Vol. 28, No. 1
OF CLINICAL MICROBIOLOGY, Jan. 1990, p. 159-162 0095-1137/90/010159-04$02.00/0 Copyright © 1990, American Society for Microbiology
JOURNAL
Rapid Quantitation of DNA Spot Hybridization by Flatbed Scintillation Counting TIMO HYYPIA,l* EEVA AUVINEN,l SATU KOVANEN,2 AND TOM H. STÂHLBERG2 Department of Virology, University of Turku, SF-20520 Turku,' and Wallac Oy, SF-20101 Turku,2 Finland Received 19 June 1989/Accepted 25 September 1989
The flatbed scintillation counting system (Betaplate) was used for quantitative measurement of the radioactive hybridization signal in detection of adenovirus and papillomavirus DNA in clinical specimens. In this method, 96 samples on a nylon membrane can be handled as a single entity throughout the hybridization and counting procedure. The technique is sensitive, rapid, and convenient in routine use when compared with conventionally applied methods for the numerical analysis of hybridization results. The assay principle allows simultaneous testing of large numbers of specimens.
from female patients with condyloma acuminata or other lesions of the genital tract were proteinase treated and phenol extracted as described earlier (E. Auvinen, V. Hukkanen, and P. Arstila, Mol. Cell. Probes, in press). Adenovirus type 2 (Ad2) DNA, herpes simplex virus type 1 (HSV-1) DNA, and purified inserts of cloned HPV-6, HPV11, HPV-16, and HPV-18 DNA, as well as pBR322 plasmid DNA, were used as controls. A manifold apparatus in the Betaplate format (Wallac Oy, Turku, Finland) was used for spotting the samples to nylon membranes (pore size, 0.45 ,um; Wailac) printed with black screening rings to mark the spot and to reduce possible scintillation from the adjacent samples. Cross talk (the interaction with the neighbor sample) for 32p with these membranes was less than 0.015%. The adenovirus DNA probe was labeled in a nick translation reaction (7) using either 32p, 35S, 125i, or 3H precursors (specific activities of the probes were 118 x 106, 142 x 106, 94 x 106, and 4.5 x 10' cpm/,ug, respectively), whereas random priming (1) with 32p was used for labeling of the HPV probes (specific activity of the HPV-16 probe was 320 x 106 cpm/,ug). The hybridization reactions and the elution of probes were carried out as described previously (3; Auvinen et al., in press). After 16 to 72 h of autoradiography, the dry membrane was sealed in a thin plastic bag and counted after addition of Betaplate Scint liquid (Wallac). When elution of the probe was necessary (HPV), the wet membrane was directly counted. The sample bag was placed in a cassette, and the bound radioactivity was detected in a Betaplate flatbed scintillation counter (Wallac). We first tested the sensitivity and specificity of the flatbed method in quantitative measurement of homologous Ad2 DNA and nonrelated HSV-1 DNA after hybridization with Ad2 DNA probe labeled with 32p, 35S, 1251 or 3H (Fig. 1; Table 1). The results were revealed both by counting and by autoradiography (except 3H). High concentrations of HSV-1 DNA gave a weak, unspecific hybridization signal which was detected by both of the methods. In the Betaplate system, the 32P-labeled probe gave a signal with 100 pg of homologous DNA, while 35S- and 125I-labeled probes detected clearly 1 ng of target Ad2 DNA and values exceeding the background were already observed with 100 pg of target DNA (Table 1). The 3H probe gave a detectable signal with 10 ng of adenovirus DNA, and some reactivity was seen with 1 ng. In autoradiography, a strong reaction with 100 pg and
Nucleic acid hybridization methods are currently used for specific laboratory diagnosis of numerous microbial diseases caused by viruses, bacteria, and parasites (4). These methods are also widely applied for research purposes in laboratories studying molecular microbiology. 32P is the most frequently used isotope for labeling of probes to high specific activity. Other isotopes and nonradioactive labels may have clinical applications in the future for selected purposes, but their disadvantage is the lower sensitivity in identification of target sequences. Detection of radioactive signals after hybridization is usually carried out by autoradiography. 32P can be revealed after overnight exposure, but for other isotopes several weeks may be needed. Interpretation of the signal intensity is made visually and is therefore subjective, especially when quantitative analysis is required. Investigators have tried to solve this problem by using either densitometry of the autoradiograms or conventional scintillation counting of the individual samples cut out from the membrane. Both these methods are laborious and prone to technical errors when a large number of samples are subjected to analysis. The flatbed scintillation counting system (6) was originally developed for analysis of labeled cells deposited on glass fiber filter mats (5). It has also been successfully used for membrane receptor analysis and certain enzyme assays (e.g., reverse transcriptase). The counting is based on flatbed geometry, in which multiple samples are applied within discrete areas on a filter sheet. The photomultiplier tubes are close together on a plate over a sample area which gives low background and high counting efficiency. Throughout the process no vials are used. Instead, the filter mat or nylon membrane is put in a sample bag into which a very small quantity of scintillant is added. We have evaluated the applicability of the Betaplate technique in detection of the DNA spot hybridization signal with different isotopes and have used the method for the analysis of adenovirus and papillomavirus DNA in clinical specimens. Nasopharyngeal samples, previously tested for the presence of adenovirus hexon antigen by an immunoassay (2), were digested with proteinase K (3) and applied directly to membranes prior to denaturation. For detection of human papillomaviruses (HPV), 18 biopsy and smear specimens
*
Corresponding author. 159
J. CLIN. MICROBIOL.
NOTES
160
TABLE 2. Comparison of Betaplate counting with autoradiography in detection of adenoviruses in nasopharyngeal
_L
_J-';
e
.
4
i'E;
specimens
Specimen no.
:
.1 .:
-'-
FIG. 1. Detection of Ad2 and HSV-1 DNA by spot hybridization with the Ad2 DNA probe. The probe was labeled by nick translation with 32p, 35S, and 1251 precursors. Results were revealed by autoradiography on an X-ray film.
a faint one with 10 pg were observed when the 32P-labeled probe was used, while the sensitivities with 35S and 1251 were 0.1 to 1 ng and 1 ng, respectively (Fig. 1). To evaluate the applicability of the Betaplate system for diagnostic routine use, we analyzed 43 coded nasopharyngeal specimens known to be positive or negative for adenovirus hexon antigen (Table 2). The same 22 of the 23 immunoassay-positive samples were positive by flatbed scintillation counting and by autoradiography. Detection of human papillomaviruses is the most widely used application of nucleic acid hybridization in clinical virology. Therefore, we wanted to evaluate the flatbed scintillation method in routine detection of HPV-6, HPV-11, HPV-16, and HPV-18, which are the types most frequently associated with genital dysplasia. Eighteen smear and biopsy specimens from the female genital tract were analyzed for the presence of the four HPV types by counting and by autoradiography (Fig. 2, Table 3). In all the samples positive by autoradiography, values (counts per minute) exceeded those observed in the negative specimens. One of the specimens (no. 16; Table 3) was positive for HPV-6, and nine samples contained HPV-16 (specimens 1, 2, 4, 6, 7, 8, 13, 14, and 17). In some cases, cross-reactivity between different HPV types was seen; e.g., specimen 7 gave a faint signal with the HPV-18 probe, while specimen 16, which gave a
TABLE 1. Comparison of different isotopes in detection of the hybridization signal by flatbed scintillation counting Isotope detected (cpm) Ad2
100 ng 170,783 10 ng 36,825 1 ng 3,505 100 pg 548 161 10pg 105 1 pg
125i
35S
32p
DNA
HSV-1
Ad2
381 124 116 121 131 135
45,743 16,041 1,325 302 216 204
HSV-1
Ad2
264 16,401 184 6,384 222 719 94 164 34 196 188 38
3H
HSV-1 Ad2 HSV-1
42 48 35 28 22 26
2,037 402 37 9 8 8
6 13 13 15 9 17
a Various amounts of Ad2 and HSV-1 DNA were spotted to nylon membranes and hybridized by using Ad2 DNA probe labeled with 32p, 35S, 125I, and 3H. The results can be compared with autoradiography results in Fig. 1.
1 2 3 4 5 6 7 8 9 10 il 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
cpm
DNA (ng)
ARb
6,315
0.5
81 66 2,110 178 80
+ -
0.2
+
3,191 66 44,108 4,969 1,920
0.3 4 0.4 0.2
-
50
-
2,483 3,149 831 89 187 2,426 297 143 3,039 106 127 1,970 968 59 2,545 65 141 800 7,130 1,189 120
0.2 0.3
0.09 0.2 -
+ + + + + + + +
0.3 0.2 0.09 0.2 0.08 0.6 0.1 -
72
-
1,576 38,771 328 16,242 66 359 5,939 103 2,297
0.1 3
+ + + +
+ + + + +
-
1
+
-
0.5
+
-
0.2
+
a The amount of virus DNA was calculated on the basis of standards tested in parallel. -, Samples below the cutoff value (375 cpm), which was determined as three times the average counts per minute of HSV-1 control DNA. Specimens shown in boldface type were positive by immunoassay for adenovirus hexon antigen. b AR, Autoradiography.
very strong reaction with HPV-6, reacted also with the other probes. The use of nucleic acid probes for detection of microorganisms is becoming a routine method in diagnostic laboratories. The advantages of these techniques include a possibility of detecting persistent infections and rapid identification of microbial pathogens with slow growth rates. In addition, the new in vitro amplification methods, e.g., polymerase chain reaction (8), enable sensitive detection of specific sequences by a simple spot hybridization procedure. Autoradiography is commonly used for the detection of the signal after hybridization. In the case of 32P, this procedure can usually be carried out overnight, which often is an acceptable delay. However, when the results should be
VOL. 28, 1990
NOTES
161
k-t~i p AiK
ré! ES ;5
1b 18 CL iNIA Il 322 6 C û ENS DNA DNADNADNADN'A SPE L -T ~ooT-kb1ô 010 ^ fl 1010 O
gn9nr3g ng ng'rn ig
n..+
n: ,q
,
nr-
1 t..z
4
4
0
*
,_*
ng
~~ci
1 foi '
DEX
n~~~~~~~~~~~~~~~~~~~~. .....!
0
ib
Lu9
1
FIG. 2. Reactivity of pBR322 plasmid DNA; HPV DNA from nucleic acid hybridization test with 32P-labeled HPV-16 probe.
types 6, 11, 16, and 18; and DNA isolated
expressed immediately or quantitative measurement of the target sequence in the sample is required, direct counting of individual spots is preferable. In addition, for screening the need for high-throughput methods is obvious. The spots can be cut out from the membrane and subjected to conventional scintillation counting (3), which is a cumbersome procedure and has reduced sensitivity compared with autoradiography. This problem can be partially overcome in the flatbed scintillation counting system, in which the detectors are localized close to the membrane, giving a high sensitivity. Another advantage of this method is that the whole membrane can be processed simultaneously and counted easily either without or with only 3 ml of scintillation liquid. 32P-labeled probes can be counted directly without adding any scintillant to the sample bag, enabling rehybridization of the membrane. Counting time for 96 specimens is purposes
from clinical specimens in
a
less than 20 min, thus enabling rapid processing of large numbers of specimens. We compared the Betaplate method with autoradiography for analysis of the presence of adenovirus and papillomavirus DNA directly in clinical specimens. In spite of the slightly lower detection level of the counting procedure, all the clinical samples gave virtually equal results by the two methods in detection of adenovirus and papillomavirus DNA. Therefore, it can be expected that in routine diagnostic use the results obtained by the flatbed scintillation system will be comparable with those obtained by autoradiography and will be obtained within 20 min instead of 16 to 72 h. Especially in laboratories where a flatbed scintillation counter is already available for other applications, it can easily be used for rapid and quantitative reading of nucleic acid hybridization tests.
TABLE 3. Comparison of hybridization resuIts obtained by Betaplate counting and by autoradiography in detection of HPV in clinical specimens
HPV6
Specimen no.'
cpm
1 2 3 4 5 6 7 8
37 41 23 47 51 49 39 27
9
21
10 il 12 13 14 15 16 17 18
36 95 42 81 37 35 4,570 48 37
-
-
AR
cpm
-
159 547 52 131 49 108 351 116
ng
1 40
AR
cpm
+ ++
59 55 65 101 70 93 126 47
ng
AR
-
-
27
-
58
-
57
-
-
32 41 49 47 38 22 145 35 43
-
57 43 55 169 133 62 142 137 82
-
74 79 102 100 75 44 115 80 68
-
-
>100 -
ng
29 29 27 55 29 50 30 30
-
-
cpm
HPV-18
HPV-16
HPV-11 ARC
ngb
+++
-
-
-
-
+
-
-
0.5
+
-
0.25 8 0.3
+ ++ +
1 0.5
+ +
0.6
a The sample was considered to contain the HPV type which had the strongest reactivity in the hybridization assay. b Nanograms are calculated on the basis of a standard curve. -, Samples below the cutoff value. c AR, Autoradiography.
+ +
-
-
+-
-
-
-
-
-
+
-
-
162
NOTES
The excellent technical assistance of Marita Maaronen and Mena Virtanen is greatly acknowledged. We thank Yolanda Bullen for revising the language of the manuscript. This study was supported by the Sigrid Jusélius Foundation. LITERATURE CITED 1. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. 2. Halonen, P., C. Bonfanti, T. Lovgren, I. Hemmila, and E. Soini. 1985. Detection of viral antigens by time-resolved fluoroimmunoassay, p. 429-437. In K.-O. Habermehl (ed.), Rapid methods and automation in microbiology and immunology. SpringerVerlag, Heidelberg, Federal Republic of Germany. 3. Hyypia, T. 1985. Detection of adenovirus in nasopharyngeal specimens by radioactive and nonradioactive DNA probes. J. Clin. Microbiol. 21:730-733.
J. CLIN. MICROBIOL. 4. Pettersson, U., and T. Hyypia. 1985. Nucleic acid hybridizationan alternative tool in diagnostic microbiology. Immunol. Today 6:268-272. 5. Potter, C. G., F. Gotch, G. T. Warner, and J. Oestrup. 1987. Lymphocyte proliferation and cytotoxic assays using flat-bed scintillation counting. J. Immunol. Methods 105:171-177. 6. Potter, C. G., G. T. Warner, T. Yrjonen, and E. Soini. 1986. A liquid scintillation counter specifically designed for samples deposited on a flat matrix. Phys. Med. Biol. 31:361-369. 7. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. 1977. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113:237-251. 8. Saiki, R. K., D. H. Gelfand, S. Soffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primer-directed enzymatic amplification of DNA with thermostable DNA polymerase. Science 239:487-491.
OF CLINICAL MICROBIOLOGY, Jan. 1990, p. 159-162 0095-1137/90/010159-04$02.00/0 Copyright © 1990, American Society for Microbiology
JOURNAL
Rapid Quantitation of DNA Spot Hybridization by Flatbed Scintillation Counting TIMO HYYPIA,l* EEVA AUVINEN,l SATU KOVANEN,2 AND TOM H. STÂHLBERG2 Department of Virology, University of Turku, SF-20520 Turku,' and Wallac Oy, SF-20101 Turku,2 Finland Received 19 June 1989/Accepted 25 September 1989
The flatbed scintillation counting system (Betaplate) was used for quantitative measurement of the radioactive hybridization signal in detection of adenovirus and papillomavirus DNA in clinical specimens. In this method, 96 samples on a nylon membrane can be handled as a single entity throughout the hybridization and counting procedure. The technique is sensitive, rapid, and convenient in routine use when compared with conventionally applied methods for the numerical analysis of hybridization results. The assay principle allows simultaneous testing of large numbers of specimens.
from female patients with condyloma acuminata or other lesions of the genital tract were proteinase treated and phenol extracted as described earlier (E. Auvinen, V. Hukkanen, and P. Arstila, Mol. Cell. Probes, in press). Adenovirus type 2 (Ad2) DNA, herpes simplex virus type 1 (HSV-1) DNA, and purified inserts of cloned HPV-6, HPV11, HPV-16, and HPV-18 DNA, as well as pBR322 plasmid DNA, were used as controls. A manifold apparatus in the Betaplate format (Wallac Oy, Turku, Finland) was used for spotting the samples to nylon membranes (pore size, 0.45 ,um; Wailac) printed with black screening rings to mark the spot and to reduce possible scintillation from the adjacent samples. Cross talk (the interaction with the neighbor sample) for 32p with these membranes was less than 0.015%. The adenovirus DNA probe was labeled in a nick translation reaction (7) using either 32p, 35S, 125i, or 3H precursors (specific activities of the probes were 118 x 106, 142 x 106, 94 x 106, and 4.5 x 10' cpm/,ug, respectively), whereas random priming (1) with 32p was used for labeling of the HPV probes (specific activity of the HPV-16 probe was 320 x 106 cpm/,ug). The hybridization reactions and the elution of probes were carried out as described previously (3; Auvinen et al., in press). After 16 to 72 h of autoradiography, the dry membrane was sealed in a thin plastic bag and counted after addition of Betaplate Scint liquid (Wallac). When elution of the probe was necessary (HPV), the wet membrane was directly counted. The sample bag was placed in a cassette, and the bound radioactivity was detected in a Betaplate flatbed scintillation counter (Wallac). We first tested the sensitivity and specificity of the flatbed method in quantitative measurement of homologous Ad2 DNA and nonrelated HSV-1 DNA after hybridization with Ad2 DNA probe labeled with 32p, 35S, 1251 or 3H (Fig. 1; Table 1). The results were revealed both by counting and by autoradiography (except 3H). High concentrations of HSV-1 DNA gave a weak, unspecific hybridization signal which was detected by both of the methods. In the Betaplate system, the 32P-labeled probe gave a signal with 100 pg of homologous DNA, while 35S- and 125I-labeled probes detected clearly 1 ng of target Ad2 DNA and values exceeding the background were already observed with 100 pg of target DNA (Table 1). The 3H probe gave a detectable signal with 10 ng of adenovirus DNA, and some reactivity was seen with 1 ng. In autoradiography, a strong reaction with 100 pg and
Nucleic acid hybridization methods are currently used for specific laboratory diagnosis of numerous microbial diseases caused by viruses, bacteria, and parasites (4). These methods are also widely applied for research purposes in laboratories studying molecular microbiology. 32P is the most frequently used isotope for labeling of probes to high specific activity. Other isotopes and nonradioactive labels may have clinical applications in the future for selected purposes, but their disadvantage is the lower sensitivity in identification of target sequences. Detection of radioactive signals after hybridization is usually carried out by autoradiography. 32P can be revealed after overnight exposure, but for other isotopes several weeks may be needed. Interpretation of the signal intensity is made visually and is therefore subjective, especially when quantitative analysis is required. Investigators have tried to solve this problem by using either densitometry of the autoradiograms or conventional scintillation counting of the individual samples cut out from the membrane. Both these methods are laborious and prone to technical errors when a large number of samples are subjected to analysis. The flatbed scintillation counting system (6) was originally developed for analysis of labeled cells deposited on glass fiber filter mats (5). It has also been successfully used for membrane receptor analysis and certain enzyme assays (e.g., reverse transcriptase). The counting is based on flatbed geometry, in which multiple samples are applied within discrete areas on a filter sheet. The photomultiplier tubes are close together on a plate over a sample area which gives low background and high counting efficiency. Throughout the process no vials are used. Instead, the filter mat or nylon membrane is put in a sample bag into which a very small quantity of scintillant is added. We have evaluated the applicability of the Betaplate technique in detection of the DNA spot hybridization signal with different isotopes and have used the method for the analysis of adenovirus and papillomavirus DNA in clinical specimens. Nasopharyngeal samples, previously tested for the presence of adenovirus hexon antigen by an immunoassay (2), were digested with proteinase K (3) and applied directly to membranes prior to denaturation. For detection of human papillomaviruses (HPV), 18 biopsy and smear specimens
*
Corresponding author. 159
J. CLIN. MICROBIOL.
NOTES
160
TABLE 2. Comparison of Betaplate counting with autoradiography in detection of adenoviruses in nasopharyngeal
_L
_J-';
e
.
4
i'E;
specimens
Specimen no.
:
.1 .:
-'-
FIG. 1. Detection of Ad2 and HSV-1 DNA by spot hybridization with the Ad2 DNA probe. The probe was labeled by nick translation with 32p, 35S, and 1251 precursors. Results were revealed by autoradiography on an X-ray film.
a faint one with 10 pg were observed when the 32P-labeled probe was used, while the sensitivities with 35S and 1251 were 0.1 to 1 ng and 1 ng, respectively (Fig. 1). To evaluate the applicability of the Betaplate system for diagnostic routine use, we analyzed 43 coded nasopharyngeal specimens known to be positive or negative for adenovirus hexon antigen (Table 2). The same 22 of the 23 immunoassay-positive samples were positive by flatbed scintillation counting and by autoradiography. Detection of human papillomaviruses is the most widely used application of nucleic acid hybridization in clinical virology. Therefore, we wanted to evaluate the flatbed scintillation method in routine detection of HPV-6, HPV-11, HPV-16, and HPV-18, which are the types most frequently associated with genital dysplasia. Eighteen smear and biopsy specimens from the female genital tract were analyzed for the presence of the four HPV types by counting and by autoradiography (Fig. 2, Table 3). In all the samples positive by autoradiography, values (counts per minute) exceeded those observed in the negative specimens. One of the specimens (no. 16; Table 3) was positive for HPV-6, and nine samples contained HPV-16 (specimens 1, 2, 4, 6, 7, 8, 13, 14, and 17). In some cases, cross-reactivity between different HPV types was seen; e.g., specimen 7 gave a faint signal with the HPV-18 probe, while specimen 16, which gave a
TABLE 1. Comparison of different isotopes in detection of the hybridization signal by flatbed scintillation counting Isotope detected (cpm) Ad2
100 ng 170,783 10 ng 36,825 1 ng 3,505 100 pg 548 161 10pg 105 1 pg
125i
35S
32p
DNA
HSV-1
Ad2
381 124 116 121 131 135
45,743 16,041 1,325 302 216 204
HSV-1
Ad2
264 16,401 184 6,384 222 719 94 164 34 196 188 38
3H
HSV-1 Ad2 HSV-1
42 48 35 28 22 26
2,037 402 37 9 8 8
6 13 13 15 9 17
a Various amounts of Ad2 and HSV-1 DNA were spotted to nylon membranes and hybridized by using Ad2 DNA probe labeled with 32p, 35S, 125I, and 3H. The results can be compared with autoradiography results in Fig. 1.
1 2 3 4 5 6 7 8 9 10 il 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
cpm
DNA (ng)
ARb
6,315
0.5
81 66 2,110 178 80
+ -
0.2
+
3,191 66 44,108 4,969 1,920
0.3 4 0.4 0.2
-
50
-
2,483 3,149 831 89 187 2,426 297 143 3,039 106 127 1,970 968 59 2,545 65 141 800 7,130 1,189 120
0.2 0.3
0.09 0.2 -
+ + + + + + + +
0.3 0.2 0.09 0.2 0.08 0.6 0.1 -
72
-
1,576 38,771 328 16,242 66 359 5,939 103 2,297
0.1 3
+ + + +
+ + + + +
-
1
+
-
0.5
+
-
0.2
+
a The amount of virus DNA was calculated on the basis of standards tested in parallel. -, Samples below the cutoff value (375 cpm), which was determined as three times the average counts per minute of HSV-1 control DNA. Specimens shown in boldface type were positive by immunoassay for adenovirus hexon antigen. b AR, Autoradiography.
very strong reaction with HPV-6, reacted also with the other probes. The use of nucleic acid probes for detection of microorganisms is becoming a routine method in diagnostic laboratories. The advantages of these techniques include a possibility of detecting persistent infections and rapid identification of microbial pathogens with slow growth rates. In addition, the new in vitro amplification methods, e.g., polymerase chain reaction (8), enable sensitive detection of specific sequences by a simple spot hybridization procedure. Autoradiography is commonly used for the detection of the signal after hybridization. In the case of 32P, this procedure can usually be carried out overnight, which often is an acceptable delay. However, when the results should be
VOL. 28, 1990
NOTES
161
k-t~i p AiK
ré! ES ;5
1b 18 CL iNIA Il 322 6 C û ENS DNA DNADNADNADN'A SPE L -T ~ooT-kb1ô 010 ^ fl 1010 O
gn9nr3g ng ng'rn ig
n..+
n: ,q
,
nr-
1 t..z
4
4
0
*
,_*
ng
~~ci
1 foi '
DEX
n~~~~~~~~~~~~~~~~~~~~. .....!
0
ib
Lu9
1
FIG. 2. Reactivity of pBR322 plasmid DNA; HPV DNA from nucleic acid hybridization test with 32P-labeled HPV-16 probe.
types 6, 11, 16, and 18; and DNA isolated
expressed immediately or quantitative measurement of the target sequence in the sample is required, direct counting of individual spots is preferable. In addition, for screening the need for high-throughput methods is obvious. The spots can be cut out from the membrane and subjected to conventional scintillation counting (3), which is a cumbersome procedure and has reduced sensitivity compared with autoradiography. This problem can be partially overcome in the flatbed scintillation counting system, in which the detectors are localized close to the membrane, giving a high sensitivity. Another advantage of this method is that the whole membrane can be processed simultaneously and counted easily either without or with only 3 ml of scintillation liquid. 32P-labeled probes can be counted directly without adding any scintillant to the sample bag, enabling rehybridization of the membrane. Counting time for 96 specimens is purposes
from clinical specimens in
a
less than 20 min, thus enabling rapid processing of large numbers of specimens. We compared the Betaplate method with autoradiography for analysis of the presence of adenovirus and papillomavirus DNA directly in clinical specimens. In spite of the slightly lower detection level of the counting procedure, all the clinical samples gave virtually equal results by the two methods in detection of adenovirus and papillomavirus DNA. Therefore, it can be expected that in routine diagnostic use the results obtained by the flatbed scintillation system will be comparable with those obtained by autoradiography and will be obtained within 20 min instead of 16 to 72 h. Especially in laboratories where a flatbed scintillation counter is already available for other applications, it can easily be used for rapid and quantitative reading of nucleic acid hybridization tests.
TABLE 3. Comparison of hybridization resuIts obtained by Betaplate counting and by autoradiography in detection of HPV in clinical specimens
HPV6
Specimen no.'
cpm
1 2 3 4 5 6 7 8
37 41 23 47 51 49 39 27
9
21
10 il 12 13 14 15 16 17 18
36 95 42 81 37 35 4,570 48 37
-
-
AR
cpm
-
159 547 52 131 49 108 351 116
ng
1 40
AR
cpm
+ ++
59 55 65 101 70 93 126 47
ng
AR
-
-
27
-
58
-
57
-
-
32 41 49 47 38 22 145 35 43
-
57 43 55 169 133 62 142 137 82
-
74 79 102 100 75 44 115 80 68
-
-
>100 -
ng
29 29 27 55 29 50 30 30
-
-
cpm
HPV-18
HPV-16
HPV-11 ARC
ngb
+++
-
-
-
-
+
-
-
0.5
+
-
0.25 8 0.3
+ ++ +
1 0.5
+ +
0.6
a The sample was considered to contain the HPV type which had the strongest reactivity in the hybridization assay. b Nanograms are calculated on the basis of a standard curve. -, Samples below the cutoff value. c AR, Autoradiography.
+ +
-
-
+-
-
-
-
-
-
+
-
-
162
NOTES
The excellent technical assistance of Marita Maaronen and Mena Virtanen is greatly acknowledged. We thank Yolanda Bullen for revising the language of the manuscript. This study was supported by the Sigrid Jusélius Foundation. LITERATURE CITED 1. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. 2. Halonen, P., C. Bonfanti, T. Lovgren, I. Hemmila, and E. Soini. 1985. Detection of viral antigens by time-resolved fluoroimmunoassay, p. 429-437. In K.-O. Habermehl (ed.), Rapid methods and automation in microbiology and immunology. SpringerVerlag, Heidelberg, Federal Republic of Germany. 3. Hyypia, T. 1985. Detection of adenovirus in nasopharyngeal specimens by radioactive and nonradioactive DNA probes. J. Clin. Microbiol. 21:730-733.
J. CLIN. MICROBIOL. 4. Pettersson, U., and T. Hyypia. 1985. Nucleic acid hybridizationan alternative tool in diagnostic microbiology. Immunol. Today 6:268-272. 5. Potter, C. G., F. Gotch, G. T. Warner, and J. Oestrup. 1987. Lymphocyte proliferation and cytotoxic assays using flat-bed scintillation counting. J. Immunol. Methods 105:171-177. 6. Potter, C. G., G. T. Warner, T. Yrjonen, and E. Soini. 1986. A liquid scintillation counter specifically designed for samples deposited on a flat matrix. Phys. Med. Biol. 31:361-369. 7. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. 1977. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113:237-251. 8. Saiki, R. K., D. H. Gelfand, S. Soffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primer-directed enzymatic amplification of DNA with thermostable DNA polymerase. Science 239:487-491.
