Laboratory tests
Human Milk Analyzer, Virus/Bacer
CMV culture and PCR in urine and HM: CMV culture and PCR were checked in urine samples within 7 days and at 4, 8, and 12 weeks. CMV culture and PCR in HM samples in each group after preparation were checked within 7 days and at 3, 6, 9, and 12 weeks.Quantification of CMV PCR: Paired samples of fresh-frozen, frozen-low temperature pasturized HM, frozen-high temperature pasturized HM were sent for CMV PCR quantitative analysis. CMV DNA load (copies/mL) was measured using quantitative PCR assay. CMV DNA extraction was performed using commercially available QIAsymphony Virus/Bacer Kits (QIAGEN, Hilden, Germany). CMV PCR quantitative test was performed using the Artus® R CMV PCR test (QIAGEN) in Rotor-gen (QIAGEN).CMV glycoprotein B (gB) PCR for genotyping: Genetic analysis of viral mutations was performed in CMV-infected infants. When entering the CMV strain, the gB gene was amplified using the following initiator: 5’’-CAA GAR GTG AAC ATG TCC GA-3’, 5’’-GTC ACG CAG CTG GCC AG-3’. Primary PCR amplification products were double-PCR (nested PCR) with 5′′-TGG AAC TGG AAC GTT TGG C-3′, 5′′-GAA ACG CGC GGC AAT CGG-3′. The PCR conditions were described by Binder et al. [CMV strain typing: The CMV gB genotype was sorted by the ClustalW program using the AD 169 strain (GenBank Accession No. FJ527563), Toledo strain (GU937742), Towne strain (FJ616285), and MEGA 7 (Arizona State University, Arizona, USA). Phylogenetic analysis was performed by the joining method.Macronutrient changes in HM: Nutritional components in HM in each group after preparations were measured using the Miris® Human Milk Analyzer (Miris, Uppsala, Sweden).