An Open One-Step RT-qPCR for SARS-CoV-2 detection.

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, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. 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.

An Open One-Step RT-qPCR for SARS-CoV-2 detection.

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.

Determinación cuantitativa del ß-sitosterol presente en vegetales de la dieta: posibles implicancias para su uso preventivo en poblaciones susceptibles / Quantitative determination of ß-sitosterol in dietary vegetables: possible implications for its preventive use in susceptible populations

El ß-sitosterol es probablemente el esterol de plantas más abundante y ampliamente distribuido. Diversos estudios han demostrado su efectividad clínica como agente capaz de disminuir los niveles de colesterol, así como en el tratamiento de la hiperplasia prostática benigna. Los vegetales de la dieta constituyen unafuente diaria de ß-sitosterol. En el presente trabajo se comparó el contenido de ß-sitosterol en diferentes vegetales constituyentes de la dieta occidental, utilizando cromatografía en capa fina y posteriormente se cuantificó su contenido a través de HPLC con detección espectrofotométrica. El análisis de ocho vegetales de consumo habitual en la población chilena, mostró que garbanzos, lentejas, porotos y arvejas presentaron los mayores contenidos de ß-sitosterol. De acuerdo a nuestros resultados y considerando la utilidad terapéutica que se ha descrito para los esteroles de plantas, parecería recomendable como medida profiláctica, incrementar el consumo de algunos de estos vegetales en poblaciones susceptibles. Adicionalmente, se presenta una metodología analítica modificada para la medición directa de ß-sitosterol en extractos vegetales.

Biotransformation of natural and synthetic isoflavonoids by two recombinant microbial enzymes.

Isolation and synthesis of isoflavonoids has become a frequent endeavor, due to their interesting biological activities. The introduction of hydroxyl groups into isoflavonoids by the use of enzymes represents an attractive alternative to conventional chemical synthesis. In this study, the capabilities of biphenyl-2,3-dioxygenase (BphA) and biphenyl-2,3-dihydrodiol 2,3-dehydrogenase (BphB) of Burkholderia sp. strain LB400 to biotransform 14 isoflavonoids synthesized in the laboratory were investigated by using recombinant Escherichia coli strains containing plasmid vectors expressing the bphA1A2A3A4 or bphA1A2A3A4B genes of strain LB400. The use of BphA and BphB allowed us to biotransform 7-hydroxy-8-methylisoflavone and 7-hydroxyisoflavone into 7,2',3'-trihydroxy-8-methylisoflavone and 7,3',4'-trihydroxyisoflavone, respectively. The compound 2'-fluoro-7-hydroxy-8-methylisoflavone was dihydroxylated by BphA at ortho-fluorinated and meta positions of ring B, with concomitant dehalogenation leading to 7,2',3',-trihydroxy-8-methylisoflavone. Daidzein (7,4'-dihydroxyisoflavone) was biotransformed by BphA, generating 7,2',4'-trihydroxyisoflavone after dehydration. Biotransformation products were analyzed by gas chromatography-mass spectrometry and nuclear magnetic resonance techniques.