Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2

Mohon, Abu Naser; Oberding, Lisa; Hundt, Jana; Marle, Guido van; Pabbaraju, Kanti; Berenger, Byron M.; Lisboa, Luiz F.; Griener, Thomas P.; Czub, Markus; Doolan, Cody; Servellita, Venice; Chiu, Charles Y.; Greninger, Alexander L.; Jerome, Keith R.; Pillai, Dylan R.

doi:10.1016/j.jviromet.2020.113972

Cited by 42 publications

(42 citation statements)

References 24 publications

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“…The included studies consist of quite a variety of samples, of which there are five studies from China; two studies from Germany (12,13); and one study each from Hongkong (8), China (6), Japan (14), Malaysia (15), South Korea (7), Canada (16), Australia (17), and USA (4). As for the index test, there are seven studies using RT-LAMP Kit (1,4,7–9,14,15,18), while other studies used Warmstart RT-LAMP Assay Kit (n=2) (5,16), Variplex RT-LAMP Assay Kit (n=1) (12), Loopamp RNA Amplification Kit (n=1) (6), RT-LAMP Mastermix kit (n=1) (17), and specific colorimetric and fluorescence RT-LAMP Kit (n=1) (13). As for the reference test, most studies used the qRT-PCR Kit with two studies specified the used kit (BGI qRT-PCR Kit (19) and NPMA RT-PCR Kit (18)), while several studies used LightMix E-Gene RT-PCR Kit (n=3) (12,13,17).…”

Section: Resultsmentioning

confidence: 99%

Reverse Transcriptase Loop Mediated Isothermal Amplification (RT-LAMP) for COVID-19 Diagnosis: A Systematic Review and Meta-Analysis

Subali

Wiyono

2021

Preprint

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BackgroundCoronavirus Disease 2019 (COVID-19) has caused a severe outbreak and become a global public health priority. Rapid increment of infection number along with significant deaths have placed the virus as a serious threat to human health. Rapid, reliable, and simple diagnostic methods are critically essential for disease control. While Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) is the current diagnostic gold standard, Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) appears as a compelling alternative diagnostic test due to its more simplicity, shorter time to result, and lower cost. This study examined RT-LAMP application for rapid identification of SARS-CoV-2 infection compared to RT-PCR assay.MethodsA systematic review and meta-analysis (2020) was conducted in 6 scientific databases following the PRISMA Guideline. Original published studies on human clinical samples in English were included. Articles evaluated sensitivity and specificity of RT-LAMP relative to RT-PCR were considered eligible. Quality assessment of bias and applicability was examined based on QUADAS-2.ResultsA total of 351 studies were found based on the keywords and search queries. 14 eligible case control studies fitted the respective criteria. Quality assessment using QUADAS-2 indicated low risk bias in all included studies. All case studies, comprises 2,112 samples, had the cumulative sensitivity of 95.5% (CI 97.5%=90.8-97.9%) and cumulative specificity of 99.5% (CI 97.5%=97.7-99.9%).ConclusionRT-LAMP assay could be suggested as a reliable alternative COVID-19 diagnostic method with reduced cost and time compared to RT-PCR. RT-LAMP could potentially be utilized during the high-throughput and high-demand critical situations.

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Section: Resultsmentioning

confidence: 99%

Reverse Transcriptase Loop Mediated Isothermal Amplification (RT-LAMP) for COVID-19 Diagnosis: A Systematic Review and Meta-Analysis

Subali

Wiyono

2021

Preprint

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“…The applicability of RT-LAMP for the detection of SARS-CoV-2 has been shown to be robust by numerous authors [16] [17], undergone clinical validation [18], and has been granted Emergency Use Authorization by the FDA (Color, Atilla Biosciences and Mammoth Biosciences). As noted by Kellner et al (2020) “There are, however, several remaining challenges, especially with respect to the world-wide distribution and access to RT-LAMP reagents.…”

Section: Discussionmentioning

confidence: 99%

Accessible LAMP-Enabled Rapid Test (ALERT) for detecting SARS-CoV-2

Bektaş¹,

Covington²,

Aidelberg

et al. 2021

Preprint

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The COVID-19 pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. PCR-based diagnostic tests are expensive, reliant on expensive centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests, that bind and detect the surface proteins of a virus, are rapid and inexpensive but suffer from high false negative rates. To address this problem, we have created an inexpensive, simple, and robust 60-minute Do-It-Yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/μl) and specificity (>97% True Negative Rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP). Our workflow, ALERT (Accessible LAMP-Enabled Rapid Test), incorporates the following features: 1) Increased shelf-life and ambient temperature storage by using wax layers to isolate enzymes from reaction, 2) Improved specificity by using sequence-specific QUASR reporters, 3) Increased sensitivity through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal, 4) Quality control with a nasopharyngeal-specific mRNA target, and 5) Co-detection of other respiratory viruses, such as Influenza B, by duplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for centralized labs and hospitals. With minimal effort, SARS-CoV-2-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms or nucleic acid-based markers.

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“…An important area of ongoing development for point-of-care nucleic acid tests is rapid RNA extraction. Standard laboratory RNA extractions are very time-consuming; however, replacing an RNA puri cation step with a simple inactivation step can compromise assay sensitivity 14,23,24 . RNA puri cations result in the concentration of viral RNA and the removal of ampli cation inhibitors, both of which increase sensitivity.…”

Section: Discussionmentioning

confidence: 99%

“…The lyophilized RT-LAMP reagents for the ampli cation of SARS-CoV-2 on the Biobox were obtained from Illucidx Inc. (Calgary, Canada). Lyophilized reactions consisted of the master mix described previously 14 but employed the dye mix described above. A proprietary excipient mix was also added.…”

Section: Saliva-dry Lamp Performed On the Bioboxmentioning

confidence: 99%

Saliva-Dry LAMP: A Rapid Near-Patient Detection System for SARS-CoV-2.

Toppings¹,

Mohon²,

Lee³

et al. 2021

Preprint

Self Cite

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The highly infectious nature of SARS-CoV-2 necessitates the use of widespread testing to control the spread of the virus. Presently, the standard molecular testing method (reverse transcriptase-polymerase chain reaction, RT-PCR) is restricted to the laboratory, time-consuming, and costly. This increases the turnaround time for getting test results. The study sought to develop a rapid, near-patient saliva-based test for COVID-19 with similar accuracy to that of standard RT-PCR tests. A lyophilized dual-target reverse transcription-loop-mediated isothermal amplification (RT-LAMP) test with fluorometric detection by the naked eye. The assay relies on dry reagents that are room temperature stable. A device containing a centrifuge, heat block, and blue LED light system was manufactured to reduce the cost of performing the assay. This test has a limit of detection of 1 copy/µL and achieved positive percent agreement of 100% [95% CI 88.43% to 100.0%] and negative percent agreement of 96.7% [95% CI 82.78% to 99.92%] on saliva. Saliva-Dry LAMP can be completed in 105 minutes. Precision, cross-reactivity, and interfering substances analysis met international regulatory standards. The combination of ease of sample collection, dry reagents, visual detection, low capital equipment cost, and excellent analytical sensitivity make Saliva-Dry LAMP particularly useful for resource-limited settings.

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Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2

Cited by 42 publications

References 24 publications

Reverse Transcriptase Loop Mediated Isothermal Amplification (RT-LAMP) for COVID-19 Diagnosis: A Systematic Review and Meta-Analysis

Reverse Transcriptase Loop Mediated Isothermal Amplification (RT-LAMP) for COVID-19 Diagnosis: A Systematic Review and Meta-Analysis

Accessible LAMP-Enabled Rapid Test (ALERT) for detecting SARS-CoV-2

Saliva-Dry LAMP: A Rapid Near-Patient Detection System for SARS-CoV-2.

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