title: Development of multiplex PCRs for detection of common viral pathogens and agents of congenital infections.
abstract: Potential causes of congenital infection include Toxoplasma gondii and viruses such as cytomegalovirus (CMV), enterovirus, hepatitis C virus, herpes simplex virus types 1 and 2 (HSV-1 and -2), human herpesvirus types 6, 7, and 8, lymphocytic choriomeningitis virus, parvovirus, rubella virus, and varicella-zoster virus. Testing for each of these agents using nucleic acid tests is time consuming and the availability of clinical samples such as amniotic fluid or neonatal blood is often limited. The aim of this study was to develop multiplex PCRs (mPCRs) for detection of DNA and RNA agents in the investigation of congenital infection and an mPCR for the viruses most commonly requested in a diagnostic virology laboratory (CMV, Epstein-Barr virus, enterovirus, HSV-1, HSV-2, and varicella-zoster virus). The assays were assessed using known pathogen-positive tissues (cultures, placentae, plasma, and amniotic fluid) and limits of detection were determined for all the agents studied using serial dilutions of plasmid targets. Nested PCR was performed as the most sensitive assay currently available, and detection of the amplicons using hybridization to labeled probes and enzyme-linked immunosorbent assay detection was incorporated into three of the four assays. This allowed detection of 10 to 10(2) copies of each agent in the samples processed. In several patients, an unexpected infection was diagnosed, including a case of encephalitis where HSV was the initial clinical suspicion but CMV was detected. In the majority of these cases the alternative agent could be confirmed using reference culture, serology, or fluorescence methods and was of relevance to clinical care of the patient. The methods described here provide useful techniques for diagnosing congenital infections and a paradigm for assessment of new multiplex PCRs for use in the diagnostic laboratory.
authors.affiliation: Department of Microbiology, South Eastern Area Laboratory Service, Prince of Wales Hospital, New South Wales 2031, Australia.
authors.forename: C J
authors.initials: CJ
authors.lastname: McIver
authors.forename: C F H
authors.initials: CF
authors.lastname: Jacques
authors.forename: S S W
authors.initials: SS
authors.lastname: Chow
authors.forename: S C
authors.initials: SC
authors.lastname: Munro
authors.forename: G M
authors.initials: GM
authors.lastname: Scott
authors.forename: J A
authors.initials: JA
authors.lastname: Roberts
authors.forename: M E
authors.initials: ME
authors.lastname: Craig
authors.forename: W D
authors.initials: WD
authors.lastname: Rawlinson
mesh_terms: D000653:Amniotic Fluid / Q000821:virology; D001331:Automation; D001769:Blood / Q000821:virology; D004267:DNA Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004279:DNA, Viral / Q000032:analysis; D006801:Humans; D010920:Placenta / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012328:RNA Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D014776:Virus Cultivation; D014777:Virus Diseases / Q000151:congenital* / Q000175:diagnosis* / Q000821:virology
chemical_list: D004279:DNA, Viral