Verification of in-nousf molecular diagnostic assays for central nervous system infections

Abstract

Central nervous system (CNS) infections including bacterial meningitis are important diseases entity world-wide. Without rapid diagnosis and correct clinical management, the infection results in serious consequences and can be life-threatening. Thus it is emerge to propose an accurate diagnosis method for meningitis. In this thesis, I describe the verification of an internally controlled real-time PCR assay using specific primers and TaqMan probes for detection of 4 most emergent bacteria in Vietnam which are Streptococcus pneumoniae and suis serotype 2, Haemophilus influenzae type b, and Neisseria meningitidis. The verification includes analytical sensitivity, specificity, repeatability and reproducibility precision. In this diagnostic assay, an internal control animal virus (Phocine Herpes Virus [PhHV]) was spiked into cultured samples to ensure the efficiency of the whole process. The assay showed a limit of detection (LoD) of less than 3 copies per 5 μL reaction (NanoDrop Spectrophotometry) and less than 1 copy per 5 μL reaction (Fluorometric assay: Picogreen and Qubit). Also, 100% specificity of primers and probes was tested with no cross-detection of one bacterium among others at low concentration (near LoD concentration). Precision assay expressed that there were homogeneity of Ct value of each run (P-value of Levene’s test >0.05) and no significant difference between 5 continuous days of assay (P-value of F test > 0.05). KEYWORDS Central nervous system infections; Bacterial meningitis; Verification; Internally controlled real-time PCR;