Direct amplification of Bordetella pertussis DNA purified from nasopharyngeal swabs by a low-cost, fast (60-second), and equipment-free method

Authors

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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Published

2022-09-30

Issue

Section

Original Article

How to Cite

1.
Juscamayta-López E, Valdivia F, Soto MP, Horna H, Nureña B. Direct amplification of Bordetella pertussis DNA purified from nasopharyngeal swabs by a low-cost, fast (60-second), and equipment-free method. Rev Peru Med Exp Salud Publica [Internet]. 2022 Sep. 30 [cited 2024 May 28];39(3):312-20. Available from: https://rpmesp.ins.gob.pe/index.php/rpmesp/article/view/10865

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