Shraddha Suryavanshi ◽  
Glenn Johns ◽  
Soumitesh Chakravorty ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Viral Rna ◽  
Clinical Samples ◽  
Sample Collection ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Guanidinium Thiocyanate ◽  
Upper Respiratory ◽  
Polymerase Chain ◽  
The Stability

Background Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. Methods We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. Results SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. Conclusion eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

scholarly journals Rapid advancement in cancer genomic big data in the pursuit of precision oncology

2021 ◽  
Author(s):  
Tiara Bunga Mayang Permata ◽  
Sri Mutya Sekarutami ◽  
Endang Nuryadi ◽  
Angela Giselvania ◽  
Soehartati Gondhowiardjo
Keyword(s):  
Big Data ◽  
Open Access ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Genomic Data ◽  
The Cancer Genome Atlas ◽  
Clinical Samples ◽  
Precision Oncology ◽  
Cancer Data ◽  
User Friendly

In the current big data era, massive genomic cancer data are available for open access from anywhere in the world. They are obtained from popular platforms, such as The Cancer Genome Atlas, which provides genetic information from clinical samples, and Cancer Cell Line Encyclopedia, which offers genomic data of cancer cell lines. For convenient analysis, user-friendly tools, such as the Tumor Immune Estimation Resource (TIMER), which can be used to analyze tumor-infiltrating immune cells comprehensively, are also emerging. In clinical practice, clinical sequencing has been recommended for patients with cancer in many countries. Despite its many challenges, it enables the application of precision medicine, especially in medical oncology. In this review, several efforts devoted to accomplishing precision oncology and applying big data for use in Indonesia are discussed. Utilizing open access genomic data in writing research articles is also described.

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