Performance verification of pre-PCR and real-time PCR step in the molecular diagnosis of pneumococcal meningitis cases

¹ Department of Epidemic Diseases, National Institute of Hygiene, Rabat, Morocco
^2. Department of Biology and Health, Faculty of Science, Ibn Tofael University, Kenitra, Morocco.

^* Corresponding author: soumayachaiboub@gmail.com

Abstract

Isolation and determination of s.pneumoniae by culture and serological methods can be time consuming or indeterminate. Molecular diagnosis by real-time PCR is independent of the growth of the pathogen causing meningitis, and is not diminished with non-viable organisms. The aim of this study was to evaluate the performance criteria of pre-PCR-TR DNA extraction step and PCR-TR step by targeting two genes encoding s.pneumoniae. In this study we evaluated the inter-sample contamination of the pre-PCR-TR step, the intermediate fidelity and the repeatability of the DNA essay. PCR-TR verification was performed by two genes targeting s. pneumoniae the Lyt A and SP 2038 gene; sensitivity, specificity and LLD were determined. Contamination rate had a value of less than 0%, which is in agreement with an absence of inter-sample contamination; the repeatability and intermediate fidelity have a cv˂7%. The evaluation of the sensitivity and specificity of the RT-PCR assays targeted 100% the Lyt A gene and the SP 2038 gene. The standard curve generated detected less than 10copies for the Lyt A gene and less than 100copies for the SP 2038 gene. This study showed that the pre-PCR and PCR-TR assays met the performance criteria targeted in this study.