title: Quantitative monitoring of HCMV DNAlactia in human milk by real time PCR assay: Implementation of internal control contributes to standardization and quality control.
abstract: For cytomegalovirus screening of breastfeeding mothers of preterm infants under risk, we present a rapid, quantitative real-time PCR protocol using the hybridization format of the viral gB target region. For quantification, we used an external gB fragment cloned into a vector system. For standardization, we created an internal control-plasmid by site-directed mutagenesis with an exchange of 9 nucleotides. Spiked with internal control, patient wildtype amplicons could be discriminated from internal controls by hybridization probes using two-channel fluorescence detection. Potential bias of formerly reported false nucleotide sequence data of gB-hybridization probes was excluded. Using this approach, we could demonstrate excellent analytical performance and high reproducibility of HCMV detection during lactation. This assay shows very good correlation with a commercial quantitative HCMV DNA PCR and may help to identify rapidly HCMV shedding mothers of very low birth weight preterm infants to prevent HCMV transmission. On the other hand, negative DNA amplification results allow feeding of milk samples of seropositive mothers to their preterm infants under risk (<30 weeks of gestational age, <1000g birth weight) during the onset and late stage of HCMV shedding during lactation.
authors: Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital of Tübingen, 72076 Tübingen, Germany; University Children's Hospital Tübingen, 72076 Tübingen, Germany.
authors: Steffen
authors: S
authors: Hartleif
authors: Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital of Tübingen, 72076 Tübingen, Germany.
authors: Katharina
authors: K
authors: Göhring
authors: University Children's Hospital Tübingen, 72076 Tübingen, Germany.
authors: Rangmar
authors: R
authors: Goelz
authors: Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital of Tübingen, 72076 Tübingen, Germany.
authors: Gerhard
authors: G
authors: Jahn
authors: Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital of Tübingen, 72076 Tübingen, Germany. Electronic address: klaus.hamprecht@med.uni-tuebingen.de.
authors: Klaus
authors: K
authors: Hamprecht
mesh_terms: D001942:Breast Feeding; D003587:Cytomegalovirus / Q000235:genetics* / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000821:virology; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D018445:Infectious Disease Transmission, Vertical / Q000517:prevention & control; D007774:Lactation; D008297:Male; D008895:Milk, Human / Q000821:virology*; D016297:Mutagenesis, Site-Directed; D011786:Quality Control; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D019562:Viral Load; D017201:Virus Shedding
chemical_list: D015342:DNA Probes; D004279:DNA, Viral
keywords: Breastfeeding; CMV; Hybridization format; Lactation; LightCycler; gB region