Effects of fixation and varying target length on the sensitivity of polymerase chain reaction for detection of human T-cell leukemia virus type I proviral DNA in formalin-fixed tissue sections.
In this study, the fixation condition most suitable for maintaining the sensitivity of the polymerase chain reaction (PCR) was investigated by using the alpha-tubulin gene sequence, and the PCR procedure most effective for detecting human T-cell leukemia virus type I (HTLV-I) proviral sequences in fixed, embedded tissues of adult T-cell leukemia patients was explored. First, the sensitivity of the PCR targeting a 286-bp alpha-tubulin sequence was studied in tissue sections fixed in several fixatives for various periods at 25 or 4 degrees C. For histological examination, fixation with 10% buffered formalin at a lower temperature for a shorter period was found to be preferable to retain the sensitivity. And the HTLV-I sequence was detected in only 7 of 18 specimens (38.9%) when the 374-bp sequence of the gag region was targeted, but the rate increased to 77.8% (14 of 18 specimens) when the length of the target sequence was reduced to 120 bp within the same region. Therefore, the one-round PCR targeting a shorter sequence is preferable for application of PCR to archival fixed tissue specimens, the fixation condition of which may not be ideal for DNA preservation.
Honma
M
M
Department of Neurological Sciences, Tohoku University School of Medicine, Sendai, Japan.
Ohara
Y
Y
Murayama
H
H
Sako
K
K
Iwasaki
Y
Y
D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D005404:Fixatives; D005557:Formaldehyde; D015750:Genes, gag; D015368:Human T-lymphotropic virus 1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000382:microbiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013696:Temperature; D014404:Tubulin / Q000235:genetics
D004279:DNA, Viral; D005404:Fixatives; D014404:Tubulin; D005557:Formaldehyde