title: Co-detection and discrimination of six human herpesviruses by multiplex PCR-ELAHA.
abstract: BACKGROUND
  Herpesviruses are a significant cause of human morbidity. Traditional approaches to the identification of these viruses require infectious or at least antigenic virus. Multiplex PCR (mPCR) is capable of simultaneously amplifying a range of targets from a single preparation of nucleic acids and when combined with a suitable detection assay, it is capable of discriminating each of the amplicons.
  
  
  OBJECTIVE
  Several methods have been described in the literature, however, they lack one or more significant design features required to suitably control a routinely applied nucleic acid amplification assay. We aimed to design a multiplex herpesvirus PCR that could co-amplify eight human herpesvirus targets plus an internal control (IC) molecule in a single tube.
  
  
  METHODS
  Primers were designed to target the DNA polymerase genes of each of the human herpesviruses. Synthetic controls were developed to act as templates for the evaluation of assay sensitivity and specificity and for development of an in-house competitive quantitative PCR. Amplicon was discriminated using a simplified enzyme linked amplicon hybridisation assay (ELAHA).
  
  
  CONCLUSIONS
  For routine diagnostic use we reduced the number of herpesviral targets from 8 to 6 in order to maintain adequate clinical sensitivity. The ELAHA proved more sensitive than agarose gel electrophoresis. Additionally, 36 cytomegalovirus positive patients were examined with an in-house quantitative PCR-ELAHA which was developed to confirm that that the mPCR's co-detection limit of 10(2) copy of synthetic template per millilitre was relevant for use in detecting virus from clinical samples. The mPCR-ELAHA was then applied to the screening of 174 patient specimens resulting in a specificity of 98% and a sensitivity of 93%. This preliminary study demonstrated that the mPCR-ELAHA was a complete approach to the detection of herpesviruses from a range of clinical samples and disease states.
authors.affiliation: Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Australia. ian.mackay@mailbox.uq.edu.au
authors.forename: Ian M
authors.initials: IM
authors.lastname: Mackay
authors.forename: Tim
authors.initials: T
authors.lastname: Gardam
authors.forename: Katherine E
authors.initials: KE
authors.lastname: Arden
authors.forename: Suzi
authors.initials: S
authors.lastname: McHardy
authors.forename: David M
authors.initials: DM
authors.lastname: Whiley
authors.forename: Erin
authors.initials: E
authors.lastname: Crisante
authors.forename: Theo P
authors.initials: TP
authors.lastname: Sloots
mesh_terms: D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006564:Herpesviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006566:Herpesviridae Infections / Q000821:virology; D006801:Humans; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase