Optimization and validation of RT-LAMP assay for diagnosis of SARS-CoV2 including the globally dominant Delta variant.
B.1.1.7 (Alpha)
B.1.351 (Beta)
B.1.427
B.1.429 (Epsilon)
B.1.525 (Eta)
B.1.526 (Iota)
B.1.617.1 (Kappa)
B.1.617.2 (Delta)
COVID-19
Loop-mediated isothermal amplification
Molecular diagnosis
P.1 (Gamma)
P.2 (Zeta)
P.3 (Theta)
RT-LAMP
SARS-CoV-2
Journal
Virology journal
ISSN: 1743-422X
Titre abrégé: Virol J
Pays: England
ID NLM: 101231645
Informations de publication
Date de publication:
30 08 2021
30 08 2021
Historique:
received:
17
03
2021
accepted:
12
08
2021
entrez:
31
8
2021
pubmed:
1
9
2021
medline:
10
9
2021
Statut:
epublish
Résumé
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 pandemic, has infected more than 179 million people worldwide. Testing of infected individuals is crucial for identification and isolation, thereby preventing further spread of the disease. Presently, Taqman™ Reverse Transcription Real Time PCR is considered gold standard, and is the most common technique used for molecular testing of COVID-19, though it requires sophisticated equipments, expertise and is also relatively expensive. Development and optimization of an alternate molecular testing method for the diagnosis of COVID-19, through a two step Reverse Transcription Loop-mediated isothermal AMPlification (RT-LAMP). Primers for LAMP were carefully designed for discrimination from other closely related human pathogenic coronaviruses. Care was also taken that primer binding sites are present in conserved regions of SARS-CoV2. Our analysis shows that the primer binding sites are well conserved in all the variants of concern (VOC) and variants of interest (VOI), notified by World Health Organization (WHO). These lineages include B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.427/B.1.429, P.2, B.1.525, P.3, B.1.526 and B.1.617.1. Various DNA polymerases with strand displacement activity were evaluated and conditions were optimized for LAMP amplification and visualization. Different LAMP primer sets were also evaluated using synthetic templates as well as patient samples. In a double blind study, the RT-LAMP assay was validated on more than 150 patient samples at two different sites. The RT-LAMP assay appeared to be 89.2% accurate when compared to the Taqman™ rt-RT-PCR assay.
Sections du résumé
BACKGROUND
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 pandemic, has infected more than 179 million people worldwide. Testing of infected individuals is crucial for identification and isolation, thereby preventing further spread of the disease. Presently, Taqman™ Reverse Transcription Real Time PCR is considered gold standard, and is the most common technique used for molecular testing of COVID-19, though it requires sophisticated equipments, expertise and is also relatively expensive.
OBJECTIVE
Development and optimization of an alternate molecular testing method for the diagnosis of COVID-19, through a two step Reverse Transcription Loop-mediated isothermal AMPlification (RT-LAMP).
RESULTS
Primers for LAMP were carefully designed for discrimination from other closely related human pathogenic coronaviruses. Care was also taken that primer binding sites are present in conserved regions of SARS-CoV2. Our analysis shows that the primer binding sites are well conserved in all the variants of concern (VOC) and variants of interest (VOI), notified by World Health Organization (WHO). These lineages include B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.427/B.1.429, P.2, B.1.525, P.3, B.1.526 and B.1.617.1. Various DNA polymerases with strand displacement activity were evaluated and conditions were optimized for LAMP amplification and visualization. Different LAMP primer sets were also evaluated using synthetic templates as well as patient samples.
CONCLUSION
In a double blind study, the RT-LAMP assay was validated on more than 150 patient samples at two different sites. The RT-LAMP assay appeared to be 89.2% accurate when compared to the Taqman™ rt-RT-PCR assay.
Identifiants
pubmed: 34461941
doi: 10.1186/s12985-021-01642-9
pii: 10.1186/s12985-021-01642-9
pmc: PMC8404189
doi:
Types de publication
Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
178Subventions
Organisme : council of scientific and industrial research
ID : MLP11001
Informations de copyright
© 2021. The Author(s).
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