Design and validation of a multiplex PCR protocol for microsatellite typing of Candida parapsilosis sensu stricto isolates

Abstract

Abstract Background Analysis of polymorphic microsatellite markers (STR) is a helpful genotyping technique to differentiate Candida parapsilosis sensu stricto isolates. The aim of this study is to develop and perform an initial validation of an alternative protocol for the reliable and accurate microsatellite genotyping of C. parapsilosis sensu stricto isolates using high-throughput multiplex PCR. To achieve this, the results obtained using the new protocol were compared to the ones obtained using a previously described reference method. To that end, diagnostic accuracy, informativeness and discrimination parameters were estimated. Results Our results showed good concordance between both methods (Kappa index: 0.920), leading to a high sensitivity (1; CI(95%) (0.991–1)) and specificity (1; CI(95%) (0.772–1)) after the validation of the new protocol. Moreover, the electropherograms profiles obtained with the new PCR scheme showed a high signal to noise ratio (SNR). Conclusions The new multiplex protocol is valuable for the differentiation of C. parapsilosis sensu stricto, with direct clinical applications. Besides, the new protocol represents a shortening the hands-on time, reducing the sample manipulation (dismissing the possibility of cross-contamination), maintaining the quality of the results (when compared to the ones obtained with the reference method), and helping to the standardization and simplification of the genotyping scheme.

Keywords

• Candida parapsilosis sensu stricto
• Genotyping technique
• Microsatellites repeats
• Multiplex PCR
• Specificity
• Reproducibility of results