title: Designing novel and simple competitive internal amplification control for reliable PCR diagnosis of herpes simplex virus.
abstract: BACKGROUND
  PCR is a molecular technique for herpes simplex virus (HSV) detection that can cause life-threatening infections such as encephalitis and keratitis. However, the main issues, false-negative results causing by PCR inhibitors, of this technique that reduce PCR efficiency. To overcome this problem, a competitive internal amplification control (IAC) was constructed for conventional PCR using the PCR-cloning technique.
  
  
  OBJECTIVE
  The purpose of this study is the design of competitive IAC for PCR diagnosis of HSV, which in fact is the main cause of keratitis and viral encephalitis in developed countries.
  
  
  METHODS
  Composite primers for PCR amplification of Leishmania major kDNA (kinetoplast DNA) were designed and optimized to use as IAC-HSV. IAC-HSV amplified in a non-stringent condition, ligated into pTZ57R plasmid vector, and transformed into Escherichia coli JM207 and then cloned. Resulting IAC was used for 105 CSF and 78 keratitis specimens.
  
  
  RESULTS
  PCR amplicons for HSV and IAC-HSV were 454-bp and 662-bp, respectively. Detection limit of IAC was determined as 1000 plasmids per PCR reaction. IAC sensitivity for HSV detection was determined as 1000 plasmids per PCR reaction. IAC sensitivity for HSV detection was 500 copies/mL of HSV DNA. Among all specimens, 7 inhibited specimens were detected.
  
  
  CONCLUSIONS
  Indeed, using other DNA as an IAC is expected to detect false-negative results and amplification of the DNA is the key tool to examine the accuracy of amplification and detection steps. This internal amplification control is applicable for early reliable diagnosis of HSV in different loads of virus in different specimens.
authors: Department of Immunology, Tarbiat Modares University, Tehran, IR Iran.
authors: Asiye
authors: A
authors: Akbarian
authors: Department of Microbiology, Shahre Qods, Branch Islamic Azad University, Tehran, IR Iran.
authors: Mohammad Hassan
authors: MH
authors: Shahhosseiny
authors: Department of Immunology, Tarbiat Modares University, Tehran, IR Iran.
authors: Somayeh
authors: S
authors: Vafaei
authors: Department of Microbiology, Shargh Branch, Islamic Azad University, Tehran, IR Iran.
authors: Elham
authors: E
authors: Moslemi
authors: Department of Microbiology, Qom Branch, Islamic Azad University, Qom, IR Iran.
authors: Maryam
authors: M
authors: Ghahri
keywords: Herpes Simplex Virus; Leishmania Major; PCR; pTZ57R