Development of a TaqMan Real-time PCR assay for detection and quantitation of Neisseria meningitidis

Background and Aim: Neisseria meningitides, a Gram-negative aerobic bacterium, is an important pathogen of epidemic bacterial meningitis and fulminant sepsis worldwide. Rapid detection is the basis for effective management of serious infections with N. meningitidis. This study aimed at developing of a TaqMan Real-time PCR assay for rapid and specificdetection of N.meningitidis. Methods: The specific primers and CY5/BHQ1 dual labeled TaqMan probe were designed to amplify the crgA gene. ThecrgA PCR product was cloned in the pTZ57R/T plasmid to create a stable positive control plasmid (pTZ-crgA). The testsensitivity was evaluated by performing the assay on 10-fold serial dilutions of pTZ-crgA. In addition, a standard curve toquantify the crgA gene copy number in the assay was created by plotting Cycle threshold (Ct) values against log crgA copy number per reaction tube. Results: In contrast to DNA of N meningitides, negative control genomes didn’t show detectable signal in the assay. Thelowest concentration of the pTZ-crgA showing fluorescent signal was 0.089fg or 240 copies of the target gene. The depicted standard curve showed R2 value, efficiency and slope of 0.997, 95% and -3.43 respectively. Also, the dynamic range of the quantitative TaqMan assay was between 24× and 240 copies of crgA gene. Conclusions: The high sensitivity, rapidity, reproducibility and specificity of the novel crgA TaqMan assay suggest the method as a good candidate to further evaluation to reach a confirmed In Vitro Diagnostic (IVD) tool.

کلیدواژه ها:

Neisseria meningitidis ، Rapid diagnosis ، TaqMan real time PCR