CMV Polimerase Chain Reaction
CMV DNA in the samples was detected by PCR method, using an adaptated
protocol from Kumazaki and colleagues (2002)4. Two
different CMV regions were amplified: part of the early gene (IE) and
the phosphoprotein 71 gene (pp71)4. Standard
precautions, such as negative controls (using water as a template) and
two positive controls, were taken in order to avoid sample
contamination. PCR reaction was composed by the following reagents: by 5
μL of DNA template, 1.5 mM MgCl 2, 0.5 mM primers, 0.2 mM dNTP, 1x
Buffer, 1 U Taq DNA polymerase enzyme and milique water. PCR assays were
performed in a thermocycler with the following cycling conditions:
initial denaturation step at 94 ° C for 4 minutes, followed by 35 cycles
under the following conditions: 94 ° C for 30 seconds, 64 ° C for 60
seconds, 72 ° C for 60 seconds and 72 ° C for 5 minutes, followed by
final extension at 10 ° C.
Positive samples were repeated at a new reaction with positive and
negative controls for confirmation. PCR products were assessed by
electrophoresis in a 2% agarose gel stained with Ethidium Bromide and
verified by UV illumination. The positive cord blood samples were
sequenced using an ABI Prism® BigDye™ Terminator Cycle Sequencing Ready
Reaction Kit (Applied Biosystems®, Foster City, CA, USA) in an automated
ABI 3130XL analyzer (Thermo Scientific, Waltham, MA, USA).