Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab and saliva patient samples

2020 ◽  
Author(s):  
Dawn M Dudley ◽  
Christina M. Newman ◽  
Andrea M Weiler ◽  
Mitchell D. Ramuta ◽  
Ceclia G. Shortreed ◽  
...  
Keyword(s):  
Infectious Virus ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Lower Sensitivity ◽  
Acid Extraction ◽  
Short Supply ◽  
Screening Programs ◽  
Qrt Pcr ◽  
The Us

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed samples, albeit with approximately 100-fold lower sensitivity than qRT-PCR. We demonstrate that adding lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Overall, the limit of detection (LOD) of RT-LAMP using direct nasopharyngeal swab or saliva samples without RNA extraction is 1x105-1x106 copies/ml. This LOD is sufficient to detect samples from which infectious virus can be cultured. Therefore, samples that test positive in this assay contain levels of virus that are most likely to perpetuate transmission. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR and we provide a revised method to work directly with saliva as starting material. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 screening programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.

scholarly journals Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab samples

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244882
Author(s):  
Dawn M. Dudley ◽  
Christina M. Newman ◽  
Andrea M. Weiler ◽  
Mitchell D. Ramuta ◽  
Cecilia G. Shortreed ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Lower Sensitivity ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Short Supply ◽  
Qrt Pcr ◽  
The Us ◽  
Surveillance Programs

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Frequent surveillance of individuals attending work or school is currently unavailable to most people but will likely be necessary to reduce the ~50% of transmission that occurs when individuals are nonsymptomatic. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed primary samples with a limit of detection (LOD) of ~625 copies/μl, approximately 100-fold lower sensitivity than qRT-PCR. While less sensitive than extraction-based molecular methods, RT-LAMP without RNA extraction is fast and inexpensive. Here we also demonstrate that adding a lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 surveillance programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.

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 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 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 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 Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples

2021 ◽  
Author(s):  
Jennifer R. Hamilton ◽  
Elizabeth C. Stahl ◽  
Connor A. Tsuchida ◽  
Enrique Lin-Shiao ◽  
C. Kimberly Tsui ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rna Extraction ◽  
Detection Sensitivity ◽  
Nasopharyngeal Swab ◽  
Infection Prevalence ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Rapid Pcr ◽  
Large Populations ◽  
Asymptomatic Individuals

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI-FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against the gold standard, nasopharyngeal swab specimens. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

scholarly journals Clinical Validation of Automated and Rapid MariPOC SARS-CoV-2 Antigen Test

2021 ◽  
Author(s):  
Juha M. Koskinen ◽  
Petri Antikainen ◽  
Kristina Hotakainen ◽  
Anu Haveri ◽  
Niina Ikonen ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Solid Phase ◽  
High Capacity ◽  
Nasopharyngeal Swab ◽  
Nucleocapsid Proteins ◽  
Clinical Sensitivity ◽  
Qrt Pcr ◽  
Photon Excitation ◽  
Antigen Tests ◽  
Asymptomatic Individuals

Abstract COVID-19 diagnostics was quickly ramped up worldwide early 2020 based on the detection of viral RNA. However, based on the scientific knowledge for pre-existing coronaviruses, it was expected that the SARS-CoV-2 RNA will be detected from symptomatic and at significant rates also from asymptomatic individuals due to persistence of non-infectious RNA. To increase the efficacy of diagnostics, surveillance, screening and pandemic control, rapid methods, such as antigen tests, are needed for decentralized testing and to assess infectiousness. A novel automated mariPOC SARS-CoV-2 test was developed for the detection of conserved structural viral nucleocapsid proteins. The test utilizes sophisticated optical laser technology for two-photon excitation and individual detection of immunoassay solid-phase particles. We validated the new method against qRT-PCR. Sensitivity of the test was 100.0% (13/13) directly from nasopharyngeal swab specimens and 84.4% (38/45) from swab specimens in undefined transport mediums. Specificity of the test was 100.0% (201/201). The test's limit of detection was 2.7 TCID50/test. It showed no cross-reactions. Our study shows that the new test can detect infectious individuals already in 20 minutes with clinical sensitivity close to qRT-PCR. The mariPOC is a versatile platform for syndromic testing and for high capacity infection control screening of infectious individuals.

scholarly journals An optimised protocol for detection of SARS-CoV-2 in stool

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Tianqi Li ◽  
Enriqueta Garcia-Gutierrez ◽  
Daniel A. Yara ◽  
Jacob Scadden ◽  
Jade Davies ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Rna Extraction ◽  
Extraction Procedure ◽  
Nasopharyngeal Swab ◽  
Clinical Practices ◽  
Oral Transmission ◽  
Viral Particles ◽  
Stool Samples ◽  
Polymerase Chain ◽  
Reliable Methods

Abstract Background SARS-CoV-2 has been detected in stool samples of COVID-19 patients, with potential implications for faecal-oral transmission. Compared to nasopharyngeal swab samples, the complexity of the stool matrix poses a challenge in the detection of the virus that has not yet been solved. However, robust and reliable methods are needed to estimate the prevalence and persistence of SARS-CoV-2 in the gut and to ensure the safety of microbiome-based procedures such as faecal microbiota transplant (FMT). The aim of this study was to establish a sensitive and reliable method for detecting SARS-CoV-2 in stool samples. Results Stool samples from individuals free of SARS-CoV-2 were homogenised in saline buffer and spiked with a known titre of inactivated virus ranging from 50 to 750 viral particles per 100 mg stool. Viral particles were concentrated by ultrafiltration, RNA was extracted, and SARS-CoV-2 was detected via real-time reverse-transcription polymerase chain reaction (RT-qPCR) using the CDC primers and probes. The RNA extraction procedure we used allowed for the detection of SARS-CoV-2 via RT-qPCR in most of the stool samples tested. We could detect as few as 50 viral particles per 100 mg of stool. However, high variability was observed across samples at low viral titres. The primer set targeting the N1 region provided more reliable and precise results and for this primer set our method had a limit of detection of 1 viral particle per mg of stool. Conclusions Here we describe a sensitive method for detecting SARS-CoV-2 in stool samples. This method can be used to establish the persistence of SARS-CoV-2 in stool and ensure the safety of clinical practices such as FMT.

scholarly journals Clinical validation of automated and rapid mariPOC SARS-CoV-2 antigen test

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Juha M. Koskinen ◽  
Petri Antikainen ◽  
Kristina Hotakainen ◽  
Anu Haveri ◽  
Niina Ikonen ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Solid Phase ◽  
High Capacity ◽  
Nasopharyngeal Swab ◽  
Nucleocapsid Proteins ◽  
Clinical Sensitivity ◽  
Qrt Pcr ◽  
Photon Excitation ◽  
Antigen Tests ◽  
Asymptomatic Individuals

AbstractCOVID-19 diagnostics was quickly ramped up worldwide early 2020 based on the detection of viral RNA. However, based on the scientific knowledge for pre-existing coronaviruses, it was expected that the SARS-CoV-2 RNA will be detected from symptomatic and at significant rates also from asymptomatic individuals due to persistence of non-infectious RNA. To increase the efficacy of diagnostics, surveillance, screening and pandemic control, rapid methods, such as antigen tests, are needed for decentralized testing and to assess infectiousness. A novel automated mariPOC SARS-CoV-2 test was developed for the detection of conserved structural viral nucleocapsid proteins. The test utilizes sophisticated optical laser technology for two-photon excitation and individual detection of immunoassay solid-phase particles. We validated the new method against qRT-PCR. Sensitivity of the test was 100.0% (13/13) directly from nasopharyngeal swab specimens and 84.4% (38/45) from swab specimens in undefined transport mediums. Specificity of the test was 100.0% (201/201). The test's limit of detection was 2.7 TCID50/test. It showed no cross-reactions. Our study shows that the new test can detect infectious individuals already in 20 min with clinical sensitivity close to qRT-PCR. The mariPOC is a versatile platform for syndromic testing and for high capacity infection control screening of infectious individuals.

scholarly journals SARS-CoV-2 detection from nasopharyngeal swab samples without RNA extraction

2020 ◽  
Author(s):  
Carolina Beltrán-Pavez ◽  
Chantal L. Márquez ◽  
Gabriela Muñoz ◽  
Fernando Valiente-Echeverría ◽  
Aldo Gaggero ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Short Supply ◽  
Extraction Step

AbstractThe ongoing COVID-19 pandemic has reached more than 200 countries and territories worldwide. Given the large requirement of SARS-CoV-2 diagnosis and considering that RNA extraction kits are in short supply, we investigated whether two commercial RT-qPCR kits were compatible with direct SARS-CoV-2 detection from nasopharyngeal swab samples. We show that one of the tested kits is fully compatible with direct SARS-CoV-2 detection suggesting that omission of an RNA extraction step should be considered in SARS-CoV-2 diagnosis.

Sign in / Sign up

Export Citation Format

Share Document