Vol. 28, No. 6


0095-1137/90/061469-04$02.00/0 Copyright C 1990, American Society for Microbiology

Microplate Hybridization of Amplified Viral DNA Segment Department

SAKAE INOUYEl* AND RYO HONDO2 Institute of Public Health,' and Department of Pathology, Institute of Medical Science, University of Tokyo/ Shirokanedai 4-6-1, Minato-ku, Tokyo, 108, Japan

of Microbiology,

Received 19 October 1989/Accepted 12 February 1990

We have developed a simple hybridization method for a DNA segment which is amplified by the polymerase chain reaction: after heat denaturation, the amplified DNA segment with a length of more than 300 bases is adsorbed to microplate wells in the presence of 1.5 M NaCI or 0.5 M ammonium sulfate; the immobilized DNA is hybridized with a biotin-labeled DNA probe; then, the hybridization signal is detected by streptavidinconjugated P-galactosidase or peroxidase. This method has several advantages over the conventional dot blot hybridization method: (i) radioisotopes are not used, (ià) synthetic oligonucleotide for the probe is not needed, (iii) the time required for washing of the solid phase is greatly reduced, and (iv) the baking and prehybridization procedures are eliminated. By this method, we were able to detect viral genomes in vesicle specimens from patients infected with varicella-zoster virus.

The recent advent of the revolutionary method for in vitro DNA amplification, the polymerase chain reaction (PCR) (11, 15), has profound implications for the field of diagnostic virology. PCR has already been used for the direct etiologic diagnosis of many viral diseases (1, 2, 6-10, 13, 14, 16). The PCR-amplified DNA is usually identified by agarose gel electrophoresis with ethidium bromide staining (10, 13) or by dot (slot) blot hybridization (1, 2, 6, 9, 14). Agarose gel electrophoresis is a simple method of identification when only a single DNA band is seen in electrophoresis. However, when multiple bands appear or large numbers of specimens are processed, dot blot hybridization is preferred. In the latter method, amplified DNA is usually immobilized on nylon or nitrocellulose membranes and then hybridized with radioisotope-labeled synthetic oligonucleotide probes. In this study, we developed a microplate hybridization technique for identification of the amplified DNA. In this method, hybridization is carried out much more easily than in the dot blot test and as easily as in enzyme-linked immunosorbent assay. Then we applied this technique to diagnosis of varicella-zoster virus (VZV) infection. PCR amplification of a DNA segment from a VZV, strain H-Si (4), was carried out as described by Saiki et al. (15). The target sequence for amplification was a 642-base-pair (bp) segment located in the EcoRI D fragment; this segment contains restriction enzyme sites (PstI, SphI, and BamHI) for digestion (5). The two oligonucleotide (21-mer) primers for PCR were 5'-TTCAGCCAACGTGCCAATAAA-3' (plus strand) and 5'-GACGCGCTTAACGGAAGTAAC-3' (minus strand). After 30 cycles of PCR, the reaction product was electrophoresed in an agarose gel; only a VZV-specific single band was obtained (R. Hondo, S. Inouye, S. Ohsawa, and S. Itoh, submitted for publication). The DNA band was extracted from the gel for the following experiments. Biotin-labeled probe was also produced by PCR. As the DNA template, 500 ng of the 642-bp amplified segment was used. In place of 200 ,uM thymidine triphosphate, 20 ,uM biotin-il (spacer)-dUTP (Enzo Biochem, New York, N.Y.) was employed. After 10 cycles of PCR, the reaction product was treated with chloroform to remove mineral oil, and then the water phase was mixed with ammonium acetate to a final *

concentration of 0.3 M and with 2.5 volumes of ethanol. After centrifugation at 10,000 x g for 10 min, the precipitated

DNA was dissolved in 2 mM NaCl-2 mM Tris hydrochloride (pH 7.8)-0.2 mM EDTA (NTE). Hybridization was carried out as follows. After heat denaturing at 100°C for 5 min, the 642-bp segment was twofold serially diluted in Maxisorp microplate wells (Nunc, Roskilde, Denmark) in 0.1 ml of sodium' phosphate (10 mM) and EDTA (10 mM) (pH 7.0). Each well contained various concentrations of different kinds of ions as mentioned below. After being sealed with adhesive tape, the plate was immersed in a water bath at 37°C for 2 h. The wells were washed three times with phosphate-buffered saline containing 0.05% Tween 20 (PBST). Then, 0.1-ml solutions containing the heat-denatured biotin probe (20 ng/ml), 50% formamide, 0.75 M NaCl, 5 mM sodium phosphate (pH 7.0), 50 ,ug of sonicated and denatured salmon sperm DNA per ml, 0.1% Tween 20, and 5 mM EDTA were added. The plate was again sealed and then incubated in a water bath at 420C overnight. After the wells were washed as described above, 0.1 ml of streptavidin-conjugated ,-galactosidase (Zymed Laboratories, San Francisco, Calif.) or peroxidase (Sigma Chemical Co., St. Louis, Mo.), diluted 1:10,000 or 1:1,000, respectively, in PBST containing 1% bovine serum albumin and 0.1% Triton X-100 was added. The plate was shaken on a Micromixer (Sanko Junyaku Co., Tokyo, Japan) at room temperature for 1 h. The wells were again washed. For the P-galactosidase wells, 0.1 ml of 0.2 mM 4-methylumbelliferyl-,3-D-galactoside in 0.1 M NaCl-10 mM sodium phosphate (pH 7.0)-i mM MgCl2-0.1% NaN3-0.1% bovine serum albumin was added. The plate was sealed and then incubated in a water bath at 37°C for 2 h. After addition of 0.1 ml of 0.1 M glycine-NaOH (pH 10.2), fluorescence units of each well were determined on a microplate fluorometric reader (Fluoroskan; Flow Laboratories, Inc., McLean, Va.). For the peroxidase wells, 0.1 ml of a mixture of 0.012% hydrogen peroxide and 0.04% orthophenylenediamine dihydrochloride in 0.05 M sodium phosphate-0.024 M citric acid buffer (pH 5.0) was added. The plate was placed at room temperature for 30 min, and then 0.05 ml of 4 N sulfuric acid was added. A492 of each well was determined on a microplate colorimetric reader (Multiskan; Flow). First, we examined optimal ionic conditions for adsorption of DNA to the microplate for hybridization. After the

Corresponding author. 1469






500 0-












300 500 1000

Input DNA (ng/well)

1 000-

IM 5.1 5M NaCI







1 25




Input DNA (pg/well)

FIG. 1. Effect of various NaCI concentrations on hybridizable immobilization of single-stranded DNA to a microplate. After heat denaturation, twofold serial dilutions of a 642-bp DNA segment were adsorbed to a microplate in the presence of various concentrations of NaCI and then hybridized with a biotinylated probe. The hybridization signal was detected by streptavidin-conjugated ,galactosidase. Enzyme activity is expressed in fluorescence units.

amplified 642-bp DNA from the standard virus was heat denatured, twofold serial dilutions were carried out in microplate wells with 0.15, 0.5, 1.0, 2.0, and 3.0 M NaCI solutions. Then, the adsorbed DNA was hybridized with a constant amount of the heat-denatured biotin probe. Hybridization occurred at above 1.0 M NaCl, whereas it did not occur at 0.15 M NaCl (Fig. 1). We also tested ammonium sulfate and sodium thiocyanate salts and found that 0.5 M ammonium sulfate was as effective as 1.5 M NaCl for hybridizable adsorption, whereas sodium thiocyanate, even at 2.0 M, did not give any hybridization signals (data not shown). Then, we investigated how much DNA can be adsorbed to the microplate at 1.5 M NaCl. Seventy-five nanograms of the 642-bp segment was mixed with 15,000 ng of sonicated salmon sperm DNA per 0.1 ml. After heat denaturation, various amounts of the DNA were immobilized for hybridization. Hybridization was at a maximum at about 200 ng of input DNA per well (Fig. 2). This experiment indicates that there is a saturation point for maximum immobilization of DNA. Next, we investigated the minimum size of DNA segment that could be immobilized at 1.5 M NaCl without loss of efficient hybridization ability. We used equimolar concentrations of DNA fragments of various sizes for immobilization. These fragments were derived from the 642-bp segment. Hybridization of these fragments was carried out with the same biotin probe. The same hybridization curve was obtained with 642-, 356-, and 286-bp fragments, but the hybridization efficiency decreased as sizes of the fragments fell below 227 bp (Fig. 3). This result indicates that it may be

FIG. 2. Saturation of the DNA immobilization. Sonicated salmon sperm DNA was mixed with the 642-bp DNA at a ratio of 200:1. After heating, various amounts of the DNA were immobilized in the presence of 1.5 M NaCI. Then, hybridization of the 642-bp segment was done.

better for the length of the target sequence for amplification to be more than about 300 bp. Finally, we tested the feasibility of this hybridization technique for routine identification of PCR-amplified products from clinical specimens. We extracted DNA from vesicles of 19 VZV-infected patients and one herpes simplex virus-infected patient and then used them as templates for PCR. The PCR-amplified products were treated with phenol and then with chloroform. The amplified DNA were then precipitated with ethanol and dissolved with the NTE buffer. After heat denaturation, these samples were 10-fold serially diluted in a microplate with 1.5 M NaCI for immobilization. After hybridization, two methods for the signal detection, the galactosidase and peroxidase systems, were compared. Figure 4 shows the results of the galactosidase system. All specimens, except one which was from herpes simplex virus-infected vesicles, gave positive hybridization results. The peroxidase system gave the same results (data not shown), although at a higher dilution (10-5), the galactosidase system had a higher detectability for the hybridization signal than the peroxidase system. The microplate hybridization method described here has several advantages over the conventional dot blot method. First, the microplate can be handled much more easily than the dot blot membrane. Second, washing of the solid phase of microplate is done in a shorter time, much as it is in the enzyme-linked immunosorbent assay. In the dot blot method, the reagents penetrate the membrane during the reaction time. Thus, during the washing step, a waiting time is necessary for the unreacted reagents to diffuse out from the inside of the membrane. In contrast, there is no penetration of the reagents to the microplate. The third advantage is a lower background level associated with the microplate. In this microplate method, neither Denhardt solution nor prehybridization and blocking procedures were needed. Furthermore, the baking or UV irradiation procedure, which is used for firmer binding of the DNA segment to the membrane, was not needed. In this study, we also synthesized the biotinylated probe by PCR with the primers used for amplification of specimen DNA. We confirmed that this probe did not react with the

VOL. 28, 1990



642 bases 7000 356 9000


8000 -o

c M 500(



o c





o s a "








e IL






20001 49 01 10000

240 2500




Input DNA ( attomoles/well) FIG. 3. Effect of DNA size on the efficiency of hybridization. The 642-bp amplified segment was divided into three aliquots. The first aliquot was treated with a restriction enzyme, PstI, which yielded 286- and 356-bp fragments. The second was treated with a mixture of PstI, SphI, and BamHI enzymes, which yielded 237-, 216-, 140-, and 49-bp fragments. The third was untreated. Then, the three aliquots were simultaneously electrophoresed in three lanes in agarose gel. Each DNA band was extracted into equal-volume solutions to give equimolar concentrations of DNAs with various sizes. (The 237- and 216-bp bands were extracted into a doublevolume solution as 227 [mean]-bp DNA.) Then, each fragment was immobilized in the presence of 1.5 M NaCI, and hybridized with the same probe.

primers (21-mer oligonucleotides) which had been adsorbed to the solid phase; the probe reacted only with the amplified DNA. This is another advantage: there is no need to prepare another primer pair for the probe. The most crucial step in the microplate hybridization method is the adsorption of single-stranded DNA to the solid phase; the immobilized DNA must retain hybridization capability. Ideally, the DNA segment should be adsorbed to microplate at only one end (3' or 5') of the strand. We found that the presence of 1.5 M NaCI or 0.5 M (NH4)2SO4 in the adsorption buffer facilitated the hybridizable adsorption, while 0.15 M NaCI and 2 M NaSCN did not. In the latter conditions, the DNA segment did adsorb to the microplate (data not shown); probably, the DNA adsorbed along the whole length of the strand, leading to no hybridizability. We also confirmed the finding by Nagata et al. (12) and Ezaki et al. (3) that addition of 0.1 M MgCl2 to 0.15 M NaCI also facilitated the hybridizable immobilization. However, we did not prefer the Mg2" ion, since it enhances the activity of DNase. At this moment, base sequence data on increasing numbers of pathogenic viruses have been emerging. Eventually, the conventional serodiagnosis of virus infection will be replaced by the more specific etiologic diagnosis with PCR. We hope that our microplate hybridization technique, which can be done at virus-diagnostic laboratories as easily as the


i- 3

10-4 10-5 Sample Dilution after PCR FIG. 4. Detection of VZV genomes in patient vesicles. The contents of varicella, herpes zoster, or herpes simplex vesicles were extracted with syringes and then suspended in 0.5 ml of phosphatebuffered saline. A portion was inoculated into human embryonic lung cells for virus isolation and identification. Another portion was centrifuged at 20,000 x g for 1 h. The pellet was treated with 0.6% sodium dodecyl sulfate and 200 ,ug of proteinase K per ml at 55°C for 1 h and then with phenol. DNA was precipitated with ethanol from the aqueous phase and then dissolved in 0.1 ml of NTE. The samples were subjected to PCR. The PCR-amplified samples were again subjected to phenol treatment and ethanol precipitation and then

10-fold serially diluted for immobilization, after which hybridization was carried out; from varicella or zoster vesicles (0) and from a herpes simplex vesicle (O).

enzyme-linked immunosorbent assay, will facilitate identification of the PCR-amplified viral DNA. We thank S. Ohsawa, BML, Tokyo, Japan, for his technical assistance. LITERATURE CITED 1. Arthur, R. R., S. Dagostin, and K. V. Shah. 1989. Detection of BK virus and JC virus in urine and brain tissue by the polymerase chain reaction. J. Clin. Microbiol. 27:1174-1179. 2. Duggan, D. B., G. D. Ehrlich, F. P. Davey, S. Kwok, J. Sninsky, J. Goldberg, L. Baltrucki, and B. J. Poiesz. 1988. HTLV1-induced lymphoma mimicking Hodgkin's disease. Diagnosis by polymerase chain reaction amplification of specific HTLV-1 sequences in tumor DNA. Blood 71:1027-1032. 3. Ezaki, T., Y. Hashimoto, N. Takeuchi, H. Yamamoto, S.-L. Liu, H. Miura, K. Matsui, and E. Yabuuchi. 1988. Simple genetic method to identify viridans group streptococci by colorimetric dot hybridization and fluorometric hybridization in microdilution wells. J. Clin. Microbiol. 26:1708-1713. 4. Hondo, R., and Y. Yogo. 1988. Strain variation of R5 direct repeats in the right-hand portion of the long unique segment of varicella-zoster virus DNA. J. Virol. 62:2916-2921.



5. Hondo, R., Y. Yogo, M. Yoshida, A. Fujima, and S. Itoh. 1989. Distribution of varicella-zoster virus strains carrying a PstI site-less mutation in Japan and DNA change responsible for the mutation. Jpn. J. Exp. Med. 59:233-237. 6. Hsia, K., D. H. Spector, J. Lawrie, and S. A. Spector. 1989. Enzymatic amplification of human cytomegalovirus sequences by polymerase chain reaction. J. Clin. Microbiol. 27:1802-1809. 7. Kwok, S., D. H. Mack, K. B. Mullis, B. Poiesz, G. D. Ehrlich, D. Blair, A. Friedman-Kien, and J. J. Sninsky. 1987. Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection. J. Virol. 61:1690-1694. 8. Larzul, D., F. Guigue, J. J. Sninsky, D. H. Mack, C. Brechot, and J. L. Guesdon. 1988. Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification. J. Virol. Methods 20:227-237. 9. Loche, M., and B. Mach. 1988. Identification of HIV-infected seronegative individuals by a direct diagnostic test based on hybridisation to amplified viral DNA. Lancet ii:418-421. 10. Melchers, W. J. G., R. Schift, E. Stolz, J. Lindeman, and W. G. V. Quint. 1989. Human papillomavirus detection in urine samples from male patients by the polymerase chain reaction. J.


Clin. Microbiol. 27:1711-1714. 11. Mullis, K., F. Faloona, S. Scharf, R. Saiki, G. Horn, and H. Erlich. 1986. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51:263-272. 12. Nagata, Y., H. Yokota, O. Kosuda, K. Yokoo, K. Takemura, and T. Kikuchi. 1985. Quantification of picogram levels of specific DNA immobilized in microtiter wells. FEBS Lett. 183:379-382. 13. Olive, D. M., M. Simsek, and S. AI-Mufti. 1989. Polymerase chain reaction assay for detection of human cytomegalovirus. J. Clin. Microbiol. 27:1238-1242. 14. Ou, C.-Y., S. Kwok, S. W. Mitchell, D. H. Mack, J. J. Sninsky, J. W. Krebs, P. Feorino, D. Warfield, and G. Schochetman. 1988. DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science 239:295-297. 15. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491. 16. Shibata, D. K., N. Arnheim, and W. J. Martin. 1988. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J. Exp. Med. 167:225-230.

Microplate hybridization of amplified viral DNA segment.

We have developed a simple hybridization method for a DNA segment which is amplified by the polymerase chain reaction: after heat denaturation, the am...
644KB Sizes 0 Downloads 0 Views