Dados do Trabalho

Título

Development of isothermal amplification assays for the detection of infectious diseases at low temperature

Introdução

Infectious diseases caused by pathogenic organisms pose a significant global public health concern, resulting in millions of deaths each year. The prompt and accurate identification of causative agents is crucial for effective disease control, treatment, and monitoring. Real-time polymerase chain reaction (PCR) has been widely used for pathogen detection due to its effectiveness. However, its practical limitations, such as cost and dependence on specialised laboratory infrastructure, hinders its widespread application. Thus, there is an urgent need for new, rapid, and sensitive molecular diagnostic techniques that meets the ASSURED criteria: affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users.

Objetivo (s)

The objective of the research is to develop an isothermal amplification assay that combines the low-temperature setup of Recombinase Polymerase Amplification (RPA) and the amplification efficiency of Loop-mediated isothermal amplification (LAMP). The goal is to create a straightforward, fast, and sensitive diagnostic approach that can be adapted for point-of-care use, especially in resource-limited or inaccessible areas.

Material e Métodos

LAMP-like primers were designed targeting a conserved region in the genome of the Influenza A strain A/WSN/33 (H1N1), and for the proof-of-concept test, a cDNA was synthesised using SuperScript IV. The primers were incubated at 37 C in the presence of a commercially available recombinase (RecA) and a nucleotide cofactor, and amplification of the target is achieved by a polymerase with strand-displacement activity such as Bsm. The RecA-primer complex, the self-folding design of the primers and the use of a strand-displacing polymerase allow the detection of the target material in as little as one hour.

Resultados e Conclusão

The isothermal amplification at 37 C proposed allows for the detection of viral cDNA in less than one hour, and results can be read in an agarose gel or visualised under UV light after the addition of SYBR green I. This assay offers a high yield of DNA copies at the human body temperature, combining the advantages of LAMP and RPA. While this proof-of-concept has been tested only with cDNA from an RNA virus, the incorporation of a reverse-transcription step should enable the detection of RNA targets as well. The developed assays, when incorporated into portable devices, will allow for rapid and accessible point-of-care testing.

Palavras-chave

RPA, LAMP, isothermal amplification

Agradecimentos

IGPP doctoral research Fund