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Volume 20, Number 5—May 2014
Research

Bovine Leukemia Virus DNA in Human Breast Tissue

Gertrude Case BuehringComments to Author , Hua Min Shen, Hanne M. Jensen, K. Yeon Choi1, Dejun Sun, and Gerard Nuovo
Author affiliations: University of California, Berkeley, California, USA (G.C. Buehring, H.M. Shen, K.Y. Choi, D. Sun); University of California Davis Medical Center, Sacramento, California, USA (H.M. Jensen); Ohio State University Comprehensive Cancer Center, Columbus, Ohio, USA (G. Nuovo)

Main Article

Table 3

BLV primers and cycling conditions used for L-PCR, IS-PCR, and preparation of DNA for sequencing for detection of BLV in human breast tissue samples*

BLV
gene Primer pair sequences, 5′ → 3′† Location in bp‡ Nested PCR role Product length, bp L-PCR/IS-PCR§
Annealing temperature, °C Extension time, s
LTR F: TAGGAGCCGCCACCGC 23–38 Outer 329 57/53 22/120
R: GCGGTGGTCTCAGCCGA 352–336
F:AAACTGCAGCGTAAACCAGACAGAGACG 41–59 Inner 290 58/57 20/120
R: CACCCTCCAAACCGTGCTTG 331–312
gag (p24) F: AACACTACGACTTGCAATCC 1068–1087 Outer 385 54/53 28/120
R: GGTTCCTTAGGACTCCGTCG 1453–1434
F: ACCCTACTCCGGCTGACCTA 1097–1116 Inner 272 56/56 24/120
R: CTTGGACGATGGTGGACCAA 1369–1350
pol F: TAGCCTACGTACATCTAACC 3238–3257 Outer 232 52/53 22/120
R: AATCCAATTGTCTAGAGAGG 3470–3451
F: GGTCCACCCTGGTACTCTTC 3265–3284 Inner 157 57/56 18/120
R: TATGGGCTTGGCATACGAGC 3422–3403
env F: TGATTGCGAGCCCCGATG 5144–5160 Outer 264 55/53 24/120
R: TCTGACAGAGGGAACCCAGT 5408–5389
F: TGATTGCGAGCCCCGATG 5144–5160 Inner 230 55/56 22/120
R: GGAAAGTCGGGTTGAGGG 5374–5357
tax F: CTTCGGGATCCATTACCTGA 7197–7216 Outer 373 55/55 26/120
R: GCTCGAAGGGGGAAAGTGAA 7570–7551
F: ATGTCACCATCGATGCCTGG 7310–7329 Inner 1 113 55/53 15/120
R: CATCGGCGGTCCAGTTGATA 7423–7404
F: GGCCCCACTCTCTACATGC 7265–7283 Inner 2
(sequencing) 206 56 22
R: AGACATGCAGTCGAGGGAAC 7471–7452

*BLV, bovine leukemia virus; L-PCR, nested liquid-phase PCR; IS-PCR, nested in situ PCR; LTR, long terminal repeat (promoter region); F, forward primer; R, reverse primer; gag, group-specific antigen (capsid region); pol, polymerase (reverse transcription); env, envelope; tax, trans-activating region of the X gene.
†Reverse sequences are reversed and complementary to the proviral reference sequence. Primers were synthesized by Operon Biotechnologies, Huntsville, AL, USA.
‡Location according to reference sequence, GenBank accession no. EF600696.
§For both rounds of nested liquid-phase PCR, cycling conditions were as follows: 1 cycle at 95°C for 2 min; 30 cycles at 95°C for 30 s, XX°C for 30 s, 72°C for XX; and 1 cycle at 72°C for 5 min, where XX represents annealing temperature and extension times, respectively, provided for each primer pair. For both rounds of nested in situ PCR, conditions were as follows: 1 cycle at 92°C for 10 min, 1 cycle at 91°C for 2 min, XX°C for 1.5 min; then 30 cycles of 91°C for 30 s, XX°C for 1.5 min, 72°C for 2 min; and 1 cycle of 72°C for 10 min, where XX represents IS-PCR annealing temperatures indicated for each primer pair.

Main Article

1Current affiliation: Texas A&M Health Science Center College of Medicine, College Station, Texas, USA.

Page created: April 16, 2014
Page updated: April 16, 2014
Page reviewed: April 16, 2014
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