Academic journal article Iranian Journal of Public Health

Development a Taqman Real-Time Pcr Assay with an Internal Positive Control for Quantitative Detection of Neisseria Meningitides

Academic journal article Iranian Journal of Public Health

Development a Taqman Real-Time Pcr Assay with an Internal Positive Control for Quantitative Detection of Neisseria Meningitides

Article excerpt

Background: This study was development a TaqMan Real- time PCR assay for rapid and specific quantitative detection of N. meningitidis. Also, by designing and using an Internal Positive Control (IPC) to monitor the assay, we were able to distinguish the false negative results caused by PCR failure.

Methods: The specific primer pair and a CY5/BHQ2 dual labeled TaqMan probe were designed based on the crgA gene. The TaqMan reaction was set up on genomic DNA of N. meningitides (ATCC 13060). The crgA PCR product was cloned into pTZ57R/T plasmid and a standard positive control plasmid (pTZ-crgA) was resulted. Performing the assay on various bacterial genomes, its specificity was evaluated. The sensitivity was determined by performing assay on 10-fold diluting pTZ-crgA, which the first provided concentration was 220ng/µl. To quantify the crgA gene copy number in the assay, a standard curve was generated by plotting Cycle threshold (Ct) values relative to log crgA copy number for each reaction tube. Also, to design a competitive IPC, a 100 bp fragment of AOX1 gene of Pichia pastoris, was inserted into the crgA PCR product and replaced with some nucleo- tides between the primer pair annealing sequences. …

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