An inexpensive and rapid diagnostic method for detection of SARS-CoV-2 RNA by loop-mediated isothermal amplification (LAMP)

Prakash, Shyam; Priyatma, Priyatma; Aasarey, R; Pandey, P K; Mathur, Purva; Arulselvi, S

doi:10.1016/j.mex.2023.102011

Cited by 8 publications

(8 citation statements)

References 34 publications

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“…Other LAMP systems use colorimetric indicators, such as hydroxynaphthol blue, a metal indicator that turns from violet to blue as free Mg 2+ concentration decreases when positive samples are successfully amplified. Nevertheless, this method has very low sensitivity levels, approximately 10-100ng of RNA (Hongjaisee et al, 2021), and low specificity values, around 80%, compared to gold standard qRT-PCR (Prakash et al, 2023). Phenol-or cresol-red are also pH indicators used as a read-out for LAMP-based diagnosis (Huang et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

Development of a Fast and Affordable Diagnostic System for RNA Viruses Using Loop-Mediated Isothermal Amplification and DNA-Nanoprobe Detection

Cea,

Arca,

Gomez-Lucia

et al. 2023

Preprint

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Airbone viral pathogens can rapidly spread and become a global menace, including both human and animal viruses which can trigger important socioeconomical and health effects. Therefore, to prevent and contain potential epidemic outbreaks, accurate, fast, and affordable diagnostic point-of-care tests (POCT) are required. As a proof of concept, we have developed a molecular detection system based in Loop-mediated isothermal AMPlification technique (LAMP) for two different RNA airborne viruses: the well-known human SARS-CoV-2, and the avian metapneumovirus (aMPV), the aetiologic agent of a communicable disease infecting mainly turkeys and chickens. To obtain a POC diagnostic system, we coupled the LAMP technique to DNA-functionalized gold nanoparticles detection. Validation of this system was carried out in 140 pharyngeal swabs from COVID-19 symptomatic and asymptomatic patients, and in 50 different samples (pharyngeal swabs and tracheal tissue samples) collected from aMPV infected chickens and turkeys. The system allows viral detection in about 60 minutes by the naked eye with 100% specificity, and 97.22% and 87.88% sensitivity for SARS-CoV-2 and aMPV, respectively. In summary, this novel detection system based on the coupling of RT-LAMP to DNA-nanoprobes allows suitable virus testing in the field, with accurate levels very close to conventional qRT-PCR based diagnosis.

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Section: Discussionmentioning

confidence: 99%

Development of a Fast and Affordable Diagnostic System for RNA Viruses Using Loop-Mediated Isothermal Amplification and DNA-Nanoprobe Detection

Cea,

Arca,

Gomez-Lucia

et al. 2023

Preprint

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“…Despite this inherent limitation to RNA-based detection methods, several groups have been able to prove its viability and increased diagnostic potential as a useful detection target. For instance, in 2023, Prakash et al proposed a loop-mediated isothermal amplification (LAMP) method for the detection of SARS-CoV-2 RNA [84]. This LAMP method utilizes RNA extraction from blood samples through the TRIzol method, which amplifies a gene in the form of dsDNA through a single-step reaction with a mixture of incubated primers; a reaction contains dNTPs, HNB (Hydroxy Naphthol Blue), Betaine, MgSO 4 , Bst DNA polymerase, and the target substrate [84].…”

Section: Implementation Of Microfluidics Towards Future Preparedness ...mentioning

confidence: 99%

“…For instance, in 2023, Prakash et al proposed a loop-mediated isothermal amplification (LAMP) method for the detection of SARS-CoV-2 RNA [84]. This LAMP method utilizes RNA extraction from blood samples through the TRIzol method, which amplifies a gene in the form of dsDNA through a single-step reaction with a mixture of incubated primers; a reaction contains dNTPs, HNB (Hydroxy Naphthol Blue), Betaine, MgSO 4 , Bst DNA polymerase, and the target substrate [84]. This preparation step allows the normally reactive single-stranded viral RNA to be paired into a complimentary DNA (cDNA) phase, which in turn, facilitates downstream processing by creating synthetic DNA strands that are far less susceptible to degradation and dehydration, compared to RNA [84,85].…”

Section: Implementation Of Microfluidics Towards Future Preparedness ...mentioning

confidence: 99%

Microfluidic Device-Based Virus Detection and Quantification in Future Diagnostic Research: Lessons from the COVID-19 Pandemic

Escobar,

Diab-Liu,

Bosland

et al. 2023

Biosensors

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The global economic and healthcare crises experienced over the past three years, as a result of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has significantly impacted the commonplace habits of humans around the world. SARS-CoV-2, the virus responsible for the coronavirus 2019 (COVID-19) phenomenon, has contributed to the deaths of millions of people around the world. The potential diagnostic applications of microfluidic devices have previously been demonstrated to effectively detect and quasi-quantify several different well-known viruses such as human immunodeficiency virus (HIV), influenza, and SARS-CoV-2. As a result, microfluidics has been further explored as a potential alternative to our currently available rapid tests for highly virulent diseases to better combat and manage future potential outbreaks. The outbreak management during COVID-19 was initially hindered, in part, by the lack of available quantitative rapid tests capable of confirming a person’s active infectiousness status. Therefore, this review will explore the use of microfluidic technology, and more specifically RNA-based virus detection methods, as an integral part of improved diagnostic capabilities and will present methods for carrying the lessons learned from COVID-19 forward, toward improved diagnostic outcomes for future pandemic-level threats. This review will first explore the context of the COVID-19 pandemic and how diagnostic technology was shown to have required even greater advancements to keep pace with the transmission of such a highly infectious virus. Secondly, the historical significance of integrating microfluidic technology in diagnostics and how the different types of genetic-based detection methods may vary in their potential practical applications. Lastly, the review will summarize the past, present, and future potential of RNA-based virus detection/diagnosis and how it might be used to better prepare for a future pandemic.

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“…One of these dyes is hydroxynaphthol blue (HNB), which shifts from violet to sky blue as the LAMP reaction progresses [ 20 ]. This dye has been used to detect SARS-CoV-2 viral RNA in blood samples extracted by the TRIzol method [ 21 ]. Positive LAMP reactions can also be visually identified using calcein dye, another metal ion indicator, which shifts color from orange to fluorescent green due to a reduction in the Mg 2+ ions concentration, particularly used in SARS-CoV-2 detection [ 5 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

Colorimetric RT-LAMP Detection of Multiple SARS-CoV-2 Variants and Lineages of Concern Direct from Nasopharyngeal Swab Samples without RNA Isolation

Werbajh,

Larocca,

Carrillo

et al. 2023

Viruses

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Since, during the Coronavirus disease 19 (COVID-19) pandemic, a large part of the human population has become infected, a rapid and simple diagnostic method has been necessary to detect its causative agent, the Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2), and control its spread. Thus, in the present study, we developed a colorimetric reverse transcription-loop-mediated isothermal amplification (RT-LAMP) kit that allows the detection of SARS-CoV-2 from nasopharyngeal swab samples without the need for RNA extraction. The kit utilizes three sets of LAMP primers targeting two regions of ORF1ab and one region in the E gene. The results are based on the colorimetric change of hydroxynaphthol blue, which allows visual interpretation without needing an expensive instrument. The kit demonstrated sensitivity to detect between 50 and 100 copies of the viral genome per reaction. The kit was authorized by the National Administration of Drugs, Food and Technology (ANMAT) of Argentina after validation using samples previously analyzed by the gold standard RT-qPCR. The results showed a sensitivity of 90.6% and specificity of 100%, consistent with conventional RT-qPCR. In silico analysis confirmed the recognition of SARS-CoV-2 variants of concern (B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.427, and B.1.429), and lineages of the Omicron variant (B.1.1.529) with 100% homology. This rapid, simple, and sensitive RT-LAMP method paves the way for a large screening strategy to be carried out at locations lacking sophisticated instrumental and trained staff, as it particularly happens in regional hospitals and medical centers from rural areas.

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An inexpensive and rapid diagnostic method for detection of SARS-CoV-2 RNA by loop-mediated isothermal amplification (LAMP)

Cited by 8 publications

References 34 publications

Development of a Fast and Affordable Diagnostic System for RNA Viruses Using Loop-Mediated Isothermal Amplification and DNA-Nanoprobe Detection

Development of a Fast and Affordable Diagnostic System for RNA Viruses Using Loop-Mediated Isothermal Amplification and DNA-Nanoprobe Detection

Microfluidic Device-Based Virus Detection and Quantification in Future Diagnostic Research: Lessons from the COVID-19 Pandemic

Colorimetric RT-LAMP Detection of Multiple SARS-CoV-2 Variants and Lineages of Concern Direct from Nasopharyngeal Swab Samples without RNA Isolation

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