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Accurate, fast and cost-effective simultaneous detection of bacterial meningitis by qualitative PCR with

Ivano de Filippis1, Aline Carvalho de Azevedo1, Irene de Oliveira Lima1

  • 1Laboratório de Microrganismos de Referência, Instituto Nacional de Controle de Qualidade em Saúde (INCQS), Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ, 21040-900, Brazil.

Biotechniques
|February 27, 2023
PubMed

Insights

A new high-resolution melting qualitative PCR method enables simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae. This sensitive, probe-free assay is a cost-effective tool for diagnosing meningitis in developing countries.

Area of Science:

  • Microbiology
  • Molecular Diagnostics
  • Infectious Diseases

Background:

  • Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae are primary causes of meningitis and invasive bacterial infections.
  • Conventional diagnostic methods for bacterial pathogens are often labor-intensive and lack the sensitivity of molecular techniques.
  • Polymerase Chain Reaction (PCR) based methods offer high sensitivity, specificity, and high-throughput capabilities for pathogen detection.

Purpose of the Study:

  • To evaluate a high-resolution melting (HRM) qualitative PCR assay for the simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae.
  • To optimize the HRM-PCR assay for accurate identification of these three key etiological agents from clinical samples.
  • To assess the sensitivity and cost-effectiveness of the HRM-PCR method compared to existing real-time PCR systems.

Main Methods:

  • Development and optimization of a multiplex qualitative PCR assay targeting species-specific genes of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae.
  • Utilized high-resolution melting (HRM) analysis for simultaneous detection and differentiation of the three pathogens.
  • Validated the assay using clinical samples to determine its diagnostic accuracy.

Main Results:

  • The developed HRM-qualitative PCR assay successfully detected and differentiated Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae simultaneously.
  • The assay demonstrated high sensitivity and specificity in identifying the etiological agents from clinical specimens.
  • The probe-free HRM-PCR method was found to be more cost-effective than the real-time PCR TaqMan® system.

Conclusions:

  • High-resolution melting qualitative PCR is a sensitive, specific, and cost-effective method for the simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae.
  • This assay provides an accurate diagnostic tool for invasive bacterial diseases, particularly suitable for public health laboratories in resource-limited settings.
  • The probe-free nature of the HRM-PCR assay contributes to its affordability and potential for widespread implementation in developing countries.
Keywords:
bacterial meningitisinvasive diseasemultiplex qPCR-HRMrapid diagnosis

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