Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications

Moore, Keith J. M.; Cahill, Jeremy; Aidelberg, Guy; Aronoff, Rachel; Bektaş, Ali; Bezdan, Daniela; Butler, Daniel; Chittur, Sridar V.; Codyre, Martin; Federici, Fernán; Tanner, Nathan A.; Tighe, Scott; True, Randy; Ware, S.B.; Wyllie, Anne L.; Afshin, Evan E.; Bendesky, Andrés; Chang, Cindy J.; Rosa, Richard Dela; Elhaik, Eran; Erickson, David; Goldsborough, Andrew S.; Grills, George S.; Hadasch, Kathrin; Hayden, Andrew; Her, Seong-Young; Karl, Julie A.; Kim, Chang Hee; Kriegel, Alison J.; Kunstman, Thomas; Landau, Zeph; Land, Kevin; Langhorst, Bradley W.; Lindner, Ariel B.; Mayer, Benjamin E.; McLaughlin, Lee A; McLaughlin, Matthew T.; Molloy, Jenny; Mozsary, Christopher; Nadler, Jerry L.; D’silva, Melinee; Ng, David Chee Eng; O’Connor, David H.; Ongerth, Jerry E.; Osuolale, Olayinka; Pinharanda, Ana; Plenker, Dennis; Ranjan, Ravi; Rosbash, Michael; Rotem, Assaf; Segarra, Jacob; Schürer, Stephan C.; Sherrill-Mix, Scott; Solo‐Gabriele, Helena M.; To, Shaina; Vogt, Merly C.; Yu, Albert D.; Mason, Christopher E.

doi:10.7171/jbt.21-3203-017

Cited by 30 publications

(26 citation statements)

References 240 publications

(248 reference statements)

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“…Previously, only four primers were included in ACTB primer set to detect ACTB gene, which prolonged the reaction (Huang et al, 2020 ; Poon et al, 2005 ). The reaction time was significantly shortened by adding loop primers (Avelar, 2019 ; Jamwal et al, 2021 ; Moore et al, 2021 ; Zhang et al, 2020 ). Therefore, we designed a new set of primers, ACTB‐n, which also detected the ACTB sequence with the additional loop primers.…”

Section: Resultsmentioning

confidence: 99%

A quantitative RT‐qLAMP for the detection of SARS‐CoV‐2 and human gene in clinical application

Zhou

Li³

et al. 2022

Microbial Biotechnology

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Reverse transcription (RT) – loop‐mediated isothermal amplification (LAMP) assay is a rapid and one‐step method to detect SARS‐CoV‐2 in the pandemic. Quantitative estimation of the viral load of SARS‐CoV‐2 in patient samples could help physicians make decisions on clinical treatment and patient management. Here, we propose to use a quantitative LAMP (qLAMP) method to evaluate the viral load of SARS‐CoV‐2 in samples. We used threshold time (TT) values of qLAMP, the isothermal incubation time required for the fluorescent or colorimetric signal to reach the threshold, to indicate the viral load of clinical samples. Similar to the cycle threshold ( C t ) values in conventional qPCR, TT values of qLAMP show a linear relationship to the copy numbers of SARS‐CoV‐2. The higher the viral loadings, the lower qLAMP TT values are. The RT‐qLAMP assay was demonstrated to quantify the viral loads of synthesized full‐length RNA, inactivated viral particles (BBIBP‐CorV), and clinical samples within 15 min by fluorescent reading and 25 min by colorimetric reading. The RT‐qLAMP has been applied to detect Alpha, Beta, Kappa, Delta, and Omicron variants of SARS‐CoV‐2, as well as the human beta‐actin gene, and their TT values showed the linear patterns. The RT‐qLAMP assays were evaluated by 64 clinical samples (25 positives and 39 negatives) for the assessment of viral loads, and it was also used to quantify the human beta‐actin gene, which was used as a control and an indicator of sampling quality in clinical swab samples. The result of RT‐qLAMP was in good agreement with the result of RT‐qPCR. The RT‐qLAMP assay detected all clinical samples, including those with C t = 35, within 10 min using fluorescent reading.

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Section: Resultsmentioning

confidence: 99%

A quantitative RT‐qLAMP for the detection of SARS‐CoV‐2 and human gene in clinical application

Zhou

Li³

et al. 2022

Microbial Biotechnology

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“…Faced with the challenge of the SARS-CoV-2 pandemic, laboratories worldwide have been applying a variety of strategies and methods to meet the exceptionally high testing demand (18, 19). For molecular testing, these include rRT-PCR performed directly on crude samples without nucleic acid extraction (20)(21)(22)(23)(24), isothermal ampli cation techniques (25,26) and CRISPR-based nucleic acid detection assays (27)(28)(29). In our approach, we maintained a standard rRT-PCR testing work ow and the associated high detection sensitivity and speci city by combining three pragmatic and straightforward measures to stepwise increase our testing capacity, generating several options to exibly adapt sample throughput according to the current demand.…”

Section: Discussionmentioning

confidence: 99%

Flexible upscaling of laboratory PCR testing capacity at the Robert Koch Institute during the SARS-CoV-2 pandemic

Krause

Michel

Puyskens

et al. 2022

Preprint

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Background Over the course of the COVID-19 pandemic, laboratories worldwide have been facing an unprecedented increase in demand for PCR testing because of the high importance of diagnostics for prevention and control of virus spread. Moreover, testing demand has been varying considerably over time, depending on the epidemiological situation, rendering efficient resource allocation difficult. Here, we present a scalable workflow which we implemented in our laboratory to increase PCR testing capacity while maintaining high flexibility regarding the number of samples to be processed. Methods We compared the performance of five automated extraction instruments, using dilutions of SARS-CoV-2 cell culture supernatant as well as clinical samples. To increase PCR throughput, we combined the two duplex PCR reactions of our previously published SARS-CoV-2 PCR assay into one quadruplex reaction and compared their limit of detection as well as their performance on the detection of low viral loads in clinical samples. Furthermore, we developed a sample pooling protocol with either two or four samples per pool, combined with a specifically adapted SARS-CoV-2 quadruplex PCR assay, and compared the diagnostic sensitivity of pooled testing and individual testing. Results All tested automated extraction instruments yielded comparable results regarding the subsequent sensitivity of SARS-CoV-2 detection by PCR. While the limit of detection of the quadruplex SARS-CoV-2 PCR assay (E-Gene assay: 28.7 genome equivalents (ge)/reaction, orf1ab assay: 32.0 ge/reaction) was slightly higher than that of our previously published duplex PCR assays (E-Gene assay: 9.8 ge/reaction, orf1ab assay: 6.6 ge/reaction), the rate of correctly identified positive patient samples was comparable for both assays. Sample pooling with optimized downstream quadruplex PCR showed no loss in diagnostic sensitivity compared to individual testing. Conclusion Specific adaptation of PCR assays can help overcome the potential loss of sensitivity due to higher levels of PCR multiplexing or sample dilution in pooled testing. Combining these adapted PCR assays with different sample processing strategies provides a simple and highly adjustable workflow for resource-efficient SARS-CoV-2 diagnostics. The presented principles can easily be adopted in a variety of laboratory settings as well as be adapted to pathogens other than SARS-CoV-2, making it feasible for any laboratory that conducts PCR diagnostics.

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“…However, unlike LAMP, RPA often produces non-specific amplification even in water controls, an effect that has been reported by the original inventors of the technology ( 54 ). Finally, it should be noted that RPA kits are currently commercialized by a single company, TwistDx [now part of Abbott Laboratories, US ( 66 )] which has full control over the price and availability of the kits. RPA has not yet been approved by the food and drug administration (FDA), and for now is intended for research-only applications ( 65 , 67 ).…”

Section: Polymerase Chain Reaction Loop Mediated Isothermal Amplifica...mentioning

confidence: 99%

Recent progress in diagnosis and treatment of Human African Trypanosomiasis has made the elimination of this disease a realistic target by 2030

et al. 2022

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Human African Trypanosomiasis (HAT) is caused by unicellular flagellated protozoan parasites of the genus Trypanosoma brucei. The subspecies T. b. gambiense is mainly responsible for mostly chronic anthroponotic infections in West- and Central Africa, accounting for roughly 95% of all HAT cases. Trypanosoma b. rhodesiense results in more acute zoonotic infections in East-Africa. Because HAT has a two-stage pathogenesis, treatment depends on clinical assessment of patients and the determination whether or not parasites have crossed the blood brain barrier. Today, ultimate confirmation of parasitemia is still done by microscopy analysis. However, the introduction of diagnostic lateral flow devices has been a major contributor to the recent dramatic drop in T. b. gambiense HAT. Other techniques such as loop mediated isothermal amplification (LAMP) and recombinant polymerase amplification (RPA)-based tests have been published but are still not widely used in the field. Most recently, CRISPR-Cas technology has been proposed to improve the intrinsic diagnostic characteristics of molecular approaches. This will become crucial in the near future, as preventing the resurgence of HAT will be a priority and will require tools with extreme high positive and negative predicted values, as well as excellent sensitivity and specificity. As for treatment, pentamidine and suramin have historically been the drugs of choice for the treatment of blood-stage gambiense-HAT and rhodesiense-HAT, respectively. For treatment of second-stage infections, drugs that pass the blood brain barrier are needed, and melarsoprol has been effectively used for both forms of HAT in the past. However, due to the high occurrence of post-treatment encephalopathy, the drug is not recommended for use in T. b. gambiense HAT. Here, a combination therapy of eflornithine and nifurtimox (NECT) has been the choice of treatment since 2009. As this treatment requires IV perfusion of eflornithine, efforts were launched in 2003 by the drugs for neglected disease initiative (DNDi) to find an oral-only therapy solution, suitable for rural sub-Saharan Africa treatment conditions. In 2019 this resulted in the introduction of fexinidazole, with a treatment regimen suitable for both the blood-stage and non-severe second-stage T. b. gambiense infections. Experimental treatment of T. b. rhodesiense HAT has now been initiated as well.

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Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications

Cited by 30 publications

References 240 publications

A quantitative RT‐qLAMP for the detection of SARS‐CoV‐2 and human gene in clinical application

A quantitative RT‐qLAMP for the detection of SARS‐CoV‐2 and human gene in clinical application

Flexible upscaling of laboratory PCR testing capacity at the Robert Koch Institute during the SARS-CoV-2 pandemic

Recent progress in diagnosis and treatment of Human African Trypanosomiasis has made the elimination of this disease a realistic target by 2030

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