JOURNAL OF VIROLOGY, Nov. 1990, p. 5290-5294 Vol. 64, No. 110022-538X/90/115290-05$02.00/0Copyright 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 MononuclearCells of Blood Donors by the Nested Double Polymerase ChainReaction Method: Comparison with HTLV-I Antibody TestsCHIEKO 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 fromperipheral mononuclear cells (PBMC) of anti-HTLV-I antibody-positive Japanese blood donors was detectedby the nested double polymerase chain reaction (PCR) method. This procedure consists of a first amplificationand 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 gelelectrophoresis of the nested double PCR products, with our primers, revealed three bands with excessamounts of template DNA, two bands with moderate amounts, and a single band with limited amounts. Theamount of provirus in PBMC was roughly estimated from the results of the nested double PCR. Particleagglutination (PA) assays and indirect immunofluorescence testing (IF) with mixed MT-2 cells and Molt-4 cellsas targets to detect anti-HTLV-I antibody were performed, and the results were compared with those of thenested double PCR of the pX region. None of the 101 PA-negative samples were positive in either the IF or PCRtest. 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 IFmethod is most appropriate for confirmation of the PA assay currently used in most diagnostic laboratories andblood centers.

To prevent posttransfusion infection by human T-cellleukemia virus type I (HTLV-I), anti-HTLV-I antibody hasbeen used since 1986 to screen donated blood in all JapaneseRed Cross blood centers in a particle agglutination (PA)assay. However, some samples were judged to be antibodynegative by indirect immunofluorescence testing (IF) in spiteof a positive PA assay (11, 29), particularly low-titer sera.Therefore, it was necessary to find some means of determin-ing the presence of HTLV-I in such sera. Accordingly, weattempted to detect HTLV-I provirus DNA by the polymer-ase chain reaction (PCR) directly.PCR was first reported by Mullis et al. (8) and Saiki et al.

(23), and since then many modifications and applicationshave been described (2, 6, 15, 25). This method has beenused for detection of viral genomes of human immunodefi-ciency 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 Thermusaquaticus polymerase and one pair of primers. Next, aportion of the products was amplified again with another pairof primers that were located inside the first pair. After thesecond amplification, almost all nonspecific backgroundobserved at the first amplification disappeared, and thedesired product was confirmed by using the inner primers.Finally, the amplified products, even if they had originatedfrom a very small number of templates, were visualized bypolyacrylamide gel electrophoresis (PAGE) stained withethidium bromide.

* Corresponding author.

The results of PCR detection of the HTLV-I provirus inperipheral mononuclear cells (PBMC) obtained from do-nated blood are compared with the results of antibodytesting of sera by the PA and IF methods in this report.

MATERIALS AND METHODS

Synthesis of oligonucleotides. Oligonucleotides were syn-thesized on an Applied Biosystems 381A DNA synthesizerby the phosphoramidate method and purified with oligonu-cleotide purification cartridges (Applied Biosystems Inc.).

Source and isolation of DNA. DNA from two human T-celllines or PBMC was isolated by the phenol-chloroformmethod. One of the cell lines, CEM, was HTLV-I nonin-fected, and the other, HUT 102, was HTLV-I infected (21).PBMC were obtained from voluntary blood donors in ourblood center.

Nested double PCR. The pX region of the HTLV-I genomewas amplified as shown in Fig. 1 by using DNA sequenceinformation from Seiki et al. (24). The first amplification wascarried out with primers 1 (5'-AGGGTTTGGACAGAGTCTT-3') and 2 (5'-AAGGACCTTGAGGGTCTTAG-3'). The second amplification was carried out with primers 3(5'-CTTTTCGGATACCCAGTCTAC-3') and 4 (5'-GGTTCTCTGGGTGGGGAAGGAG-3') and 10 ,ul of the first-ampli-fication products as template DNA. All reactions wereperformed in a volume of 100 ,ul containing 50 pmol of eachprimer, 50 mM Tris hydrochloride (pH 8.8), 10 mM MgCl2,10 mM (NH4)2SO4, template DNA, 2.5 U of T. aquaticuspolymerase (Perkin-Elmer Cetus), and 1.5 mM each dATP,dCTP, dGTP, and dTTP. Reactions were carried out for 30cycles at an annealing temperature of 60°C for 1 min, apolymerization temperature of 72°C for 2 min, and a heat

5290

NESTED DOUBLE PCR TO DETECT HTLV-I PROVIRUS

73a a -

primer 1(731 2-7330)

prirr(733 1-7

312I 7567

_ *a -

-job,I. primer 2ner 3 :3~qwl L (7548-7567)ner 3= -h-

'351); primer 4(7525-7546)

_ 216 -235237 - "

I_ ~ 256

FIG. 1. Amplification of the HTLV-I pX region by the nesteddouble PCR method. The first amplification was carried out withprimers 1 and 2. A portion of the amplified product was amplifiedagain with primers 3 and 4. The distance between primers is shown(in bases).

denaturation temperature of 94°C for 1 min on a Perkin-Elmer Cetus DNA thermal cycler.

Analysis of the PCR products. A portion (4 IL) from each ofthe completed PCR reactions was mixed with 4 ,ul of loadingbuffer and subjected to PAGE on 5% polyacrylamide gels.PAGE was performed in Tris-borate buffer (pH 8.0). Aftercompletion of PAGE, the gel was stained with ethidiumbromide and photographed under UV transillumination.

Detection of anti-HTLV-I antibody. (i) PA assay. A PAassay was used for mass screening for anti-HTLV-I antibod-ies in sera from donors from all Red Cross blood centers inJapan. A Serodia-ATLA kit (Fujirebio Inc., Tokyo, Japan)was used. A final serum dilution of 1:16 or higher that causedagglutination of the antigen-coated particle was consideredpositive.

(ii) IF test. IF was performed by the method of Hinuma et

a Sinqle PCR)4_ ; F a 11I0ii

256 -bp

al. (7) with some modifications, with mixed targets ofHTLV-I-infected and noninfected cells. Mixtures of care-fully maintained MT-2 cells, an HTLV-I-infected T-cell linederived from cord blood cells which were cocultured withT-cells bearing adult T-cell leukemia (17), and Molt-4 cells,used as a noninfected human T-cell line, were fixed with coldacetone and used as antibody target cells. The ratio of MT-2cells to Molt-4 cells was 1:3. Fixed cells were incubated withserum samples for 30 min at 37°C, and after the cells werewashed with phosphate-buffered saline, pH 7.2, fluoresceinisothiocyanate-conjugated rabbit anti-human immunoglobu-lin G was added. Incubation was continued for 30 min at37°C, followed by washing with phosphate-buffered salineand water. Stained cells were observed under a fluorescencemicroscope. When only MT-2 cells, i.e., one-fourth of thecell population, were stained, this result was judged to be IFpositive. When both MT-2 cells and Molt-4 cells werestained, this was judged to be nonspecific.

RESULTSDetection of HTLV-I genome in HUT 102 cell DNA. PAGE

patterns of the product of the first 30 cycles of PCR withprimers 1 and 2, expected to be 256 bp in length, are shownin Fig. 2a. A band corresponding to 256 bp was obtained with130 pg of HUT 102 cell DNA. Nonspecific background wasobserved in the upper region. The products of repeated PCRwith the same primers, 1 and 2, in the second amplificationare shown in Fig. 2b. Only 256-bp bands with severalnonspecific background bands were observed with an end-point of 15 pg of HUT 102 cell DNA, which was nine timesmore sensitive than that obtained with single PCR. The

b o(Repeated fPCHjl . _ ;_ 7 4 ;r 1

256 -bp

c (Nested Double PCOR1 2 3 54 ;- A -

256I-25-237-

6

2 1 6 /-

bp

FIG. 2. PAGE of PCR products. (a) HUT 102 cell and CEM cell DNAs were amplified for 30 cycles with primers 1 and 2 (single PCR).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). Ineach amplification, 1 ,ug of CEM cell DNA plus 0, 3.3 x 104 pg, 1.1 x 104 pg, 3.7 x 103 pg, 1.2 x 103 pg, 4 x 102 pg, 1.3 x 102 pg, 4.5 x 101pg, 1.5 x 101 pg, 5 pg, or 1.6 pg of HUT 102 cell DNA (lanes 1 to 11, respectively) were used as template DNA.

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5292 MATSUMOTO ET AL.

1 2 3 4 5 6 7 8 9 10

2 1 6 -

bp

FIG. 3. PAGE of the nested double PCR products. HUT 102 celland CEM cell DNAs were first amplified for 30 cycles with primers1 and 2. In the second step, a pair of interior primers, 3 and 4, were

used and amplified 30 times. HUT 102 cell DNA (0.5 pg) and 1 jig ofCEM cell DNA were used as template DNA in every lane.

products of nested double PCR with the inner-positionprimers 3 and 4 (216 bp in length) are shown in Fig. 2c. Adistinct 216-bp band without background was observed with1.6 pg of HUT 102 cell DNA, showing a sensitivity morethan 80 times higher than single PCR. With 15 to 130 pg ofHUT 102 cell DNA, two bands (216 and 235 to 237 bp inlength) were observed, and with more than 400 pg of cellDNA, three bands (216, 235 to 237, and 256 bp in length)were observed.The results of nested double PCR with 0.5 pg of HUT 102

cell DNA as a starting template are shown in Fig. 3. Four of10 PCR tests carried out with 0.5 pg of HUT 102 cell DNAshowed distinct single bands of 216 bp without nonspecificbackground. One HUT 102 cell contains 8 to 10 copies of thepX region of HTLV-I (26), and 150 human diploid cellscontain 1 ng of genomic DNA (assuming a haploid genomesize of 3 x 109 bp). It was calculated that 0.5 pg of HUT 102cell DNA contains one or no molecules of the pX regionDNA, and it was thereby determined that one template DNAcan be detected by the nested double PCR.Comparison of nested double PCR for detection of the

HTLV-I genome and anti-HTLV-I antibody testing in donatedblood. Detection in blood donor sera of HTLV-I provirusDNA in PBMC by nested double PCR and anti-HTLV-Iantibody by the PA and IF methods was carried out in a

blind test, and the results were compared. DNA (1 ,ug) wasextracted from PBMC (1.5 x 105 cells) from each blooddonor and used for the nested double PCR test (some ofthem are shown in Fig. 4). Of the 256 blood donors, 101 were

negative for anti-HTLV-I antibody by both PA and IF, andno HTLV-I provirus DNA was detected.The results of PCR and IF for the other 155 samples which

were positive by PA testing are summarized in Table 1 andare classified by PA titers. Fifty-seven samples were anti-body positive by IF, and provirus was detected in the same57 samples without exception. Almost all serum sampleswith antibody titers lower than 64 in the PA assay were

negative for antibody by IF, and provirus was not detectedin the PBMC of these individuals. Two subjects in this groupwere positive for antibody by IF and for provirus by thenested double PCR. Four of eight serum samples with a titerof 128 by PA were both IF and provirus positive. Fifty-onecases with a titer of 256 and higher were positive in both IFfor serum antibody detection and the nested double PCR testfor HTLV-I provirus detection. The results of IF forHTLV-I antibody coincided completely with the results of

1 2 3 4 5 6 7 9 0o

2 16-bP

FIG. 4. Detection of HTLV-I provirus in HUT 102 cells andPBMC from blood donors by the nested double PCR method.Products of nested double PCR were observed by PAGE (5%polyacrylamide) CEM cell DNA (1 ,ug) plus HUT 102 cell DNA at 4,40, 400, 2,000, or 0 pg (lanes 1 to 5, respectively) were used ascontrol templates, and 1 ,ug of cellular DNA obtained from PBMC offive blood donors (lanes 6 to 10) was subjected to amplification.

the nested double PCR for detection of provirus DNA inPBMC.

Estimation of the number of HTLV-I proviruses. Thenested double PCR DNA samples were titrated on the basisof the number of bands which appeared on PAGE gels (Table2). As shown in Fig. 2c, a single band (216 bp) was obtainedwith 1.6 to 5 pg of HUT 102 cell DNA, two bands (216 and235 to 237 bp) were obtained with 15 to 45 pg, and threebands (216, 235 to 237, and 256 bp) were obtained with morethan 130 pg. It was calculated that 1,000 pg of HUT 102 cellDNA contains 1,200 to 1,500 molecules of pX region DNA.Therefore, it was estimated that one, two, and three bandscame from 2 to 7, 22 to 70, and more than 200 proviruses,respectively.The nested double PCR for detection of provirus was

carried out with 1 Fg each of DNA prepared from approxi-mately 1.5 x 105 PBMC from 57 individuals who wereanti-HTLV-I antibody positive by IF. Of these, 2 showed asingle band (216 bp), 7 showed two bands (216 bp and 235 to237 bp), and 48 showed three bands (216, 235 to 237, and 256bp). Therefore, the majority (about 84%) of HTLV-I-anti-body-positive samples contained more than 200 HTLV-Iproviruses in 1.5 x 105 PBMC and only a minor proportion

TABLE 1. Comparison of HTLV-I antibody and provirusdetection by PCR and anti-HTLV-I antibody testing by

PA and IF'

PA titer No. of samples No. of samples positive by:(final serum dilution) (n = 256) IF (antibody) PCR (provirus)

---8 101 0 016 55 1 132 29 1 164 8 0 0128 8 4 4256 14 12 12512 10 10 10

1,024 11 10 102,048 12 11 114,096 2 2 2-8,192 6 6 6

"The HTLV-I pX region was amplified by nested double PCR with T.aquanticus polymerase. Samples tested by IF and PCR were identical.

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NESTED DOUBLE PCR TO DETECT HTLV-1 PROVIRUS

TABLE 2. Number of bands in PAGE gels of nested double PCR products and antibody titer of HTLV-I in adult carriers

No. of No. of samples with PA titer": HUT 102 Estimated copiesbands ~~~~~~~~ ~~~~~~~~~~~cellDNA ofbands 16 32 64 128 256 512 1,024 2,048 4,096 -8,192 (pg) pX regionb1 2 1.6-5 2-72 1 1 3 1 1 15-45 22-703 1 3 7 10 10 10 2 5 -130 .200

a The PA titer represents the final serum dilution.b Estimated from data in Fig. 2.

(3.5%) contained fewer than 10 proviruses in the samenumber of PBMC.

DISCUSSIONThe method described here as nested double PCR was

used for detection of HTLV-I provirus in HUT 102 cells andfound to be extremely sensitive. It can be used to detect asingle H'rLV-I genome in cellular DNA with almost nononspecific background.Wit, moderate amounts (22 to 70 genomes) of template

provirus DNA, two bands, 216 and 235 to 237 bp in length,were observed. The 216-bp band corresponds to the regionbetween primers 3 and 4, which were added in the nestedsecond amplification (Fig. 1). In this amplification, moderateaniounts of 256-base products (corresponding to the distancebetween primers 1 and 2) might be produced in the firstamplification, and these were annealed with only primer 3 or4 in the second amplification. Annealing of primer 3 to256-base products of the first amplification produced 237-base plus-strand products, and annealing of primer 4 to256-base products produced 235-base minus-strand prod-ucts. The amounts of both the 237-base plus-strand and235-base minus-strand products were expected to be 30-foldgreater than the amount of template 256-base products usedin the second 30-cycle amplification. Under these condi-tions, in addition to the 216-base plus- and minus-strandproducts, 237-base plus-strand products coupled with 216-base minus-strand products and 235-base minus-strand prod-ucts coupled with 216-base plus-strand products might beproduced. Distinguishing between the 235-base product and237-base product was difficult, and they appeared as a singleband on PAGE. Therefore, two bands (216 bp and partiallysingle-stranded 235 and 237 bases) were observed on PAGE.With a limited amount (one to seven genomes) of template

provirus DNA, only one band, 216 bp in length, was ob-served. We assume that the first amplification with a smallamount of template DNA produced only a limited amount of256-bp-length products. In addition, in the second amplifi-cation, 237-base plus-strand and 235-base minus-strandproducts might have been produced in amounts 30-foldgreater than the template 256-base products, but their quan-tities were not high enough to be detected by PAGE.Accordingly, only the 216-bp plus- and minus-strand prod-ucts in the second amplification were observed by PAGE.With excess DNA from 200 provirus genomes or more,

large amounts of 256-bp DNA were produced in the firstamplification, and they annealed with only primer 3 or 4. Alarge number of 237-base plus-strand/216-base minus-strandand 235-base minus-strand/216-base plus-strand productsmight have been produced, as postulated above. If so,237-base plus strands would be easily coupled with 235-baseminus strands and partially single-stranded 256-base prod-ucts composed of 237-base plus and 235-base minus strandsmight be formed. Therefore, in addition to 216-bp products

and 235- and 237-base products, a third band of 256 basesshould appear, and three bands would be detected.We amplified the portion of the HTLV-I provirus pX

region at positions 7312 to 7567. It overlaps the coding regionof p40far and p27rex, which regulate replication of HTLV-I atthe transcriptional and posttranscriptional levels, respec-tively (3, 5, 10). Therefore, the region is considered indis-pensable for HTLV-I provirus multiplication in vivo. It isbelieved that the region we amplified is adequate for detect-ing HTLV-I provirus in PBMC of HTLV-I-infected individ-uals.

Although the PA test has been widely used in bloodscreening as one of the simplest, speediest, and most specifictests with high sensitivity (8, 12), false-positive results canbe a problem, especially when using samples with lowantibody titers. Therefore, tests for detecting the presence ofHTLV-I in blood donors are urgently required to rule out thepossibility of transfusing infected blood. In addition, whenthe presence of this virus can be determined with certainty,blood donors can be notified about their status with respectto HTLV-I infection. For these purposes, cocultivation withHTLV-I-noninfected T cells (16, 27) and detection of theHTLV-I genome (14, 28) have been considered. Our nesteddouble PCR test has now been demonstrated to be the mostsensitive and specific test for detection of the HTLV-Igenome in PBMC of donors. However, at present, bothcocultivation and the PCR test are time-consuming, compli-cated, and costly as routine confirmation tests.As described in this report, the results of our IF method

with carefully maintained MT-2 cells and Molt-4 cells aspositive and negative targets, respectively, for HTLV-Iantibody testing coincided completely with those of thenested double PCR test regardless of the antibody titerdetermined by PA. As preliminary tests, IF tests with coldacetone-fixed Molt-4 cells as the target were carried out with100 serum samples which were proven to be positive forHTLV-I antibody by both PA and IF against HTLV-I-infected MT-2 cells. The results were all negative againstMolt-4 cells. Therefore, when Molt-4 cells were stained, thiswas judged to be nonspecific. Although Molt-4 cells aresmaller than MT-2 cells, sometimes it is difficult to distin-guish them only by size. Therefore, mixtures of MT-2 cellsand Molt-4 cells at a 1:3 ratio were prepared and used asantibody target cells for the routine confirmation test. Whenone-fourth of the larger cells were stained, this was judged tobe IF positive as described above. The end titer of the PAtest and IF test for five anti-HTLV-I antibody-positiveserum samples did not show any difference between the twomethods, showing that the PA assay is no more sensitivethan IF tests. Therefore, serum samples giving low antibodytiters on the PA assay and negative results for IF and PCRare considered false-positive samples. Therefore, at present,the IF method described here is now considered the most

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5294 MATSUMOTO ET AL.

appropriate confirmatory test for most diagnostic laborato-ries and blood centers.

ACKNOWLEDGMENTS

We thank T. Juji, K. Tokunaga, S. Kuwata, and E. Tokunaga foruseful suggestions on PCR and K. Nakamura for technical collabo-ration.

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