Enhancing Colorimetric Loop-mediated Isothermal Amplification Speed and Sensitivity with Guanidine Chloride

Zhang, Yinhua; Ren, Guoping; Buss, Jackson; Barry, Andrew; Patton, Gregory C.; Tanner, Nathan A.

doi:10.2144/btn-2020-0078

Cited by 165 publications

(234 citation statements)

References 14 publications

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“…Again, we only used 0.5 μl UTM for each reaction. In addition, we added guanidine hydrochloride as a classical RNAse inhibitor and LAMP enhancer [ 18 ] to the multiplexed reaction at 60°C. These modifications in sample preparation combined with our new multiplexed assay were used to analyze a set of 102 clinical samples consisting of 74 SARS-CoV-2 positive and 28 negative swabs ( Fig 2 ).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2020

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Background Rapid 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. Methods To 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). Results Whilst 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. Conclusion In summary, this approach reveals one-step multiplexed RT-LAMP assays as a prime-option for the development of easy and cheap POC test kits.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2020

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“…The copyright holder for this this version posted September 17, 2020. ; https://doi.org/10.1101/2020.08.30.20184796 doi: medRxiv preprint in S1 Table (16,17,(25)(26)(27)(28)(29). Of note, the Color-…”

Section: Rt-lampmentioning

confidence: 99%

“…The copyright holder for this this version posted September 17, 2020. ; https://doi.org/10.1101/2020.08.30.20184796 doi: medRxiv preprint Next, we compared the Gene-N-A, Color-N, Color-E, and Color-N/E combination to second generation primers described in Zhang et al (25) (Gene-N2, Gene-E1 and As1e). We found that As1e, originally published by Rabe et al (26) yielded the lowest Cq value followed closely by a combination of As1e with the two primers targeting the Gene-N2 and Gene-E1 genes designed by Zhang et al (Fig 3B).…”

Section: Alternative Primers Improve Efficiencymentioning

confidence: 99%

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

Dudley

Newman

Weiler

et al. 2020

Preprint

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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 achieves a similar LOD to qRT-PCR. 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.

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“…S1).RNA was extracted from swabs used to sample the surface of the candy wrappers with the Omega MagBind Viral DNA/RNA kit on the Kingfisher platform, followed by RT-qPCR using the Thermo TaqPath COVID-19 Multiplex assay on the Quantstudio 7 Pro platform. Loop-mediated isothermal amplification (LAMP) was performed in multiplex using NEB WarmStart Colorimetric LAMP Master Mix with UDG(1)(2)(3).Surprisingly, coughing and extensive handling candies yielded the same SARS-CoV-2 positivity rate as unwashed handling (5 of 10 untreated candies, 6 of 30 total). Hand washing prior to handling greatly reduced positives (1 of 10 untreated candies, 2 of 30 total) (Fig.…”

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confidence: 99%

Handwashing and Detergent Treatment Greatly Reduce SARS-CoV-2 Viral Load on Halloween Candy Handled by COVID-19 Patients

et al. 2020

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Due to the COVID-19 pandemic and potential public health implications, we are publishing this peer-reviewed manuscript in its accepted form. The final, copyedited version of the paper will be available at a later date. Although SARS-CoV-2 is primarily transmitted by respiratory droplets and aerosols, transmission by fomites remains plausible. During Halloween, a major event for children in numerous countries, SARS-CoV-2 transmission risk via candy fomites worries many parents. To address this concern, we enrolled 10 recently diagnosed asymptomatic or mildly/moderately symptomatic COVID-19 patients to handle typical Halloween candy (pieces individually wrapped) under three conditions: normal handling with unwashed hands, deliberate coughing and extensive touching, and normal handling following handwashing. We then used a factorial design to subject the candies to two post-handling treatments: no washing (untreated) and household dishwashing detergent. We measured SARS-CoV-2 load by RT-qPCR and LAMP. From the candies not washed post-handling, we detected SARS-CoV-2 on 60% of candies that were deliberately coughed on, 60% of candies normally handled with unwashed hands, but only 10% of candies handled after hand washing. We found that treating candy with dishwashing detergent reduced SARS-CoV-2 load by 62.1% in comparison to untreated candy. Taken together, these results suggest that although the risk of transmission of SARS-CoV-2 by fomites is low even from known COVID-19 patients, viral RNA load can be reduced to near zero by the combination of handwashing by the infected patient and ≥1 minute detergent treatment after collection. We also found that the inexpensive and fast LAMP protocol was more than 80% concordant with RT-qPCR. IMPORTANCE The COVID-19 pandemic is leading to important tradeoffs between risk of SARS-CoV-2 transmission and mental health due to deprivation from normal activities, with these impacts being especially profound in children. Due to the ongoing pandemic, Halloween activities will be curtailed as a result of the concern that candy from strangers might act as fomites. Here we demonstrate that these risks can be mitigated by ensuring that prior to handling candy, the candy giver washes their hands, and by washing collected candy with household dishwashing detergent. Even in the most extreme case, with candy deliberately coughed on by known COVID-19 patients, viral load was reduced dramatically after washing with household detergent. We conclude that with reasonable precautions, even if followed only by either the candy giver or the candy recipient, the risk of viral transmission by this route is very low.

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Enhancing Colorimetric Loop-mediated Isothermal Amplification Speed and Sensitivity with Guanidine Chloride

Cited by 165 publications

References 14 publications

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

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

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

Handwashing and Detergent Treatment Greatly Reduce SARS-CoV-2 Viral Load on Halloween Candy Handled by COVID-19 Patients

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