The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, and the kit showed comparable sensitivity to approved commercial kits. The One-Step RT-qPCR was then tested on clinical samples and demonstrated similar performance to commercial kits in terms of positive and negative calls. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.
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http://dx.doi.org/10.1101/2021.11.29.21267000 | DOI Listing |
Parasit Vectors
December 2024
Global Health and Tropical Medicine (GHTM), Associate Laboratory in Translation and Innovation Towards Global Health (LA-REAL), Instituto de Higiene e Medicina Tropical (IHMT), Universidade NOVA de Lisboa (UNL), Rua da Junqueira 100, 1349-008, Lisbon, Portugal.
Background: Culex quinquefasciatus plays a crucial role as a vector of West Nile virus (WNV). This mosquito species is widely distributed in Cape Verde, being found in all inhabited islands of the archipelago. However, no data are currently available on the susceptibility of the local mosquito population to WNV.
View Article and Find Full Text PDFBMC Infect Dis
December 2024
Laboratory of Molecular Epidemiology and Experimental Pathology, LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, LR16IPT04, Tunisia.
Background: The COVID-19 has put emphasis on pivotal needs for diagnosis and surveillance worldwide, with the subsequent shortage of diagnostic reagents and kits. Therefore, it has become strategic for the countries to access diagnostics, expand testing capacity, and develop their own diagnostic capabilities and alternative rapid accurate nucleic acid diagnostics that are at lower costs. Here, we propose a visual SARS-CoV-2 detection using a one-step fast multiplex reverse transcription-PCR (RT-PCR) amplification coupled to lateral flow immunoassay detection on a PCRD device (Abingdon Health, UK).
View Article and Find Full Text PDFInt J Mol Sci
December 2024
Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Porcine reproductive and respiratory syndrome virus (PRRSV) is the pathogen that causes porcine reproductive and respiratory syndrome (PRRS), leading to abortion of sows and the manifestation of respiratory diseases in piglets. PRRSV strains are categorized into two distinct genotypes: PRRSV-1 and PRRSV-2. PRRSV-2 can be further classified into several lineages, including sub-lineage 1.
View Article and Find Full Text PDFPlants (Basel)
November 2024
CREA Research Centre for Plant Protection and Certification, Via C.G. Bertero 22, 00156 Rome, Italy.
Plum pox virus (PPV) is the etiological agent of sharka, the most important viral disease of stone fruit worldwide. In this study, a one-step reverse transcription real-time PCR test (RT-qPCR) was modified and translated as a one-step RT-droplet digital PCR (RT-ddPCR) for sensitive, direct, and accurate detection and quantification of PPV. The modified RT-qPCR and RT-ddPCR PPV detection tests were validated using both plant purified total RNA (TRNA) and crude extract as templates.
View Article and Find Full Text PDFFood Environ Virol
November 2024
ICBAS - School of Medicine and Biomedical Sciences, Porto University, Porto, Portugal.
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