Vol. 64, No.

11

JOURNAL OF VIROLOGY, Nov. 1990, p. 5290-5294
0022-538X/90/115290-05$02.00/0
Copyright C 1990, American Society for Microbiology

Detection of Human T-Cell Leukemia Virus Type I (HTLV-I)
Provirus in an Infected Cell Line and in Peripheral Mononuclear
Cells of Blood Donors by the Nested Double Polymerase Chain
Reaction Method: Comparison with HTLV-I Antibody Tests
CHIEKO MATSUMOTO,* SHIGEKI MITSUNAGA, TAKASHI OGUCHI, YOSHITADA MITOMI,
TOORU SHIMADA, AKIKO ICHIKAWA, JUNNOSUKE WATANABE, AND KUSUYA NISHIOKA
The Japanese Red Cross Central Blood Center, Hiroo 4-1-31, Shibuya-ku, Tokyo 150, Japan
Received 30 May 1990/Accepted 27 July 1990

Human T-cell leukemia virus type I (HTLV-I) provirus DNA from the cultured cell line HUT 102 and from
peripheral mononuclear cells (PBMC) of anti-HTLV-I antibody-positive Japanese blood donors was detected
by the nested double polymerase chain reaction (PCR) method. This procedure consists of a first amplification
and a second amplification with the products of the first amplification and primers interior to the first primers.
Using this method, we demonstrated that it is possible to detect single-template DNA. Polyacrylamide gel
electrophoresis of the nested double PCR products, with our primers, revealed three bands with excess
amounts of template DNA, two bands with moderate amounts, and a single band with limited amounts. The
amount of provirus in PBMC was roughly estimated from the results of the nested double PCR. Particle
agglutination (PA) assays and indirect immunofluorescence testing (IF) with mixed MT-2 cells and Molt-4 cells
as targets to detect anti-HTLV-I antibody were performed, and the results were compared with those of the
nested double PCR of the pX region. None of the 101 PA-negative samples were positive in either the IF or PCR
test. Of the 155 samples that were antibody positive by the PA assay, 57 were positive by both PCR and IF.
Furthermore, the results of the IF and PCR tests coincided completely. It was therefore concluded that the IF
method is most appropriate for confirmation of the PA assay currently used in most diagnostic laboratories and
blood centers.

The results of PCR detection of the HTLV-I provirus in
peripheral mononuclear cells (PBMC) obtained from donated blood are compared with the results of antibody
testing of sera by the PA and IF methods in this report.

To prevent posttransfusion infection by human T-cell
leukemia virus type I (HTLV-I), anti-HTLV-I antibody has
been used since 1986 to screen donated blood in all Japanese
Red Cross blood centers in a particle agglutination (PA)
assay. However, some samples were judged to be antibody
negative by indirect immunofluorescence testing (IF) in spite
of a positive PA assay (11, 29), particularly low-titer sera.
Therefore, it was necessary to find some means of determining the presence of HTLV-I in such sera. Accordingly, we
attempted to detect HTLV-I provirus DNA by the polymerase chain reaction (PCR) directly.
PCR was first reported by Mullis et al. (8) and Saiki et al.
(23), and since then many modifications and applications
have been described (2, 6, 15, 25). This method has been
used for detection of viral genomes of human immunodeficiency virus (20), cytomegalovirus (8), hepatitis B virus (13),
and HTLV-I (1, 4, 22).
We adopted the nested double PCR method, in which two
amplifications are used under nonradioactive conditions (18,
19). We began by amplifying a DNA sample with Thermus
aquaticus polymerase and one pair of primers. Next, a
portion of the products was amplified again with another pair
of primers that were located inside the first pair. After the
second amplification, almost all nonspecific background
observed at the first amplification disappeared, and the
desired product was confirmed by using the inner primers.
Finally, the amplified products, even if they had originated
from a very small number of templates, were visualized by
polyacrylamide gel electrophoresis (PAGE) stained with
ethidium bromide.

MATERIALS AND METHODS
Synthesis of oligonucleotides. Oligonucleotides were synthesized on an Applied Biosystems 381A DNA synthesizer
by the phosphoramidate method and purified with oligonucleotide purification cartridges (Applied Biosystems Inc.).
Source and isolation of DNA. DNA from two human T-cell
lines or PBMC was isolated by the phenol-chloroform
method. One of the cell lines, CEM, was HTLV-I noninfected, and the other, HUT 102, was HTLV-I infected (21).
PBMC were obtained from voluntary blood donors in our
blood center.
Nested double PCR. The pX region of the HTLV-I genome
was amplified as shown in Fig. 1 by using DNA sequence
information from Seiki et al. (24). The first amplification was
carried out with primers 1 (5'-AGGGTTTGGACAG
AGTCTT-3') and 2 (5'-AAGGACCTTGAGGGTCTTAG3'). The second amplification was carried out with primers 3
(5'-CTTTTCGGATACCCAGTCTAC-3') and 4 (5'-GGTTC
TCTGGGTGGGGAAGGAG-3') and 10 ,ul of the first-amplification products as template DNA. All reactions were
performed in a volume of 100 ,ul containing 50 pmol of each
primer, 50 mM Tris hydrochloride (pH 8.8), 10 mM MgCl2,
10 mM (NH4)2SO4, template DNA, 2.5 U of T. aquaticus
polymerase (Perkin-Elmer Cetus), and 1.5 mM each dATP,
dCTP, dGTP, and dTTP. Reactions were carried out for 30
cycles at an annealing temperature of 60°C for 1 min, a
polymerization temperature of 72°C for 2 min, and a heat

* Corresponding author.
5290

The first amplification was carried out with primers 1 and 2.2. 3. A band corresponding to 256 bp was obtained with 130 pg of HUT 102 cell DNA. (ii) IF test. The ratio of MT-2 cells to Molt-4 cells was 1:3. 5 pg. PAGE was performed in Tris-borate buffer (pH 8. The products of repeated PCR with the same primers. expected to be 256 bp in length. used as a noninfected human T-cell line. PAGE patterns of the product of the first 30 cycles of PCR with primers 1 and 2. (a) HUT 102 cell and CEM cell DNAs were amplified for 30 cycles with primers 1 and 2 (single PCR). Detection of anti-HTLV-I antibody. When only MT-2 cells. A portion of the amplified product was amplified again with primers 3 and 4. Portions of these were amplified for a further 30 cycles with the same primers 1 and 2 (b) or with the inner-position primers 3 and 4 (c). 3.. A Serodia-ATLA kit (Fujirebio Inc.5 x 101 pg. Mixtures of carefully maintained MT-2 cells. were fixed with cold acetone and used as antibody target cells. The b o(Repeated fPCHj Sinqle PCR) a - bp 4.Pll. 1990 I NESTED DOUBLE PCR TO DETECT HTLV-I PROVIRUS 73312 7567 *a a a - primer 1 _ - -job. fluorescein isothiocyanate-conjugated rabbit anti-human immunoglobulin G was added.0). Incubation was continued for 30 min at 37°C. with mixed targets of HTLV-I-infected and noninfected cells. 4 x 102 pg. 1. primer 2 (731 2-7330) ner 3 :3 ~qwl L (7548-7567) prirrner 3= -hprimer 4 (733 1-7 '351). Amplification of the HTLV-I pX region by the nested double PCR method. i. 1.5 x 101 pg. (i) PA assay. 1. After completion of PAGE. Fixed cells were incubated with serum samples for 30 min at 37°C.- A - 56I6 25-237- .3 x 102 pg.ug of CEM cell DNA plus 0. and Molt-4 cells..3 x 104 pg. in the second amplification are shown in Fig.VOL. 5291 F a ii 1 1I0 l . 64. followed by washing with phosphate-buffered saline and water. PAGE of PCR products. 2b.6 pg of HUT 102 cell DNA (lanes 1 to 11. A final serum dilution of 1:16 or higher that caused agglutination of the antigen-coated particle was considered positive. pH 7. are shown in Fig. this result was judged to be IF positive. the gel was stained with ethidium bromide and photographed under UV transillumination. (7) with some modifications. were stained. I. this was judged to be nonspecific. 1 .e. 2a. IF was performed by the method of Hinuma et _ 256 al.2 x 103 pg. . an HTLV-I-infected T-cell line derived from cord blood cells which were cocultured with T-cells bearing adult T-cell leukemia (17). 4. 1. one-fourth of the cell population.ul of loading buffer and subjected to PAGE on 5% polyacrylamide gels. A PA assay was used for mass screening for anti-HTLV-I antibodies in sera from donors from all Red Cross blood centers in Japan. (7525-7546) 216 235 237 _ I_ ~ " - 256 FIG. respectively) were used as template DNA._ 7 4 . When both MT-2 cells and Molt-4 cells were stained. 1 and 2. A portion (4 IL) from each of the completed PCR reactions was mixed with 4 .1 x 104 pg. and after the cells were washed with phosphate-buffered saline. or 1. In each amplification. Analysis of the PCR products. The distance between primers is shown (in bases). Only 256-bp bands with several nonspecific background bands were observed with an endpoint of 15 pg of HUT 102 cell DNA. 1. denaturation temperature of 94°C for 1 min on a PerkinElmer Cetus DNA thermal cycler. _ . 2. RESULTS Detection of HTLV-I genome in HUT 102 cell DNA.7 x 103 pg. Stained cells were observed under a fluorescence microscope. Tokyo.l 2 1 6 /- bp FIG. Japan) was used. Nonspecific background was observed in the upper region. which was nine times more sensitive than that obtained with single PCR.r 1 256 bp c (Nested Double 1 2 3 4 5 2 PCOR .

The nested double PCR DNA samples were titrated on the basis of the number of bands which appeared on PAGE gels (Table 2). MATSUMOTO ET AL. and provirus was detected in the same 57 samples without exception. 4).5292 J. 1 2 3 4 5 6 7 8 9 10 1 products of nested double PCR with the inner-position primers 3 and 4 (216 bp in length) are shown in Fig. and 256 bp) were obtained with more than 130 pg. and the results were compared. Of the 256 blood donors.000 pg of HUT 102 cell DNA contains 1. 235 to 237.5 x 105 cells) from each blood donor and used for the nested double PCR test (some of them are shown in Fig.5 x 105 PBMC from 57 individuals who were anti-HTLV-I antibody positive by IF. and three bands (216. and 256 bp). 2c.200 to 1.ug of cellular DNA obtained from PBMC of five blood donors (lanes 6 to 10) was subjected to amplification. Detection of HTLV-I provirus in HUT 102 cells and PBMC from blood donors by the nested double PCR method. VIROL. showing a sensitivity more than 80 times higher than single PCR. Four of eight serum samples with a titer of 128 by PA were both IF and provirus positive.5 x 105 PBMC and only a minor proportion TABLE 1. Almost all serum samples with antibody titers lower than 64 in the PA assay were negative for antibody by IF. As shown in Fig.5 pg of HUT 102 cell DNA as a starting template are shown in Fig.5 pg of HUT 102 cell DNA contains one or no molecules of the pX region DNA. Fifty-seven samples were antibody positive by IF. and with more than 400 pg of cell DNA. HUT 102 cell and CEM cell DNAs were first amplified for 30 cycles with primers 1 and 2. a pair of interior primers. Products of nested double PCR were observed by PAGE (5% polyacrylamide) CEM cell DNA (1 .ug) plus HUT 102 cell DNA at 4. DNA (1 . of samples (n = 256) IF (antibody) PCR (provirus) 101 55 29 8 8 14 10 11 12 2 6 0 1 1 0 4 12 10 10 11 2 6 0 1 1 0 4 12 10 10 11 2 6 1. 2c.192 "The HTLV-I pX region was amplified by nested double PCR with T. 40. it was estimated that one. and more than 200 proviruses.5 pg of HUT 102 cell DNA showed distinct single bands of 216 bp without nonspecific background. and 48 showed three bands (216. Detection in blood donor sera of HTLV-I provirus DNA in PBMC by nested double PCR and anti-HTLV-I antibody by the PA and IF methods was carried out in a blind test. It was calculated that 0. three bands (216. Four of 10 PCR tests carried out with 0. respectively. 101 were negative for anti-HTLV-I antibody by both PA and IF. Estimation of the number of HTLV-I proviruses. and 1 . 22 to 70. The results of PCR and IF for the other 155 samples which were positive by PA testing are summarized in Table 1 and are classified by PA titers. 4. and no HTLV-I provirus DNA was detected. of samples positive by: No. and three bands came from 2 to 7.024 2. 7 showed two bands (216 bp and 235 to 237 bp). the nested double PCR for detection of provirus DNA in PBMC.6 pg of HUT 102 cell DNA. were used and amplified 30 times. It was calculated that 1. two. two bands (216 and 235 to 237 bp) were obtained with 15 to 45 pg. HUT 102 cell DNA (0. 2 showed a single band (216 bp). and provirus was not detected in the PBMC of these individuals. 400. a single band (216 bp) was obtained with 1. or 0 pg (lanes 1 to 5. the majority (about 84%) of HTLV-I-antibody-positive samples contained more than 200 HTLV-I proviruses in 1. and 150 human diploid cells contain 1 ng of genomic DNA (assuming a haploid genome size of 3 x 109 bp). Comparison of nested double PCR for detection of the HTLV-I genome and anti-HTLV-I antibody testing in donated blood. Therefore.048 4. 2. In the second step. .ug) was extracted from PBMC (1. Samples tested by IF and PCR were identical. aquanticus polymerase. 2 bP FIG. 3. Fifty-one cases with a titer of 256 and higher were positive in both IF for serum antibody detection and the nested double PCR test for HTLV-I provirus detection. Two subjects in this group were positive for antibody by IF and for provirus by the nested double PCR. One HUT 102 cell contains 8 to 10 copies of the pX region of HTLV-I (26).5 pg) and 1 jig of CEM cell DNA were used as template DNA in every lane.6 to 5 pg of HUT 102 cell DNA. and 256 bp in length) were observed. Of these. 235 to 237. two bands (216 and 235 to 237 bp in length) were observed. 3.096 -8. With 15 to 130 pg of HUT 102 cell DNA. and it was thereby determined that one template DNA can be detected by the nested double PCR. 3 and 4. respectively) were used as control templates. The nested double PCR for detection of provirus was carried out with 1 Fg each of DNA prepared from approximately 1. Therefore. A distinct 216-bp band without background was observed with 1. The results of nested double PCR with 0. Comparison of HTLV-I antibody and provirus detection by PCR and anti-HTLV-I antibody testing by PA and IF' PA titer (final serum dilution) ---8 16 32 64 128 256 512 No.500 molecules of pX region DNA.000. PAGE of the nested double PCR products. The results of IF for HTLV-I antibody coincided completely with the results of 3 4 5 6 7 9 0o 2 16- 216 bp FIG. 235 to 237.

especially when using samples with low antibody titers. in the second amplification. Therefore.096 -8. showing that the PA assay is no more sensitive than IF tests. and three bands would be detected. 10). The 216-bp band corresponds to the region between primers 3 and 4. Although the PA test has been widely used in blood screening as one of the simplest. Therefore. was observed. blood donors can be notified about their status with respect to HTLV-I infection. when the presence of this virus can be determined with certainty. 27) and detection of the HTLV-I genome (14. and they appeared as a single band on PAGE.VOL. speediest.200 DNA of ~~~~~~~~ ~~~~~~~~~~cell ~ pX regionb (pg) a The PA titer represents the final serum dilution. Therefore. 237-base plus-strand products coupled with 216base minus-strand products and 235-base minus-strand products coupled with 216-base plus-strand products might be produced. large amounts of 256-bp DNA were produced in the first amplification.6-5 15-45 -130 2-7 22-70 . at present. With excess DNA from 200 provirus genomes or more. 1). The results were all negative against Molt-4 cells. this was judged to be IF positive as described above. false-positive results can be a problem. which regulate replication of HTLV-I at the transcriptional and posttranscriptional levels. in addition to 216-bp products and 235. DISCUSSION The method described here as nested double PCR was used for detection of HTLV-I provirus in HUT 102 cells and found to be extremely sensitive. Distinguishing between the 235-base product and 237-base product was difficult. and costly as routine confirmation tests. 64. a third band of 256 bases should appear. but their quantities were not high enough to be detected by PAGE. 28) have been considered. only the 216-bp plus. two bands (216 bp and partially single-stranded 235 and 237 bases) were observed on PAGE. of samples with PA 128 256 512 titer": 1. at present. It overlaps the coding region of p40far and p27rex. 12). Therefore. this was judged to be nonspecific. which were added in the nested second amplification (Fig. Accordingly. 1990 NESTED DOUBLE PCR TO DETECT HTLV-1 PROVIRUS 5293 TABLE 2.048 10 1 10 4. respectively. both cocultivation and the PCR test are time-consuming. In this amplification. We assume that the first amplification with a small amount of template DNA produced only a limited amount of 256-bp-length products. 2. complicated. only one band. Wit.024 2. Therefore. moderate amounts (22 to 70 genomes) of template provirus DNA. (3. respectively (3. 216 and 235 to 237 bp in length. for HTLV-I antibody testing coincided completely with those of the nested double PCR test regardless of the antibody titer determined by PA. and these were annealed with only primer 3 or 4 in the second amplification. The amounts of both the 237-base plus-strand and 235-base minus-strand products were expected to be 30-fold greater than the amount of template 256-base products used in the second 30-cycle amplification. moderate aniounts of 256-base products (corresponding to the distance between primers 1 and 2) might be produced in the first amplification. IF tests with cold acetone-fixed Molt-4 cells as the target were carried out with 100 serum samples which were proven to be positive for HTLV-I antibody by both PA and IF against HTLV-Iinfected MT-2 cells. b Estimated from data in Fig. the region is considered indispensable for HTLV-I provirus multiplication in vivo. As described in this report.5%) contained fewer than 10 proviruses in the same number of PBMC. A large number of 237-base plus-strand/216-base minus-strand and 235-base minus-strand/216-base plus-strand products might have been produced. We amplified the portion of the HTLV-I provirus pX region at positions 7312 to 7567. 237-base plus strands would be easily coupled with 235-base minus strands and partially single-stranded 256-base products composed of 237-base plus and 235-base minus strands might be formed. mixtures of MT-2 cells and Molt-4 cells at a 1:3 ratio were prepared and used as antibody target cells for the routine confirmation test. and most specific tests with high sensitivity (8. and annealing of primer 4 to 256-base products produced 235-base minus-strand products. It is believed that the region we amplified is adequate for detecting HTLV-I provirus in PBMC of HTLV-I-infected individuals. were observed. two bands. Therefore.and 237-base products. The end titer of the PA test and IF test for five anti-HTLV-I antibody-positive serum samples did not show any difference between the two methods. In addition. 216 bp in length. For these purposes.and minus-strand products in the second amplification were observed by PAGE. 5. when Molt-4 cells were stained. tests for detecting the presence of HTLV-I in blood donors are urgently required to rule out the possibility of transfusing infected blood. sometimes it is difficult to distinguish them only by size. In addition. Therefore. as postulated above. 237-base plus-strand and 235-base minus-strand products might have been produced in amounts 30-fold greater than the template 256-base products. It can be used to detect a single H'rLV-I genome in cellular DNA with almost no nonspecific background. Therefore. However. With a limited amount (one to seven genomes) of template provirus DNA. serum samples giving low antibody titers on the PA assay and negative results for IF and PCR are considered false-positive samples. Number of bands in PAGE gels of nested double PCR products and antibody titer of HTLV-I in adult carriers No. As preliminary tests. Our nested double PCR test has now been demonstrated to be the most sensitive and specific test for detection of the HTLV-I genome in PBMC of donors. Under these conditions. in addition to the 216-base plus.and minus-strand products. and they annealed with only primer 3 or 4. cocultivation with HTLV-I-noninfected T cells (16.192 2 1 5 2 1 1 1 3 3 7 10 HUT 102 Estimated copies 1. When one-fourth of the larger cells were stained. Annealing of primer 3 to 256-base products of the first amplification produced 237base plus-strand products. the results of our IF method with carefully maintained MT-2 cells and Molt-4 cells as positive and negative targets. If so. the IF method described here is now considered the most . of bands bands 32 64 16 1 2 3 No. Although Molt-4 cells are smaller than MT-2 cells.

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