title: Construction and Evaluation of a Novel Internal Positive Control (IPC) for Detection of Coxiella burnetii by PCR.
abstract: BACKGROUND
  Due to the limitations of the classical methods to detect Coxiella burnetii, direct diagnosis of the pathogen using PCR techniques is still the preferable approach. However, false negative results owing to the presence of PCR inhibitors are troublesome.
  
  
  OBJECTIVE
  In order to identify the inhibitors during PCR assay, an internal positive control (IPC) was designed based on 16SrRNA gene of C. burnetii.
  
  
  METHODS
  In the current study, the initial and ending parts of the target gene in an external positive control plasmid (pTZ57R/T-16S) amplified using internal primers which had a BglII restriction site on the 5´ends. Both PCR products (fragments 1 and 2) were cloned into pTZ57R/T vector. Following BglII enzyme digestion, the two obtained linear plasmids were ligated. The ligation product was transformed into Escherichia coli Top10 F'. Screening of the desired recombinant clone was carried out using colony PCR.
  
  
  RESULTS
  The size of the PCR product was equal to the sum of the first and second fragments. Sequencing confirmed the presence of the desire insert (IPC sequence) in recombinant plasmid. Consequently, the IPC fragment was longer than the target gene while both ends had similar attachments to the same primer pair.
  
  
  CONCLUSIONS
  The results showed that direct fusion of the recombinant plasmids containing the initial and ending parts of the target gene are simple and cost-effective techniques for increasing the length of the fragment and constructing IPC.
authors.affiliation: Tasnim Biotechnology of Research Center (TBRC), Faculty of Medicine, AJA University of Medical Sciences, Tehran, IR Iran ; Cancer Genetics Research Group, Breast Cancer Research Center (BCRC), ACECR, Tehran, IR Iran.
authors.forename: Keivan
authors.initials: K
authors.lastname: Majidzadeh
authors.affiliation: Tasnim Biotechnology of Research Center (TBRC), Faculty of Medicine, AJA University of Medical Sciences, Tehran, IR Iran.
authors.forename: Amirhossein
authors.initials: A
authors.lastname: Mohseni
authors.affiliation: Tasnim Biotechnology of Research Center (TBRC), Faculty of Medicine, AJA University of Medical Sciences, Tehran, IR Iran.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Soleimani
keywords: Coxiella burnetii; Molecular Detection; Polymerase Chain Reaction; Q Fever