scholarly journals Phenol-chloroform-based RNA purification for detection of SARS-CoV-2 by RT-qPCR: comparison with automated systems

2020 ◽  
Author(s):  
Henrik Dimke ◽  
Sanne L Larsen ◽  
Marianne N Skov ◽  
Hanne Larsen ◽  
Gitte N Hartmeyer ◽  
...  
Keyword(s):  
Low Cost ◽  
Rna Extraction ◽  
Diagnostic System ◽  
Nasopharyngeal Swab ◽  
Analytical Sensitivity ◽  
Automated Systems ◽  
Suitable Alternative ◽  
Fully Integrated ◽  
Rna Purification ◽  
Sensitivity Specificity

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly reached pandemic levels. Sufficient testing for SARS-CoV-2 remains essential for tracking and containing the rapid spread of the virus. However, due to increased global demand, kits and proprietary reagents for RNA extraction are limited, which markedly reduce SARS-CoV-2 testing capabilities in many countries. Here, we explore the use of conventional acid guanidinium thiocyanate-phenol-chloroform (AGPC)-based RNA purification as an alternative to commercial automated systems for detection of SARS-CoV-2 by RT-qPCR. 87 clinical oropharyngeal or nasopharyngeal swab specimens were extracted by AGPC and compared to the commercial platforms, the Maxwell® RSC 48 instrument for automated RNA extraction and the fully integrated diagnostic system, the Cobas®6800 apparatus. Our results show that RNA extracted using the AGPC method is fully comparable to modern automated systems regarding analytical sensitivity, specificity and accuracy with respect to detection of SARS-CoV-2 as evaluated by RT-qPCR. Moreover, we find that the AGPC method is easily scalable and implemented in conventional laboratories. Taken together, these data identify conventional AGPC-based RNA extraction as a low cost and suitable alternative to automated systems for the detection of SARS-CoV-2, when automated systems, kits and reagents are not readily available.

scholarly journals Phenol-chloroform-based RNA purification for detection of SARS-CoV-2 by RT-qPCR: Comparison with automated systems

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247524
Author(s):  
Henrik Dimke ◽  
Sanne L. Larsen ◽  
Marianne N. Skov ◽  
Hanne Larsen ◽  
Gitte N. Hartmeyer ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Sensitivity And Specificity ◽  
Low Cost ◽  
Rna Extraction ◽  
Diagnostic System ◽  
Analytical Sensitivity ◽  
Automated Systems ◽  
Suitable Alternative ◽  
Fully Integrated ◽  
Rna Purification

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic levels. Sufficient testing for SARS-CoV-2 has remained essential for tracking and containing the virus. SARS-CoV-2 testing capabilities are still limited in many countries. Here, we explore the use of conventional RNA purification as an alternative to automated systems for detection of SARS-CoV-2 by RT-qPCR. 87 clinical swab specimens were extracted by conventional phenol-chloroform RNA purification and compared to commercial platforms for RNA extraction and the fully integrated Cobas®6800 diagnostic system. Our results show that the conventional RNA extraction is fully comparable to modern automated systems regarding analytical sensitivity and specificity with respect to detection of SARS-CoV-2 as evaluated by RT-qPCR. Moreover, the method is easily scalable and implemented in conventional laboratories as a low cost and suitable alternative to automated systems for the detection of SARS-CoV-2.

scholarly journals Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 615
Author(s):  
Allen Wing-Ho Chu ◽  
Cyril Chik-Yan Yip ◽  
Wan-Mui Chan ◽  
Anthony Chin-Ki Ng ◽  
Dream Lok-Sze Chan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Suitable Alternative ◽  
Pcr Inhibitors ◽  
Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

scholarly journals Self-Collection of Saliva Specimens as a Suitable Alternative to Nasopharyngeal Swabs for the Diagnosis of SARS-CoV-2 by RT-qPCR

2021 ◽  
Vol 10 (2) ◽  
pp. 299
Author(s):  
Camino Trobajo-Sanmartín ◽  
Marta Adelantado ◽  
Ana Navascués ◽  
María J. Guembe ◽  
Isabel Rodrigo-Rincón ◽  
...  
Keyword(s):  
Chronic Conditions ◽  
Saliva Sample ◽  
Virus Detection ◽  
Kappa Coefficient ◽  
Nasopharyngeal Swab ◽  
Personal Protection ◽  
Personal Protection Equipment ◽  
Suitable Alternative ◽  
Lysis Buffer ◽  
Sensitivity Specificity

A nasopharyngeal swab is a sample used for the diagnosis of SARS-CoV-2 infection. Saliva is a sample easier to obtain and the risk of contagion for the professional is lower. This study aimed to evaluate the utility of saliva for the diagnosis of SARS-CoV-2 infection. This prospective study involved 674 patients with suspected SARS-CoV-2 infection. Paired nasopharyngeal and saliva samples were processed by RT-qPCR. Sensitivity, specificity, and kappa coefficient were used to evaluate the results from both samples. We considered the influence of age, symptoms, chronic conditions, and sample processing with lysis buffer. Of the 674 patients, 636 (94.4%) had valid results from both samples. The virus detection in saliva compared to a nasopharyngeal sample (gold standard) was 51.9% (95% CI: 46.3%–57.4%) and increased to 91.6% (95% CI: 86.7%–96.5%) when the cycle threshold (Ct) was ≤ 30. The specificity of the saliva sample was 99.1% (95% CI: 97.0%–99.8%). The concordance between samples was 75% (κ = 0.50; 95% CI: 0.45–0.56). The Ct values were significantly higher in saliva. In conclusion, saliva sample utility is limited for clinical diagnosis, but could be a useful alternative for the detection of SARS-CoV-2 in massive screening studies, when the availability of trained professionals for sampling or personal protection equipment is limited.

scholarly journals Development and Validation of Two In-house, Low-Cost SARS-CoV-2 Detection Assays

2020 ◽  
Author(s):  
Fatimah Alhamlan ◽  
Ahmed Alqahtani ◽  
Dana Bakheet ◽  
Marie Bohol ◽  
Sahar Althawadi ◽  
...  
Keyword(s):  
Low Cost ◽  
False Negative ◽  
Rna Extraction ◽  
Melting Curve ◽  
Sybr Green ◽  
Heat Processing ◽  
Nasopharyngeal Swab ◽  
Level 2 ◽  
Biosafety Level ◽  
Biosafety Level 2

Background One major challenge for detecting the virus that causes COVID19 is commercial SARSCoV2 testing kit or reagent availability. To allow every laboratory or hospital access to an inhouse assay, we developed two low cost SARSCoV2 detection assay protocols using inhouse primers and reagents equipment on hand in most biology or diagnostic laboratories a SYBR Green based RTPCR and PCR assays. RNA extraction has also become a major bottleneck due to limited supplies and the required labor. Thus, we validated alternative RNA extraction protocols. Methods SARSCoV2 genome sequences deposited into the GISAID database were retrieved to design and synthesize inhouse primers. Forty patient samples were collected by nasopharyngeal swab, coded, and used to develop and validate the assay protocols. Both assays used TRIzol and heat-processing techniques to extract RNA from patient samples and to inactivate the virus; thus, testing was conducted in a conventional biosafety level 2 laboratory. Results The sensitivity and specificity of the primers were evaluated using samples previously confirmed positive for SARSCoV2. The positive amplicons were sequenced to confirm the results. The assay protocols were developed, and the specificity of each PCR product was confirmed using melting curve analyses. The most accurate heat processing technique for primers with short amplicon lengths was 95C for 15 mins. Of 40 samples, both the SYBR Green based quantitative RTPCR assay and the PCR assay detected SARSCoV2 target genes in 28 samples, with no false positive or false-negative results. These findings were concordant with those of the diagnostic laboratory that tested the same samples using a Rotor Gene PCR cycler with an Altona Diagnostics SARSCoV2 kit (R2=0.889). Conclusions These approaches are reliable, repeatable, specific, sensitive, simple, and low cost tools for the detection of SARSCoV2 in a conventional biosafety level 2 laboratory, offering alternative approaches when commercial kits are unavailable or cost ineffective.

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

2020 ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Nucleic Acid Amplification ◽  
Nasopharyngeal Swab ◽  
Viral Transport ◽  
Extraction Step ◽  
Highly Sensitive

AbstractThe COVID-19 pandemic has resulted in an urgent global need for rapid, point-of-care diagnostic testing. Existing methods for nucleic acid amplification testing (NAAT) require an RNA extraction step prior to amplification of the viral RNA. This step necessitates the use of a centralized laboratory or complex and costly proprietary cartridges and equipment, and thereby prevents low-cost, scalable, point-of-care testing. We report the development of a highly sensitive and robust, easy-to-implement, SARS-CoV-2 test that utilizes isothermal amplification and can be run directly on viral transport media following a nasopharyngeal swab without the need for prior RNA extraction. Our assay provides visual results in 30 min with 85% sensitivity, 100% specificity, and a limit of detection (LoD) of 2.5 copies/μl, and can be run using a simple heat block.

scholarly journals A comparative evaluation of a dye-based and probe-based RT-qPCR assay for the screening of SARS-CoV-2 using individual and pooled-sample testing

2020 ◽  
Author(s):  
Claudio Verdugo ◽  
Anita Plaza ◽  
Gerardo Acosta-Jamett ◽  
Natalia Castro ◽  
Josefina Gutiérrez ◽  
...  
Keyword(s):  
Low Cost ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Detection Rates ◽  
Efficient System ◽  
Population Sampling ◽  
Effective Interventions ◽  
Surveillance Programs

ABSTRACTEffective interventions are mandatory to control the transmission and spread of SARS-CoV-2, a highly contagious virus causing devastating effects worldwide. Cost-effective approaches are pivotal tools required to increase the detection rates and escalate further in massive surveillance programs, especially in countries with limited resources that most of the efforts have focused on symptomatic cases only. Here, we compared the performance of the RT-qPCR using an intercalating dye with the probe-based assay. Then, we tested and compared these two RT-qPCR chemistries in different pooling systems: after RNA extraction (post-RNA extraction) and before RNA extraction (pre-RNA extraction) optimizing by pool size and template volume. We evaluated these approaches in 610 clinical samples. Our results show that the dye-based technique has a high analytical sensitivity similar to the probe-based detection assay used worldwide. Further, this assay may also be applicable in testing by pool systems post-RNA extraction up to 20 samples. However, the most efficient system for massive surveillance, the pre-RNA extraction pooling approach, was obtained with the probe-based assay in test up to 10 samples adding 13.5 µL of RNA template. The low cost and the potential use in pre-RNA extraction pool systems, place of this assays as a valuable resource for scalable sampling to larger populations. Implementing a pool system for population sampling results in an important savings of laboratory resources and time, which are two key factors during an epidemic outbreak. Using the pooling approaches evaluated here, we are confident that it can be used as a valid alternative assay for the detection of SARS-CoV-2 in human samples.

scholarly journals Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA

2020 ◽  
Author(s):  
Mohammad Rubayet Hasan ◽  
Faheem Mirza ◽  
Hamad Al-Hail ◽  
Sathyavathi Sundararaju ◽  
Thabisile Xaba ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Viral Rna ◽  
Standard Approach ◽  
Analytical Sensitivity ◽  
Limited Availability ◽  
Highly Correlated ◽  
Sensitivity Specificity

AbstractTo circumvent the limited availability of RNA extraction reagents, we developed a protocol for direct RT-qPCR to detect SARS-CoV-2 in nasopharyngeal swabs. Incubation of specimens at 65°C for 10 minutes along with the use of TaqPath™ 1-Step RT-qPCR Master Mix provides higher analytical sensitivity for detection of SARS-CoV-2 RNA than many other commercial and laboratory-developed methods. In 132 specimens submitted for SARS-CoV-2 testing, the sensitivity, specificity and accuracy of our optimized approach were 95%, 99% and 98.5%, respectively, with reference to results obtained by a standard approach involving RNA extraction. Also, the RT-qPCR CT values obtained by the two methods were highly correlated.

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.

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 Pre-Procedural COVID-19 Nasopharyngeal Swab Has Good Concordance with Bronchoalveolar Lavage in Patients at Low Risk for Viral Infection

Respiration ◽  
2021 ◽  
pp. 1-5
Author(s):  
Catherine L. Oberg ◽  
Reza Ronaghi ◽  
Erik E. Folch ◽  
Colleen L. Channick ◽  
Tao He ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Low Risk ◽  
Lower Airway ◽  
Nasopharyngeal Swab ◽  
Symptom Screening ◽  
The World ◽  
Good Concordance ◽  
Sensitivity Specificity ◽  
Overall Reliability ◽  
Rule Out

<b><i>Background:</i></b> The coronavirus disease 2019 (COVID-19) pandemic has drastically affected hospital and operating room (OR) workflow around the world as well as trainee education. Many institutions have instituted mandatory preoperative SARS-CoV-2 PCR nasopharyngeal swab (NS) testing in patients who are low risk for COVID-19 prior to elective cases. This method, however, is challenging as the sensitivity, specificity, and overall reliability of testing remains unclear. <b><i>Objectives:</i></b> The objective of this study was to assess the concordance of a negative NS in low risk preoperative patients with lower airway bronchoalveolar lavage (BAL) specimens obtained from the same patients. <b><i>Methods:</i></b> We prospectively sent intraoperative lower airway BAL samples collected within 48 h of a negative mandatory preoperative NS for SARS-CoV-2 PCR testing. All adult patients undergoing a scheduled bronchoscopic procedure for any reason were enrolled, including elective and nonelective cases. <b><i>Results:</i></b> One-hundred eighty-nine patients were included. All BAL specimens were negative for SARS-CoV-2 indicative of 100% concordance between testing modalities. <b><i>Conclusions:</i></b> These results are promising and suggest that preoperative nasopharyngeal SARS-CoV-2 testing provides adequate screening to rule out active COVID-19 infection prior to OR cases in a population characterized as low risk by negative symptom screening. This information can be used for both pre-procedural screening and when reintroducing trainees into the workforce.

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