Enhancing sensitivity of qPCR assays targeting Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae by using a mutant Taq DNA polymerase


Nar Otgun S., Ketre Kolukirik C. Z., Unal Sahin N., Kolukirik M., Girgin Ozgumus G., Turan M., ...More

Journal of Microbiological Methods, vol.219, 2024 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 219
  • Publication Date: 2024
  • Doi Number: 10.1016/j.mimet.2024.106899
  • Journal Name: Journal of Microbiological Methods
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, Aquatic Science & Fisheries Abstracts (ASFA), BIOSIS, Biotechnology Research Abstracts, CAB Abstracts, Environment Index, Food Science & Technology Abstracts, Veterinary Science Database
  • Keywords: Bacterial meningitis, Real-time PCR, Taq DNA polymerase
  • Istanbul Technical University Affiliated: Yes

Abstract

Aims: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae are important causes of bacterial meningitis. In this study, the DNA binding site of the wild type Taq DNA polymerase was modified to produce a mutant enzyme with enhanced DNA affinity and PCR performance. The engineered and the wild type enzymes were integrated into qPCR-based assays for molecular detection of S. pneumoniae, N. meningitidis, H. influenzae, and serogroups and serotypes of these three pathogens. Methods: Bio-Speedy® Bacterial DNA Isolation Kit (Bioeksen R&D Technologies, Turkiye) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Turkiye) and CFX96 Instrument (Biorad Inc., USA) were used for all molecular analyses. Spiked negative clinical specimens were tested using the developed qPCR assays and the culture-based conventional methods for the analytical performance evaluation. Results: All qPCR assays did not produce any positive results for the samples spiked with potential cross-reacting bacteria. Limit of detection (LOD) of the assays containing the mutant enzyme was 1 genome/reaction (10 cfu/mL sample) which is at least 3 times lower than the previously reported LOD levels for DNA amplification based molecular assays. LODs for the spiked serum and cerebrospinal fluid (CSF) samples decreased 2.3–4.7 and 1.2–3.5 times respectively when the mutant enzyme was used instead of the wild type Taq DNA polymerase. Conclusions: It is possible to enhance analytical sensitivity of qPCR assays targeting the bacterial agents of meningitis by using an engineered Taq DNA polymerase. These qPCR-based assays can be used for direct detection and serogrouping / serotyping of S. pneumoniae, N. meningitidis and H. influenzae at concentrations close to the lower limit of medical decision point.