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Initial Evaluation of a Mobile SARS-CoV-2 RT-LAMP Testing Strategy.

ABSTRACT: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control in the United States remains hampered, in part, by testing limitations. We evaluated a simple, outdoor, mobile, colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay workflow where self-collected saliva is tested for SARS-CoV-2 RNA. From July 16, 2020, to November 19, 2020, surveillance samples (n = 4704) were collected from volunteers and tested for SARS-CoV-2 at 5 sites. Twenty-one samples tested positive for SARS-CoV-2 by RT-LAMP; 12 were confirmed positive by subsequent quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, whereas 8 tested negative for SARS-CoV-2 RNA, and 1 could not be confirmed because the donor did not consent to further molecular testing. We estimated the false-negative rate of the RT-LAMP assay only from July 16, 2020, to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or fewer and testing for SARS-CoV-2 RNA by qRT-PCR. We observed a 98.8% concordance between the RT-LAMP and qRT-PCR assays, with only 5 of 421 RT-LAMP-negative pools (2493 total samples) testing positive in the more-sensitive qRT-PCR assay. Overall, we demonstrate a rapid testing method that can be implemented outside the traditional laboratory setting by individuals with basic molecular biology skills and that can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.

SUBMITTER: Newman CM

PROVIDER: S-EPMC8730517 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Initial evaluation of a mobile SARS-CoV-2 RT-LAMP testing strategy.

Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control in the United States remains hampered, in part, by testing limitations. We evaluated a simple, outdoor, mobile, colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay workflow where self-collected saliva is tested for SARS-CoV-2 RNA. From July 16 to November 19, 2020, 4,704 surveillance samples were collected from volunteers and tested for SARS-CoV-2 at 5 sites. A total of 21 samples tested positive for SARS-CoV-2 by RT-LAMP; 12 were confirmed positive by subsequent quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, while 8 were negative for SARS-CoV-2 RNA, and 1 could not be confirmed because the donor did not consent to further molecular testing. We estimated the RT-LAMP assay's false-negative rate from July 16 to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or less and testing for SARS-CoV-2 RNA by qRT-PCR. We observed a 98.8% concordance between the RT-LAMP and qRT-PCR assays, with only 5 of 421 RT-LAMP negative pools (2,493 samples) testing positive in the more sensitive qRT-PCR assay. Overall, we demonstrate a rapid testing method that can be implemented outside the traditional laboratory setting by individuals with basic molecular biology skills and can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.

| S-EPMC7924282 | biostudies-literature

Developing RT-LAMP Assays for Detection of SARS-CoV-2 in Saliva

Project description:Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a fast and convenient method to amplify and identify the transcripts of a targeted pathogen. We combined bioinformatic and experimental analyses to improve the RT-LAMP assay performance for COVID-19 diagnosis. First, we developed an improved algorithm to design LAMP primers targeting the nucleocapsid (N), membrane (M), and spike (S) genes of SARS-CoV-2. Next, we rigorously validated these new assays for their efficacy and specificity. Further, we demonstrated that multiplexed RT-LAMP assays could directly detect as low as a few copies of SARS-CoV-2 RNA in saliva, without the need of RNA isolation. Importantly, further testing using saliva samples from COVID-19 patients indicated that the new RT-LAMP assays were in total agreement in sensitivity and specificity with standard RT-qPCR. In summary, our new LAMP primer design algorithm along with the validated assays provide a fast and reliable method for the diagnosis of COVID-19 cases.

2021-04-22 | GSE173086 | GEO

SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP.

Project description:Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

| S-EPMC7472728 | biostudies-literature

A Rapid RT-LAMP Assay for SARS-CoV-2 with Colorimetric Detection Assisted by a Mobile Application.

Project description:Loop-mediated amplification has been promoted for SARS-CoV-2 screening, however, antigen tests are preferred in low-income countries and remote zones. Poor training in molecular biology, plus the need for RNA purification or reading instruments to overcome issues of sensitivity in colorimetric detection, are some of the reasons limiting the use of this technique. In this study, nasopharyngeal swabs, aspirates and saliva were amplified in an in-house LAMP assay and subject to colorimetric detection, achieved by the naked eye and by image analysis with a mobile application. Accuracy of detection by the naked eye ranged from 61-74% but improved to 75-86% when using the application. Sensitivity of the digital approach was 81% and specificity 83%, with poor positive predictive value, and acceptable negative predictive value. Additionally to the reported effect of some transport media's pH, the presence of mucus and warming up of reagents while setting up the reaction critically affected performance. Accuracy per type of sample was 55, 70 and 80%, for swabs, aspirates and saliva, respectively, suggesting potential to improve the test in saliva. This assay, carried out in a closed tube, reduces contamination, has few pipetting steps and requires minimal equipment. Strategies to improve performance and implications of the use this sort of colorimetric LAMP for massive testing are discussed.

| S-EPMC9032071 | biostudies-literature

Scalable RT-LAMP-based SARS-CoV-2 testing for infection surveillance with applications in pandemic preparedness.

Project description:Throughout the SARS-CoV-2 pandemic, limited diagnostic capacities prevented sentinel testing, demonstrating the need for novel testing infrastructures. Here, we describe the setup of a cost-effective platform that can be employed in a high-throughput manner, which allows surveillance testing as an acute pandemic control and preparedness tool, exemplified by SARS-CoV-2 diagnostics in an academic environment. The strategy involves self-sampling based on gargling saline, pseudonymized sample handling, automated RNA extraction, and viral RNA detection using a semiquantitative multiplexed colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with an analytical sensitivity comparable with RT-qPCR. We provide standard operating procedures and an integrated software solution for all workflows, including sample logistics, analysis by colorimetry or sequencing, and communication of results. We evaluated factors affecting the viral load and the stability of gargling samples as well as the diagnostic sensitivity of the RT-LAMP assay. In parallel, we estimated the economic costs of setting up and running the test station. We performed > 35,000 tests, with an average turnover time of < 6 h from sample arrival to result announcement. Altogether, our work provides a blueprint for fast, sensitive, scalable, cost- and labor-efficient RT-LAMP diagnostics, which is independent of potentially limiting clinical diagnostics supply chains.

| S-EPMC10157315 | biostudies-literature

Rapid SARS-CoV-2 testing in primary material based on a novel multiplex RT-LAMP assay.

Project description:BackgroundRapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose.MethodsTo avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (RT-LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK).ResultsWhilst specificity of standard RT-LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed RT-LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel RT-LAMP assays with SHERLOCK.ConclusionIn summary, this approach reveals one-step multiplexed RT-LAMP assays as a prime-option for the development of easy and cheap POC test kits.

| S-EPMC7605681 | biostudies-literature

Implementation of a Rapid RT-LAMP Saliva-Based SARS-CoV-2 Testing Program in the Workplace.

Project description:Rising SARS-CoV-2 cases, testing delays, and the risk of pre-symptomatic and asymptomatic transmission provided the impetus for an in-house rapid testing program. Employees and their household contacts were encouraged to self-collect saliva samples that were pooled for routine testing using an established colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. In brief, individual or a maximum of four saliva samples were pooled and heat-inactivated to render microorganisms, especially SARS-CoV-2, non-infectious prior to being added to RT-LAMP assay tubes containing either the human sample control gene, RNase P, or a region of the SARS-CoV-2 gene, ORF1ab. During the second wave of SARS-CoV-2 infections in November 2020, two samples from an employee and a member of their household tested positive via RT-LAMP within two days of each other. A delayed clinical qRT-PCR test confirmation of both individuals 5 days later underscored the power of routine rapid testing with within-the-hour turnaround times. Workplace rapid testing programs using RT-LAMP are flexible in their design, have a reduced cost compared to qRT-PCR, may involve non-invasive self-saliva collection for increased safety for the testing personnel, and can be performed with minimal training.

| S-EPMC8871197 | biostudies-literature

Clinical testing on SARS-CoV-2 swab samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP).

Project description:BackgroundHigh cost of commercial RNA extraction kits limits the testing efficiency of SARS-CoV-2. Here, we developed a simple nucleic acid extraction method for the detection of SARS-CoV-2 directly from nasopharyngeal swab samples.MethodsA pH sensitive dye was used as the end point detection method. The obvious colour changes between positive and negative reactions eliminates the need of other equipment.ResultsClinical testing using 260 samples showed 92.7% sensitivity (95% CI 87.3-96.3%) and 93.6% specificity (95% CI 87.3-97.4%) of RT-LAMP.ConclusionsThe simple RNA extraction method minimizes the need for any extensive laboratory set-up. We suggest combining this simple nucleic acid extraction method and RT-LAMP technology as the point-of care diagnostic tool.

| S-EPMC9387413 | biostudies-literature

Electromechanical RT-LAMP device for portable SARS-CoV-2 detection.

Project description:Rapid point-of-care tests for infectious diseases are essential, especially in pandemic conditions. We have developed a point-of-care electromechanical device to detect SARS-CoV-2 viral RNA using the reverse-transcription loop-mediated isothermal amplification (RT-LAMP) principle. The developed device can detect SARS-CoV-2 viral RNA down to 103 copies/mL and from a low amount of sample volumes (2 μL) in less than an hour of standalone operation without the need for professional labor and equipment. Integrated Peltier elements in the device keep the sample at a constant temperature, and an integrated camera allows automated monitoring of LAMP reaction in a stirring sample by using colorimetric analysis of unfocused sample images in the hue/saturation/value color space. This palm-fitting, portable and low-cost device does not require a fully focused sample image for analysis, and the operation could be stopped automatically through image analysis when the positive test results are obtained. Hence, viral infections can be detected with the portable device produced without the need for long, expensive, and labor-intensive tests and equipment, which can make the viral tests disseminated at the point-of-care.

| S-EPMC9733968 | biostudies-literature

RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2.

Project description:The pandemic coronavirus SARS-CoV-2 in the world has caused a large infected population suffering from COVID-19. To curb the spreading of the virus, WHO urgently demanded an extension of screening and testing; thus, a rapid and simple diagnostic method is needed. We applied a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) to achieve the detection of SARS-CoV-2 in 30 min. We designed four sets of LAMP primers (6 primers in each set), targeting the viral RNA of SARS-CoV-2 in the regions of orf1ab, S gene and N gene. A colorimetric change was used to report the results, which enables the outcome of viral RNA amplification to be read by the naked eye without the need of expensive or dedicated instrument. The sensitivity can be 80 copies of viral RNA per ml in a sample. We validated the RT-LAMP method in a hospital in China, employing 16 clinic samples with 8 positives and 8 negatives. The testing results are consistent with the conventional RT-qPCR. In addition, we also show that one-step process without RNA extraction is feasible to achieve RNA amplification directly from a sample. This rapid, simple and sensitive RT-LAMP method paves a way for a large screening at public domain and hospitals, particularly regional hospitals and medical centres in rural areas.

| S-EPMC7264870 | biostudies-literature

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