Amplificación directa de ADN de Bordetella pertussis purificado de hisopados nasofaríngeos por un método de bajo costo, rápido (60-segundos) y libre de equipos

Eduardo Juscamayta-López; Faviola Valdivia; María Pía Soto; Helen Horna; Brenda Nureña

doi:10.17843/rpmesp.2022.393.10865

Authors

Eduardo Juscamayta-López Laboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, Perú https://orcid.org/0000-0001-6843-3206
Faviola Valdivia Laboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, Perú https://orcid.org/0000-0001-8347-6853
María Pía Soto Laboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, Perú https://orcid.org/0000-0002-7885-2866
Helen Horna Laboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, Perú https://orcid.org/0000-0003-1020-6910
Brenda Nureña Laboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, Perú https://orcid.org/0000-0003-4433-6019

DOI:

https://doi.org/10.17843/rpmesp.2022.393.10865

Keywords:

Point-of-Care Testing, Isolation & Purification, DNA, Bordetella pertussis, Cellulose, Molecular Diagnostic Techniques, Real-Time Polymerase Chain Reaction, LAMP loop-mediated isothermal amplification, Whooping Cough

Abstract

Objective. Develop and evaluate a low-cost cellulose-based method for the fast purification and direct amplification of Bordetella pertussis DNA from nasopharyngeal swabs. Materials and methods. Different parameters of the cellulose paper discs (lysis/wash buffers, number of discs and DNA elution) were evaluated. The method for DNA purification was coupled to direct amplification by real-time PCR (qPCR) and its performance was assessed using nasopharyngeal swabs positive (n=100) and negative (n=50) for B. pertussis in which the DNA was obtained with silica column-based method. The degree of agreement, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated. The feasibility of the fast method to be coupled to a loop-mediated isothermal amplification (LAMP) colorimetric assay was tested. Results. The fast method for DNA purification using a cellulose paper disc using lysis and wash buffer containing PVP-40 and Tween 20, respectively, showed to be able to purify amplifiable B. pertussis DNA. The method presented a sensitivity of 89.0% (95% CI, 80.2%-94.9%) and a specificity of 98.5% (95% CI, 92.1%-100.0%), with a good degree of agreement (Kappa=0.867; 95% CI, 0.788–0.946), compared to the reference method. PPV and NPV were 98.6% (95% CI, 92.7%-100.0%) and 88.2% (95% CI, 78.7%-94.4%), respectively. A successful amplification by LAMP was evidenced, obtaining comparable results with the silica column method. Conclusion. The developed method is simple, low-cost, and equipment-free for rapid (60 second) DNA purification at point-of-care and can be implemented to several molecular techniques for timely diagnosis and epidemiological studies of whooping cough.

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