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Revista Peruana de Medicina Experimental y Salud Publica
versão impressa ISSN 1726-4634
Resumo
JUSCAMAYTA-LOPEZ, Eduardo et al. Direct amplification of Bordetella pertussis DNA purified from nasopharyngeal swabs by a low-cost, fast (60-second), and equipment-free method. Rev. perú. med. exp. salud publica [online]. 2022, vol.39, n.3, pp.312-320. Epub 30-Set-2022. ISSN 1726-4634. http://dx.doi.org/10.17843/rpmesp.2022.393.10865.
Objective.
To develop and evaluate a low-cost cellulose-based method for rapid purification and direct amplification of Bordetella pertussis DNA from nasopharyngeal swabs.
Materials and methods.
We prepared cellulose discs and evaluated different parameters (lysis/wash buffers, number of discs and DNA elution). The method was coupled to a direct real-time PCR (qPCR) amplification and the performance was estimated using nasopharyngeal swabs that were positive (n=100) and negative (n=50) for B. pertussis DNA by qPCR, compared to the silica column-based method. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and the degree of agreement. The feasibility of the rapid method to be coupled to a loop-mediated isothermal amplification colorimetric assay (LAMP) was evaluated.
Results.
The rapid method, with a cellulose disk and lysis and wash buffer containing PVP-40 and Tween 20, respectively, showed a greater capacity to purify amplifiable DNA from B. pertussis. The method had 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. The PPV and NPV were 98.6% (95%CI: 92.7.2%-100.0%) and 88.2% (95%CI: 78.7%-94.4%), respectively. Successful amplification by LAMP was evident, and comparable results were obtained with the silica column method.
Conclusion.
The developed method is simple, low-cost and equipment-free for rapid (60 seconds) DNA collection at the point of care, and can be implemented in various molecular techniques aimed at the timely diagnosis and epidemiological study of pertussis.
Palavras-chave : 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.