DIRECT AMPLIFICATION OF Bordetella pertussis DNA PURIFIED FROM NASOPHARYNGEAL SWABS BY A LOW-COST, FAST (60-SECOND), AND EQUIPMENT-FREE METHOD

Juscamayta-López E.; Valdivia F.; Soto M.P.; Horna H.; Nureña B. · Revista Peruana de Medicina Experimental y Salud Publica (2022) · DOI: 10.17843/rpmesp.2022.393.10865

Resumen

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, speci-ficity, 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 colori-metric 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.