Quantitative monitoring of HCMV DNAlactia in human milk by real time PCR assay: Implementation of internal control contributes to standardization and quality control

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• 作者：Steffen Hartleif^a ; ^b ; Katharina Gö ; hring^a ; Rangmar Goelz^b ; Gerhard Jahn^a ; Klaus Hamprecht^a ; ^{klaus.hamprecht@med.uni-tuebingen.de" class="auth_mail" title="E-mail the corresponding author}
• 关键词：bp ; base pairs ; CACM ; Cobas Amplicor HCMV Monitor system ; DNA ; Deoxyribonucleic Acid ; gB ; Human Cytomegalovirus Glycoprotein B ; gB-F ; forward primer gB region ; gB-R ; reverse primer gB region ; gB_mut-F ; forward mismatch primer gB region ; gB_mut-R ; reverse mismatch primer gB region ; gB-Fl ; donor hybridization probe labeled with fluorescein ; gB-640 ; acceptor hybridization probe labeled LC red dye 640 ; gB-640 ; acceptor hybridization probe labeled LC red dye 705 ; HCMV ; human cytomegalovirus ; IC ; inte
• 刊名：Journal of Virological Methods
• 出版年：2016
• 出版时间：November 2016
• 年：2016
• 卷：237
• 期：Complete
• 页码：101-106
• 全文大小：1507 K

文摘

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, <1000 0;g birth weight) during the onset and late stage of HCMV shedding during lactation.