EBV, HCMV, HHV6, and HHV7 Screening in Bone Marrow Samples from Children with Acute Lymphoblastic Leukemia

Table 2

PCR cycling conditions and primers sequences.

Virus/endogenous gene	Type of PCR	Cycling conditions^(a)	Primers sequences (5′→3′)
EBV	Standard	95°C/40 sec, 57°C/1 min, 72°C/1.5 min ()	F CCATGTAAGCTTGCCTCGAG; R GCCTTAGATCTGGCTCTTTG [55]^(c)
EBV	Nested	95°C/20 sec, 57°C/30 sec, 72°C/45 sec ()	F CTTTGTCCAGATGTCAGGGG; R GCCTGAGCCTCTACTTTTGG^(b)
CMV	Standard	95°C/1 min, 55°C/1 min, 72°C/1 min ()	F AAGATGCGGTAGATGTCGTT; R CTGCGCTCTTCTTTTTCGAT^(b)
CMV	Nested	95°C/45 sec, 55°C/45 sec, 72°C/45 sec ()	F TTCTGACCCTGAACCGTAG; R CGACGAAGAACTCGTAACC^(b)
HHV6^(d)	Standard	95°C/1 min, 50°C/1 min, 72°C/1 min ()	F GTGCGCTATAAAATCGATAGC; R TGATTTCCGTTGTGTGTTTTCC^(b)
HHV6^(d)	Nested	95°C/30 sec, 56°C/30 sec, 72°C/30 sec ()	F GTCTCTTCGTATCCACGCG; R CGTTCCCGTCGAAGAAATC^(b)
HHV7	Standard	95°C/45 sec, 53°C/45 sec, 72°C/45 sec ()	F TTTTTACATTTGGCTTGCTTTTTG; R ATATTTCTGTACCTATCTTCCCAA [56]^(c)
HHV7	Nested	95°C/30 sec, 55°C/30 sec, 72°C/30 sec ()	F GAACGGTTTGCTTAGATTGC; R GCAGACCAAACTCCACAAATTC^(b)
-actin	Standard	95°C/1 min, 60°C/1 min, 72°C/1.5 min ()	F CCTAAGGCCAACCGTGAAAAG; R TCTTCATGGTGCTAGGAGCCA [57]^(c)

All amplification runs included an initial denaturation step at 95°C for 5 minutes and a final extension step at 72°C for 10 minutes. Annealing temperatures were optimized for every reaction.
^(b)These primers were designed in our laboratory using the Primer-BLAST program [25].
^(c)The specificity of the previously reported primers was corroborated using Primer-BLAST program [25].
^(d)HHV6 primers recognize both HHV6A and HHV6B subtypes.