Molecular and Cellular Probes (1990) 4, 341-352
A rapid solution hybridization method for detection of human papi I lomavi ruses Tarja Jalava, Arja Kallio,* Antti W . Leinonen and Marjut Ranki Orion Corporation, Orion Pharmaceutica, Biotechnology, Valimotie 7, SF-00380 Helsinki, Finland (Received 23 January 1990, Accepted 19 February 1990)
Nucleic acid hybridization methods in routine diagnosis of micro-organisms have been limited by the tedious assay procedures . We have previously described the sandwich hybridization method which allows convenient testing of biological specimens . In this paper we describe the adaptation of the solution hybridization method into the microtitre plate format using 35 S-isotope as label . Using 3-hour hybridization followed by 2-hour collection of the hybrids a sensitivity of 5 x 10' target DNA molecules was achieved . The method was applied for identification of human papillomaviruses in crude gynaecological specimens . A simple 1-day assay protocol was achieved with high HPV type specificity . The specificity was confirmed by testing a variety of unrelated microorganisms, none of which gave a positive signal in the test . Results, obtained as numerical values, were easy to interpret; positive and negative samples gave clearly distinguishable signals .
KEYWORDS : nucleic acid hybridization in solution, diagnostics, human papillomavirus .
INTRODUCTION There is a need for a technique providing convenient, sensitive and specific determination of human papillomavirus (HPV) DNA in infected cells . The predominant HPV types commonly found in the genital area are 6, 11, 16, 18, 31 and 33 .' -a The HPV types 6 and 11 are frequently identified in benign tumours' , ' while the types 16 and 18 account for 70% of the HPV types associated with severe dysplasias and invasive carcinoma of the cervix . 9' 10 PapiIlomaviruses cannot be cultivated and viral antigens are not reliably detected in the clinical specimens . Indirect evidence of genital HPV infection can be obtained through physical examinations and by observing the presence of characteristic cellular changes in PAP smears . The methods currently providing information on the HPV type present are the detection of viral DNA using nucleic acid hybridization techniques ." *Author to whom correspondence should be addressed .
0890-8508/90/050341 + 12 $03 .00/0
© 1990 Academic Press Limited 341
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A variety of DNA techniques are used in research for detection and typing of HPV . These include analyses which require purification of DNA (Southern blot, dotblot) or which can be performed directly on tissue sections and scrapings (in situ 12 and filter in situ hybridizations) . Nucleic acid sandwich hybridization assay," used in this study, has been previously demonstrated to be a suitable method for testing crude samples for the presence of viruses"" and bacteria . 19" In this paper we describe the development of a simple and rapid sandwich hybridization assay for detection of HPV DNA . The method takes advantage of the speed and sensitivity of hybridization in solution, utilizes 35 S label in the detection of the hybrids and microtitre plates for affinity-based hybrid collection . This assay format is suitable for routine diagnostic purposes . The analysis of HPV DNA in cervical and vaginal scrapings by this method is presented .
MATERIALS AND METHODS DNA reagents The HPV 16 DNA fragments, used as probes in this study have been previously described ." The 2 . 8 kb Pst I fragment of HPV 16 DNA, cloned in pBR 322 was the detector probe . The 1 . 7 kb and 1 . 1 kb Pst I fragments, cloned into the bacteriophage M13 mp 10 and 11, were used as capture probes . The DNA reagents for HPV types 6/11 and 18 were prepared by an analogous strategy . As control targets recombinant plasmids, i .e . pBR 322 containing the HPV genome, were used . The detector probes were labelled by nick-translation 22 using 35S-dCTP (Amersham, UK) . The probes were purified using either a Sephadex G-25 (Pharmacia LKB, Sweden) column followed by phenol extraction and ethanol precipitation, or a GeneClean kit (BIO -1 DNA 101, USA) . The specific activities of the probes were about 2 . 5 x 108 cpm ltg and the capture DNAs were chemically biotinylated (Bengtström and jungellNortamo, to be published) .
The hybridization reaction The probe DNAs were denaturated in a 0 . 2% SDS solution and the recombinant DNA targets were denatured in a solution containing 0E5% SDS, 0 . 2 M NaOH and 0. 4 mg ml -1 herring sperm DNA (HS-DNA) for 5 min at 100 ° C . The hybridizations were carried out in eppendorf tubes in a final volume of 110 .tl at 65°C for 3 h . The reaction mixture contained 0 . 66 M NaCl, 65 mm sodium-citrate, 0 . 3 mm EDTA, 0 . 1 M P0 4 -buffer (pH 6 . 6), 0 . 02% Ficoll, 0. 2% polyvinylpyrrolidone, 0 . 5% polyethyleneglycol (4000), 1 . 2 mg ml -1 BSA and the denatured and neutralized target DNA, 500,000 cpm of denatured probe DNA and 1 . 0-1 . 2 X 1010 molecules of biotinylated capture DNAs .
Collection and measurement of the hybrids After hybridization the hybrids were collected onto microtitre plates coated with straptavidin (Orion Diagnostica, Finland) . The collection was for 2 h at 37 ° C using a
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microtitre plate shaker. After collection the plates were washed with 150 Al of prewarmed (+50 ° C) washing solution for 2 min six times ." The formed hybrids were then eluted twice with 110 pl of 0 . 2 M NaOH for 5 min and the radioactivity was quantified by counting in a scintillation counter for 5 min . The results are given as the mean of five parallel determinations .
Clinical specimens Cervical and vaginal scrapes were collected with an endocervical brush . The cells were suspended in 5 ml of 0 . 05 m phosphate buffered saline (PBS) and pelleted by centrifugation . The cell pellets were stored at - 20°C . Before analysis the cells were suspended in 100 pl of PBS . Prior to hybridization the patient samples were divided in aliquots and denatured in a solution containing 0 . 5% SDS, 0 . 2 m NaOH and 0. 4 mg HS-DNA ml - ' for 5 min at 100 ° C .
RESULTS Principle of the method The method is based on sandwich hybridization in solution followed by affinitybased hybrid collection ." Target DNA is allowed to hybridize with a radioactively (35 S) labelled detector probe and a biotinylated capture DNA . The formed hybrids are subsequently collected onto microtitre wells coated with streptavidin, from which they are eluted for detection by scintillation counting (Fig. 1) . Detectable label ( 35 S) is bound to the well only if the target DNA is present in the reaction .
Stability of the probe The use of the 35S isotope as label of the probe DNA has a number of advantages as compared to 32 P . Because it has a longer half-life (87 . 2 days vs 14 . 3 days for 32 P) it is more suitable for labelling the probes . The stability of a HPV 16 DNA probe, labelled with 35 S by nick translation was tested during storage up to 13 weeks using the solution hybridization assay containing low or high amounts of HPV 16 target DNA . The input of the radioactivity was kept constant (500,000 cpm) . No significant change in the signals was observed during the period of analysis (Table 1) .
Collection of the hybrids The effect of the amount of biotinylated capture DNA on collection of the hybrids was studied . The collection rate is dependent on the concentration of the captures . However, the binding capacity of the microtitre well is limited . Therefore, increase of the HPV 16 capture concentration in the hybridization assay over the capacity threshold caused a gradual decrease in the signal (Fig . 2) . On the other hand, quantities lower than 2 X 109 molecules lowered the sensitivity of the assay . With the optimal capture DNA concentration the same sensitivity was reached as by
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(a) Hybridization in solution
capture
target
probe
hybrid
(b) Affinity-based hybrid collection
--c
--c
affinity matrix
hybrid
detectable complex
Fig. 1 . Principle of sandwich hybridization in solution . (a) Biotinylated capture DNA and radiolabelled probe are hybridized to the target DNA . (b) Hybrids are collected onto streptavidin coated affinity-matrix . Results are obtained by quantifying the label in the complex .
using avidin coated polystyrene beads, 24 which have a considerably higher biotin binding capacity . 25 The sufficient binding time of the hybrids into the microtitre wells was also studied . After 1 h approximately 70% of the maximal binding was achieved and the binding of the hybrids was almost completed in 2 h at 37 ° C . If the collections were done at room temperature, the signal levels decreased about 20% (data not shown) .
Table 1 .
Stability of the 35S-labelled probe
Storage of probe* (weeks)
Number of HPV 16 DNA moleculest 0
5 X 105
10,
0
30
78
6732
6
21
78
5800
8 12
36 26
97 81
5959 7351
13
17
77
6443
* HPV 16 probe was labelled with 35S radioisotope by nick translation, thereafter purified using a GeneClean kit and stored in TE-buffer at +4 ° C . f Each hybridization contained 500,000 cpm of probe . Results are shown as cpm .
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10000
1000 E
CL V o C o N
,
100
2 4
8
12
16
20
Number of capture DNA molecules (x 10 -9 ) Fig. 2.
The effect of the number of capture DNA molecules on hybridization signals . HPV 16 target
DNA, 108 molecules (A), 5 x 105 molecules (O) or negative control (x) were allowed to hybridize with 35S-labelled probe and varying amount of HPV 16 capture DNA molecules as described in materials and
methods .
Sensitivity of the assay A standard curve obtained for the HPV 16 DNA using 35 S-labelled probe in a solution hybridization reaction combined with affinity-based hybrid collection is presented in Fig . 3 . There is a linear relationship between the log of amount of target DNA and the log of radioactivity measured from the': collected hybrids . The sensitivity of the method is 5 x 105 molecules of target DNA, which constantly gives signals at least two times over the background level .
Kinetics of the hybridization reaction Figure 4 illustrates the rate of the solution hybridization reaction when the HPV 16 target and DNA reagents were used . In 3 h at 65 ° C 70-100% of the reaction, depending on the target concentration, was completed . The background level tended to increase when hybridization was prolonged up to 24 h .
Specificity of the reaction The specificity of the assay for detecting HPV 6/11, 16 and 18 DNAs was tested . HPV
10000
1000 E O o O C
100
0
5 x 105 106
5 x 10 6 107
5 x 107 10 8
Number of target DNA molecules Fig . 3 . A standard curve for HPV 16 DNA using the solution hybridization and the affinity-based hybrid collection method.
10000
E O o
1000
O C U
100
Hours Fig. 4 . Kinetics of the hybridization reaction . HPV 16 target DNA, 10 8 molecules ( •) and 5 X 105 molecules (O), were allowed to hybridize with biotinylated capture DNAs and 35 5-labelled probe DNA for different time periods at 65 ° C . The radioactivity with HPV DNA and of the background controls (A) are shown .
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Table 2.
347
Cross-reactivity of the HPV DNA reagents with HPV DNA of a different type Quantity of target DNA detected (molecules)*
DNA reagents
HPV 6
HPV 11
HPV 16
HPV 18
HPV 6/11 HPV16 HPV 18
5X105 5X105 3x10'
5X10' 4x10' >10'
>10' 5X105 >10'
3x10 8 3X10' 5X10'
* Results are given as the number of molecules which gave the same cpm-value as obtained by testing 5 X 10 5 molecules of the specific HPV DNA . Hybridizations were done as described in materials and methods .
DNAs of different types were cross-tested using the type-specific HPV DNA reagents in the hybridization assay . Cross reactions did not occur until the amount of nonidentical HPV DNA exceeded 3
X
10 8 molecules (Table 2) . The specificity of HPV
reagents was also confirmed by testing a variety of micro-organisms, isolated microbial DNA or cloning vectors in the hybridizations (Table 3) . No signal over the background was recorded even with high concentrations of unrelated DNA .
Effect of crude biological samples on HPV DNA detection To determine the effect of crude biological samples on the detection of the HPV DNA, cervical and vaginal scrapings were analyzed . A pool of HPV negative and the Table 3 .
Specificity of HPV DNA reagents Quantity tested
Micro-organism (a) Staphylococcus aureus Candida albicans Neisseria gonorrhoeae Bacteroides fragilis Escherichia coli Acinetobacter lwoffii Pseudomonas aeruginosa Streptococcus faecalis Lactobacillus acidophilus Gardnerella vaginalis Staphylococcus epidermis
2 2 2 2 2 2 2 2 2 2 2
sg DNA µg DNA µg DNA µg DNA µg DNA µg DNA µg DNA µg DNA µg DNA µg DNA jig DNA
(b) Serratia marcescens Escherichia coli jM 101 Escherichia coli HB 101 Salmonella typhimurium Chlamydia trachomatis
> 108 molecules > 108 molecules > 108 molecules > 108 molecules 4-0 X 10' EB
(c) Human cytomegalovirus Herpes simplex virus, type 1 Herpes simplex virus, type 2
2 .4 X 10' molecules of DNA 3 0 X 10' molecules of DNA 6 .0 X 10' molecules of DNA
(a) pBR 322 M13 RF
2 . 5 X 109 molecules of DNA 2 . 5 X 109 molecules of DNA
of of of of
DNA DNA DNA DNA
Various micro-organisms or their DNA or cloning vector DNA were added to the standard hybridization reactions . Reagents for HPV 6/11, 16 or 18 were used . Determinations were done in duplicates . (a) Purified DNA was tested . (b) From some bacteria a colony (at least 10 8 bacteria) from a culture plate was taken and lysed by boiling in SDS and thereafter subjected to the test . (c) SDS-lysates from cells infected with CMV, HSV 1 and HSV 2 were tested . The quantity of viral DNA was determined by the respective virus specific hybridization assay .
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15
4)
o0) N
a ô
5
oa
I 0
10
20
30
Cell suspension (pl) Fig. 5. The effect of cervical and vaginal cell suspension on the hybridization signals. Different volumes of both HPV 16 negative cell suspension with 10 8 molecules of HPV 16 target DNA (x) and HPV 16 positive cell suspension (A) were analyzed in the hybridization reaction . The signals obtained were converted to picogram amounts of HPV 16 DNA using a standard curve .
HPV 16 positive specimens were tested . Varying amounts (10-30 pl) of the negative pool were added to a standard hybridization reaction containing 1 . 34 ng (10 8 molecules) of HPV 16 DNA . Pretreated cells caused a moderate, 17-25%, decrease in the signal levels (Fig . 5), which is equivalent to 13-20% decrease in the quantity of HPV DNA detected . However, when analyzing varying amounts (10-30 pl) of the positive pool, the highest signals were clearly obtained with 30 pl of the infected cells .
Analysis of clinical specimens
With a recombinant plasmid containing the HPV genome, the sensitivity of the solution hybridization method was, as shown above, 5 x 10 5 molecules . This was chosen to be the cut-off value of the assay . The adequacy of the cut-off value was tested by analyzing 352 cervical or vaginal scrapings from 178 patients . Each specimen (30 pl) was assayed for the presence of HPV 16 . The ratio of each test result to the signal of the cut-off value was calculated . The distribution of the ratios is shown in Fig . 6 . Of the 324 negative specimens analyzed, 94% gave signals 10 . Similar distributions were obtained when the samples were analyzed for the presence of HPV 6/11 and 18 DNA . 26
Detection of human papi I lomavi ruses Negative specimens
349
Positive specimens
180
18 ...rte
150
15 0
U
120 w T r, C 0
o
12 G) T 0
90
9
0
o
o 2
60
6
30
3
0 .2
0 .4 0 .6
0 .8
1
2
10
Ratio (signal /cut-off) Fig. 6. Distribution of the ratio of the signal to the cut-off value obtained by testing of 352 cervical and vaginal scrapings for the presence of HPV 16 DNA . The cut-off value was the signal obtained with 5 X 105 molecules of HPV 16 DNA.
DISCUSSION Practical application of nucleic acid hybridization to the diagnosis of infectious diseases requires that the procedures be simple and rapid . Solution hybridization offers several benefits over solid phase nucleic acid hybridization assays ; it is easy to perform, requires less handling of the samples and the rate of the hybridization reaction in solution is faster than in mixed phase reactions ." We have developed a method based on sandwich hybridization in solution followed by affinity-based hybrid collection for specific detection of HPV DNA from cervical and vaginal scrapings . In this particular technique the detector probes are labelled with 355 isotope by nick-translation procedure . The use of 35 S-labelled probes is convenient because of their long half-life and weak ß-emitter nature . The probes used in this assay cover a large region of the target DNA enabling the detection even when parts of the HPV genome are lost, which often takes place during integration of the DNA . 28 The microtitre plate format in affinity-based hybrid collection, also used by others ,29,3o enable simple sample handling, particularly if compared to the coated 23,24,31,32 The test was microparticles used in other solution hybridization methods . not effected by the crudeness of the samples . However, the biotinylated capture concentration used during collection was critical . The capacity of the streptavidin coated well for binding biotin was much higher than its capacity to bind biotinylated DNA . The limited capacity might be a problem if samples containing high amounts of free biotin are analyzed . However, the sensitivity of the assay
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obtained using microtitre wells was comparable to that obtained using beads with high biotin binding capacity as the solid phase ." The sensitivity of the test was 5 X 105 molecules, which is close to the theoretical limit for scintillation counting of 35 S-labelled DNA . It was comparable to radioactive probes, labelled with 32 P and 125 1, used in many solution hybridization assays . Future improvement of this assay format includes the adaptation of a sensitive nonradioactive detection technique such as time-resolved fluorometry with europiumchelates as label . 33,34 To demonstrate the potential application of the assay described in this paper clinical samples were analyzed for the presence of HPV 16 DNA . As pretreatment, boiling the scrapings in the presence of an alkaline detergent solution was sufficient to solubilize the cells and to release the target DNA for hybridization . The decrease in the signal level, caused by crude sample, was low and the negative specimen often gave background signals less than negative buffer controls . Thus the test results, obtained as numerical values, allow unequivocal interpretation of the results . Positive samples are clearly distinguished from the negative ones . From the results obtained in this study and experiments described earlier, 13 it can be concluded that a solution hybridization format is suitable for the identification of HPV DNA from cervicovaginal scrapings . Using this method a large number of samples can be analyzed within one working day and only standard laboratory equipment is required . Based on the method presented we have developed an AffiProbe test kit for identification of the HPV types 6/11, 16 and 18 from cervical and vaginal scrapings . A clinical study for the evaluation of the AffiProbe test kit is 26 currently being conducted .
ACKNOWLEDGEMENTS We thank Drs Hans Söderlund and A .-C . Syvänen for fruitful discussions . Excellent technical assistance of Seija Lehto and Jaana Raussi is highly acknowledged . The clinical specimens were kindly provided by Dr . Pekka Nieminen from the Department of Obstetrics and Gynecology, University Central Hospital, Helsinki .
REFERENCES 1 . de Villiers, E .-M ., Wagner, D., Schneider, A . et al. (1987). Human papillomavirus infection in women with and without abnormal cervical cytology . Lancet ii, 703-6 . 2 . Syrjänen, K ., Hakama, M ., Saarikoski, S . et al. (1990). Prevalence, incidence and estimated life-time risk of cervical human papillomavirus infection in nonselected Finnish female populations . Sexually Transmitted Diseases, 17, 15-9. 3 . Gissmann, L., Wolnik, L ., lkenberg, H ., Koldovsky, U ., Schnurch, H . G . & zur Hausen, H . (1983) . Human papillomavirus types 6 and 11 DNA sequences in genital and laryngeal papillomas and in some cervical cancers . Proceedings of the National Academy of Science, USA 80, 560-3 . 4 . Gissmann, L., de Villiers, E .-M . & zur Hausen, H . (1982) . Analysis of human genital warts (condylomata acuminata) and other genital tumors for human papillomavirus type 6 DNA . International journal of Cancer 29, 143-6 . 5 . Durst, M ., Gissmann, L ., lkenberg, H . & zur Hausen, H . (1983). A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions . Proceedings of the National Academy of Science, USA 80, 3812-5 . 6 . Boshart, M ., Gissmann, L ., lkenberg, H ., Kleinheinz, A., Scheurlen, W . & zur Hausen, H . (1984) . A new
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type of papillomavirus DNA, its presence in genital cancer biopsies and in lines derived from cervical cancer . EMBO Journal 3, 1151-7 . 7. Lörincz, A . T ., Lancaster, W . D . & Temple, G . F . (1986) . Cloning and characterization of the DNA of a new human papillomavirus from a woman with dysplasia of the uterine cervix . Journal of Virology 58, 225-9 . 8 . Beaudenon, S ., Kremsdorf, D ., Croissant, O ., Jablonska, S . & Wain-Hobson, S . (1986) . A novel type of human papillomavirus associated with genital neoplasias . Nature 321, 246-9 . 9 . zur Hausen, H . (1989) . Papillomaviruses in anogenital cancer as a model to understand the role of viruses in human cancers . Cancer Research 49, 4677-81 . 10 . zur Hausen, H . & Schneider, A . (1987) . The role of papillomavirus in anogenital cancer . In The Papovaviridae 2. (Salzman, N . P., Howley, P ., eds) pp . 245-63 . New York: Plenum Publishing Corporation . 11 . Schneider, A . (1987) . Methods of identification of human papillomaviruses . In Papillomaviruses and Human Disease . (Syrjänen, K . J ., Gissmann, Z ., Koss, L . G ., eds) pp . 19-39. Berlin : Springer-Verlag . 12 . Roman, A . & Fife, K . H . (1989) . Human papillomaviruses : Are we ready to type? Clinical Microbiology Reviews 2, 166-90 . 13 . Ranki, M., Palva, A ., Virtanen, M ., Laaksonen, M. & Söderlund, H . (1983). Sandwich hybridization as a convenient method for the detection of nucleic acids in crude samples . Gene 21, 77-85 . 14 . Virtanen, M ., Laaksonen, M ., Söderlund, H ., Palva, A ., Halonen, P. & Ranki, M . (1983). Novel test for rapid viral diagnosis : detection of adenovirus in nasopharyngeal aspirates by means of nucleic acid sandwich hybridization . Lancet i, 381-3 . 15 . Virtanen, M ., Syvänen, A.-C ., Oram, J ., Söderlund, H . & Ranki, M. (1984). Cytomegalovirus in urine : Detection of viral DNA by sandwich hybridization . Journal of Clinical Microbiology 20, 1083-8 . 16. Parkkinen, S ., Mäntyjärvi, R ., Syrjänen, K . & Ranki, M . (1986) . Detection of human papillomavirus DNA by nucleic acid sandwich hybridization method from cervical scrapings . Journal of Medical Virology 20, 279-88 . 17 . Parkkinen, S . (1988). Nucleic acid sandwich hybridization in detection of HPV 16 DNA : technique and its clinical application . Journal of Virological Methods 19, 69-77 . 18 . Urdea, M . S ., Running, J . A ., Horn, T ., Clyne, J ., Ku, L . & Warner, B . D . (1987). A novel method for the rapid detection of specific nucleotide sequences in crude biological samples without blotting or radioactivity: application to the analysis of hepatitis B virus in human serum . Gene 61, 253-64. 19 . Palva, A . M . (1983). omp A gene in the detection of Escherichia coli and other Enterobacteriaceae by nucleic acid sandwich hybridization . Journal of Clinical Microbiology 18, 92-100 . 20 . Kolberg, J . A ., Besemer, D . J ., Stempier, M . M . & Urdea, M . S . (1989) . The specificity of pilin DNA sequences for the detection of pathogenic Neisseria . Molecular and Cellular Probes 3, 59-72 . 21 . Palva, A ., Jousimies-Somer, H ., Saikku, P., Väänänen, P ., Söderlund, H . & Ranki, M . (1984) . Detection of Chlamydia trachomatis by nucleic acid sandwich hybridization . FEMS Letters 23, 83-9. 22 . Ribgy, P . W . J ., Dieckmann, M., Rhodes, C . & Berg, P. (1977) . Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I . Journal of Molecular Biology 113, 237-51 . 23 . Syvänen, A .-C ., Laaksonen, M. & Söderlund, H . (1986) . Fast quantification of nucleic acid affinitybased hybrid collection . Nucleic Acids Research 14, 5037-48 . 24 . Parkkinen, S ., Mäntyjärvi, R ., Syrjänen, K ., Syvänen, A . C . & Ranki, M . (1989) . Sandwich hybridization in solution . A rapid method to screen HPV 16 DNA in cervical scrapes . Molecular and Cellular Probes 3, 1-11 . 25 . Syvänen, A .-C., Bengtström, M ., Tenhunen, J . & Söderlund, H . (1988) . Quantification of polymerase chain reaction products by affinity-based hybrid collection . Nucleic Acids Research 16, 11327-38 . 26 . Ranki, M ., Leinonen, A . W ., Jalava, T . et al. (1990) . AffiProbe HPV test kit in type-specific detection of human papillomavirus DNA from genital scrapes . Journal of Clinical Microbiology (in press) . 27 . Flavell, R . A ., Birfelder, E. J ., Sanders, P . M . & Borst, P . (1974) . DNA-DNA hybridization on nitrocellulose filters . 1 . General considerations and non-ideal kinetics . European Journal of Biochemistry 47, 535-43 . 28 . Durst, M . (1987) . Physical state of papillomavirus DNA in tumors. In Papillomaviruses and Human Disease . (Syrjänen, K. J ., Gissmann, L., Koss, L . G ., eds) . pp . 393-408 . Berlin: Springer-Verlag . 29 . Rashtchian, A ., Eldredge, J ., Ottaviani, M . et al. (1987) . Immunological capture of nucleic acid hybrids and application to nonradioactive DNA probe assay . Clinical Chemistry 33, 1526-30 . 30. Urdea, M . S ., Kolberg, J ., Clyne, J . et al . (1989) . Application of a rapid non-radioisotopic nucleic acid
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analysis system to the detection of sexually transmitted disease-causing organisms and their associated antimicrobial resistance . Clinical Chemistry 35, 1571-5 . 31 . Yehle, C . O ., Patterson, W . L ., Boguslawski, S . J ., Albarella, J . P ., Yip, K . F . & Carrico, R . J . (1987) . A solution hybridization assay for ribosomal RNA from bacteria using biotinylated DNA probes and enzyme-labeled antibody to DNA :RNA . Molecular and Cellular Probes 1, 177-93 . 32 . Thompson, J ., Solomon, R ., Pellegrino, M . et al. (1989) . A noise-free molecular hybridization procedure for measuring RNA in cell lysates . Analytical Biochemistry 181, 371-8.,' 33 . Dahlén, P ., Syvänen, A .-C ., Hurskainen, P. et al . (1987). Sensitive detection of genes by sandwich hybridization and time-resolved fluorometry. Molecular and Cellular Probes 1, 159-68 . 34 . Dahlén, P ., Hurskainen, P ., Lövgren, T. & Hyypiä, T. (1988) . Time-resolved fluorometry for the identification of viral DNA in clinical specimens . Journal of Clinical Microbiology 26, 2434-6 .
A rapid solution hybridization method for detection of human papi I lomavi ruses Tarja Jalava, Arja Kallio,* Antti W . Leinonen and Marjut Ranki Orion Corporation, Orion Pharmaceutica, Biotechnology, Valimotie 7, SF-00380 Helsinki, Finland (Received 23 January 1990, Accepted 19 February 1990)
Nucleic acid hybridization methods in routine diagnosis of micro-organisms have been limited by the tedious assay procedures . We have previously described the sandwich hybridization method which allows convenient testing of biological specimens . In this paper we describe the adaptation of the solution hybridization method into the microtitre plate format using 35 S-isotope as label . Using 3-hour hybridization followed by 2-hour collection of the hybrids a sensitivity of 5 x 10' target DNA molecules was achieved . The method was applied for identification of human papillomaviruses in crude gynaecological specimens . A simple 1-day assay protocol was achieved with high HPV type specificity . The specificity was confirmed by testing a variety of unrelated microorganisms, none of which gave a positive signal in the test . Results, obtained as numerical values, were easy to interpret; positive and negative samples gave clearly distinguishable signals .
KEYWORDS : nucleic acid hybridization in solution, diagnostics, human papillomavirus .
INTRODUCTION There is a need for a technique providing convenient, sensitive and specific determination of human papillomavirus (HPV) DNA in infected cells . The predominant HPV types commonly found in the genital area are 6, 11, 16, 18, 31 and 33 .' -a The HPV types 6 and 11 are frequently identified in benign tumours' , ' while the types 16 and 18 account for 70% of the HPV types associated with severe dysplasias and invasive carcinoma of the cervix . 9' 10 PapiIlomaviruses cannot be cultivated and viral antigens are not reliably detected in the clinical specimens . Indirect evidence of genital HPV infection can be obtained through physical examinations and by observing the presence of characteristic cellular changes in PAP smears . The methods currently providing information on the HPV type present are the detection of viral DNA using nucleic acid hybridization techniques ." *Author to whom correspondence should be addressed .
0890-8508/90/050341 + 12 $03 .00/0
© 1990 Academic Press Limited 341
3 42
T . jalava et al.
A variety of DNA techniques are used in research for detection and typing of HPV . These include analyses which require purification of DNA (Southern blot, dotblot) or which can be performed directly on tissue sections and scrapings (in situ 12 and filter in situ hybridizations) . Nucleic acid sandwich hybridization assay," used in this study, has been previously demonstrated to be a suitable method for testing crude samples for the presence of viruses"" and bacteria . 19" In this paper we describe the development of a simple and rapid sandwich hybridization assay for detection of HPV DNA . The method takes advantage of the speed and sensitivity of hybridization in solution, utilizes 35 S label in the detection of the hybrids and microtitre plates for affinity-based hybrid collection . This assay format is suitable for routine diagnostic purposes . The analysis of HPV DNA in cervical and vaginal scrapings by this method is presented .
MATERIALS AND METHODS DNA reagents The HPV 16 DNA fragments, used as probes in this study have been previously described ." The 2 . 8 kb Pst I fragment of HPV 16 DNA, cloned in pBR 322 was the detector probe . The 1 . 7 kb and 1 . 1 kb Pst I fragments, cloned into the bacteriophage M13 mp 10 and 11, were used as capture probes . The DNA reagents for HPV types 6/11 and 18 were prepared by an analogous strategy . As control targets recombinant plasmids, i .e . pBR 322 containing the HPV genome, were used . The detector probes were labelled by nick-translation 22 using 35S-dCTP (Amersham, UK) . The probes were purified using either a Sephadex G-25 (Pharmacia LKB, Sweden) column followed by phenol extraction and ethanol precipitation, or a GeneClean kit (BIO -1 DNA 101, USA) . The specific activities of the probes were about 2 . 5 x 108 cpm ltg and the capture DNAs were chemically biotinylated (Bengtström and jungellNortamo, to be published) .
The hybridization reaction The probe DNAs were denaturated in a 0 . 2% SDS solution and the recombinant DNA targets were denatured in a solution containing 0E5% SDS, 0 . 2 M NaOH and 0. 4 mg ml -1 herring sperm DNA (HS-DNA) for 5 min at 100 ° C . The hybridizations were carried out in eppendorf tubes in a final volume of 110 .tl at 65°C for 3 h . The reaction mixture contained 0 . 66 M NaCl, 65 mm sodium-citrate, 0 . 3 mm EDTA, 0 . 1 M P0 4 -buffer (pH 6 . 6), 0 . 02% Ficoll, 0. 2% polyvinylpyrrolidone, 0 . 5% polyethyleneglycol (4000), 1 . 2 mg ml -1 BSA and the denatured and neutralized target DNA, 500,000 cpm of denatured probe DNA and 1 . 0-1 . 2 X 1010 molecules of biotinylated capture DNAs .
Collection and measurement of the hybrids After hybridization the hybrids were collected onto microtitre plates coated with straptavidin (Orion Diagnostica, Finland) . The collection was for 2 h at 37 ° C using a
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microtitre plate shaker. After collection the plates were washed with 150 Al of prewarmed (+50 ° C) washing solution for 2 min six times ." The formed hybrids were then eluted twice with 110 pl of 0 . 2 M NaOH for 5 min and the radioactivity was quantified by counting in a scintillation counter for 5 min . The results are given as the mean of five parallel determinations .
Clinical specimens Cervical and vaginal scrapes were collected with an endocervical brush . The cells were suspended in 5 ml of 0 . 05 m phosphate buffered saline (PBS) and pelleted by centrifugation . The cell pellets were stored at - 20°C . Before analysis the cells were suspended in 100 pl of PBS . Prior to hybridization the patient samples were divided in aliquots and denatured in a solution containing 0 . 5% SDS, 0 . 2 m NaOH and 0. 4 mg HS-DNA ml - ' for 5 min at 100 ° C .
RESULTS Principle of the method The method is based on sandwich hybridization in solution followed by affinitybased hybrid collection ." Target DNA is allowed to hybridize with a radioactively (35 S) labelled detector probe and a biotinylated capture DNA . The formed hybrids are subsequently collected onto microtitre wells coated with streptavidin, from which they are eluted for detection by scintillation counting (Fig. 1) . Detectable label ( 35 S) is bound to the well only if the target DNA is present in the reaction .
Stability of the probe The use of the 35S isotope as label of the probe DNA has a number of advantages as compared to 32 P . Because it has a longer half-life (87 . 2 days vs 14 . 3 days for 32 P) it is more suitable for labelling the probes . The stability of a HPV 16 DNA probe, labelled with 35 S by nick translation was tested during storage up to 13 weeks using the solution hybridization assay containing low or high amounts of HPV 16 target DNA . The input of the radioactivity was kept constant (500,000 cpm) . No significant change in the signals was observed during the period of analysis (Table 1) .
Collection of the hybrids The effect of the amount of biotinylated capture DNA on collection of the hybrids was studied . The collection rate is dependent on the concentration of the captures . However, the binding capacity of the microtitre well is limited . Therefore, increase of the HPV 16 capture concentration in the hybridization assay over the capacity threshold caused a gradual decrease in the signal (Fig . 2) . On the other hand, quantities lower than 2 X 109 molecules lowered the sensitivity of the assay . With the optimal capture DNA concentration the same sensitivity was reached as by
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(a) Hybridization in solution
capture
target
probe
hybrid
(b) Affinity-based hybrid collection
--c
--c
affinity matrix
hybrid
detectable complex
Fig. 1 . Principle of sandwich hybridization in solution . (a) Biotinylated capture DNA and radiolabelled probe are hybridized to the target DNA . (b) Hybrids are collected onto streptavidin coated affinity-matrix . Results are obtained by quantifying the label in the complex .
using avidin coated polystyrene beads, 24 which have a considerably higher biotin binding capacity . 25 The sufficient binding time of the hybrids into the microtitre wells was also studied . After 1 h approximately 70% of the maximal binding was achieved and the binding of the hybrids was almost completed in 2 h at 37 ° C . If the collections were done at room temperature, the signal levels decreased about 20% (data not shown) .
Table 1 .
Stability of the 35S-labelled probe
Storage of probe* (weeks)
Number of HPV 16 DNA moleculest 0
5 X 105
10,
0
30
78
6732
6
21
78
5800
8 12
36 26
97 81
5959 7351
13
17
77
6443
* HPV 16 probe was labelled with 35S radioisotope by nick translation, thereafter purified using a GeneClean kit and stored in TE-buffer at +4 ° C . f Each hybridization contained 500,000 cpm of probe . Results are shown as cpm .
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10000
1000 E
CL V o C o N
,
100
2 4
8
12
16
20
Number of capture DNA molecules (x 10 -9 ) Fig. 2.
The effect of the number of capture DNA molecules on hybridization signals . HPV 16 target
DNA, 108 molecules (A), 5 x 105 molecules (O) or negative control (x) were allowed to hybridize with 35S-labelled probe and varying amount of HPV 16 capture DNA molecules as described in materials and
methods .
Sensitivity of the assay A standard curve obtained for the HPV 16 DNA using 35 S-labelled probe in a solution hybridization reaction combined with affinity-based hybrid collection is presented in Fig . 3 . There is a linear relationship between the log of amount of target DNA and the log of radioactivity measured from the': collected hybrids . The sensitivity of the method is 5 x 105 molecules of target DNA, which constantly gives signals at least two times over the background level .
Kinetics of the hybridization reaction Figure 4 illustrates the rate of the solution hybridization reaction when the HPV 16 target and DNA reagents were used . In 3 h at 65 ° C 70-100% of the reaction, depending on the target concentration, was completed . The background level tended to increase when hybridization was prolonged up to 24 h .
Specificity of the reaction The specificity of the assay for detecting HPV 6/11, 16 and 18 DNAs was tested . HPV
10000
1000 E O o O C
100
0
5 x 105 106
5 x 10 6 107
5 x 107 10 8
Number of target DNA molecules Fig . 3 . A standard curve for HPV 16 DNA using the solution hybridization and the affinity-based hybrid collection method.
10000
E O o
1000
O C U
100
Hours Fig. 4 . Kinetics of the hybridization reaction . HPV 16 target DNA, 10 8 molecules ( •) and 5 X 105 molecules (O), were allowed to hybridize with biotinylated capture DNAs and 35 5-labelled probe DNA for different time periods at 65 ° C . The radioactivity with HPV DNA and of the background controls (A) are shown .
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Table 2.
347
Cross-reactivity of the HPV DNA reagents with HPV DNA of a different type Quantity of target DNA detected (molecules)*
DNA reagents
HPV 6
HPV 11
HPV 16
HPV 18
HPV 6/11 HPV16 HPV 18
5X105 5X105 3x10'
5X10' 4x10' >10'
>10' 5X105 >10'
3x10 8 3X10' 5X10'
* Results are given as the number of molecules which gave the same cpm-value as obtained by testing 5 X 10 5 molecules of the specific HPV DNA . Hybridizations were done as described in materials and methods .
DNAs of different types were cross-tested using the type-specific HPV DNA reagents in the hybridization assay . Cross reactions did not occur until the amount of nonidentical HPV DNA exceeded 3
X
10 8 molecules (Table 2) . The specificity of HPV
reagents was also confirmed by testing a variety of micro-organisms, isolated microbial DNA or cloning vectors in the hybridizations (Table 3) . No signal over the background was recorded even with high concentrations of unrelated DNA .
Effect of crude biological samples on HPV DNA detection To determine the effect of crude biological samples on the detection of the HPV DNA, cervical and vaginal scrapings were analyzed . A pool of HPV negative and the Table 3 .
Specificity of HPV DNA reagents Quantity tested
Micro-organism (a) Staphylococcus aureus Candida albicans Neisseria gonorrhoeae Bacteroides fragilis Escherichia coli Acinetobacter lwoffii Pseudomonas aeruginosa Streptococcus faecalis Lactobacillus acidophilus Gardnerella vaginalis Staphylococcus epidermis
2 2 2 2 2 2 2 2 2 2 2
sg DNA µg DNA µg DNA µg DNA µg DNA µg DNA µg DNA µg DNA µg DNA µg DNA jig DNA
(b) Serratia marcescens Escherichia coli jM 101 Escherichia coli HB 101 Salmonella typhimurium Chlamydia trachomatis
> 108 molecules > 108 molecules > 108 molecules > 108 molecules 4-0 X 10' EB
(c) Human cytomegalovirus Herpes simplex virus, type 1 Herpes simplex virus, type 2
2 .4 X 10' molecules of DNA 3 0 X 10' molecules of DNA 6 .0 X 10' molecules of DNA
(a) pBR 322 M13 RF
2 . 5 X 109 molecules of DNA 2 . 5 X 109 molecules of DNA
of of of of
DNA DNA DNA DNA
Various micro-organisms or their DNA or cloning vector DNA were added to the standard hybridization reactions . Reagents for HPV 6/11, 16 or 18 were used . Determinations were done in duplicates . (a) Purified DNA was tested . (b) From some bacteria a colony (at least 10 8 bacteria) from a culture plate was taken and lysed by boiling in SDS and thereafter subjected to the test . (c) SDS-lysates from cells infected with CMV, HSV 1 and HSV 2 were tested . The quantity of viral DNA was determined by the respective virus specific hybridization assay .
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15
4)
o0) N
a ô
5
oa
I 0
10
20
30
Cell suspension (pl) Fig. 5. The effect of cervical and vaginal cell suspension on the hybridization signals. Different volumes of both HPV 16 negative cell suspension with 10 8 molecules of HPV 16 target DNA (x) and HPV 16 positive cell suspension (A) were analyzed in the hybridization reaction . The signals obtained were converted to picogram amounts of HPV 16 DNA using a standard curve .
HPV 16 positive specimens were tested . Varying amounts (10-30 pl) of the negative pool were added to a standard hybridization reaction containing 1 . 34 ng (10 8 molecules) of HPV 16 DNA . Pretreated cells caused a moderate, 17-25%, decrease in the signal levels (Fig . 5), which is equivalent to 13-20% decrease in the quantity of HPV DNA detected . However, when analyzing varying amounts (10-30 pl) of the positive pool, the highest signals were clearly obtained with 30 pl of the infected cells .
Analysis of clinical specimens
With a recombinant plasmid containing the HPV genome, the sensitivity of the solution hybridization method was, as shown above, 5 x 10 5 molecules . This was chosen to be the cut-off value of the assay . The adequacy of the cut-off value was tested by analyzing 352 cervical or vaginal scrapings from 178 patients . Each specimen (30 pl) was assayed for the presence of HPV 16 . The ratio of each test result to the signal of the cut-off value was calculated . The distribution of the ratios is shown in Fig . 6 . Of the 324 negative specimens analyzed, 94% gave signals 10 . Similar distributions were obtained when the samples were analyzed for the presence of HPV 6/11 and 18 DNA . 26
Detection of human papi I lomavi ruses Negative specimens
349
Positive specimens
180
18 ...rte
150
15 0
U
120 w T r, C 0
o
12 G) T 0
90
9
0
o
o 2
60
6
30
3
0 .2
0 .4 0 .6
0 .8
1
2
10
Ratio (signal /cut-off) Fig. 6. Distribution of the ratio of the signal to the cut-off value obtained by testing of 352 cervical and vaginal scrapings for the presence of HPV 16 DNA . The cut-off value was the signal obtained with 5 X 105 molecules of HPV 16 DNA.
DISCUSSION Practical application of nucleic acid hybridization to the diagnosis of infectious diseases requires that the procedures be simple and rapid . Solution hybridization offers several benefits over solid phase nucleic acid hybridization assays ; it is easy to perform, requires less handling of the samples and the rate of the hybridization reaction in solution is faster than in mixed phase reactions ." We have developed a method based on sandwich hybridization in solution followed by affinity-based hybrid collection for specific detection of HPV DNA from cervical and vaginal scrapings . In this particular technique the detector probes are labelled with 355 isotope by nick-translation procedure . The use of 35 S-labelled probes is convenient because of their long half-life and weak ß-emitter nature . The probes used in this assay cover a large region of the target DNA enabling the detection even when parts of the HPV genome are lost, which often takes place during integration of the DNA . 28 The microtitre plate format in affinity-based hybrid collection, also used by others ,29,3o enable simple sample handling, particularly if compared to the coated 23,24,31,32 The test was microparticles used in other solution hybridization methods . not effected by the crudeness of the samples . However, the biotinylated capture concentration used during collection was critical . The capacity of the streptavidin coated well for binding biotin was much higher than its capacity to bind biotinylated DNA . The limited capacity might be a problem if samples containing high amounts of free biotin are analyzed . However, the sensitivity of the assay
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T. Jalava et al .
obtained using microtitre wells was comparable to that obtained using beads with high biotin binding capacity as the solid phase ." The sensitivity of the test was 5 X 105 molecules, which is close to the theoretical limit for scintillation counting of 35 S-labelled DNA . It was comparable to radioactive probes, labelled with 32 P and 125 1, used in many solution hybridization assays . Future improvement of this assay format includes the adaptation of a sensitive nonradioactive detection technique such as time-resolved fluorometry with europiumchelates as label . 33,34 To demonstrate the potential application of the assay described in this paper clinical samples were analyzed for the presence of HPV 16 DNA . As pretreatment, boiling the scrapings in the presence of an alkaline detergent solution was sufficient to solubilize the cells and to release the target DNA for hybridization . The decrease in the signal level, caused by crude sample, was low and the negative specimen often gave background signals less than negative buffer controls . Thus the test results, obtained as numerical values, allow unequivocal interpretation of the results . Positive samples are clearly distinguished from the negative ones . From the results obtained in this study and experiments described earlier, 13 it can be concluded that a solution hybridization format is suitable for the identification of HPV DNA from cervicovaginal scrapings . Using this method a large number of samples can be analyzed within one working day and only standard laboratory equipment is required . Based on the method presented we have developed an AffiProbe test kit for identification of the HPV types 6/11, 16 and 18 from cervical and vaginal scrapings . A clinical study for the evaluation of the AffiProbe test kit is 26 currently being conducted .
ACKNOWLEDGEMENTS We thank Drs Hans Söderlund and A .-C . Syvänen for fruitful discussions . Excellent technical assistance of Seija Lehto and Jaana Raussi is highly acknowledged . The clinical specimens were kindly provided by Dr . Pekka Nieminen from the Department of Obstetrics and Gynecology, University Central Hospital, Helsinki .
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The specificity of pilin DNA sequences for the detection of pathogenic Neisseria . Molecular and Cellular Probes 3, 59-72 . 21 . Palva, A ., Jousimies-Somer, H ., Saikku, P., Väänänen, P ., Söderlund, H . & Ranki, M . (1984) . Detection of Chlamydia trachomatis by nucleic acid sandwich hybridization . FEMS Letters 23, 83-9. 22 . Ribgy, P . W . J ., Dieckmann, M., Rhodes, C . & Berg, P. (1977) . Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I . Journal of Molecular Biology 113, 237-51 . 23 . Syvänen, A .-C ., Laaksonen, M. & Söderlund, H . (1986) . Fast quantification of nucleic acid affinitybased hybrid collection . Nucleic Acids Research 14, 5037-48 . 24 . Parkkinen, S ., Mäntyjärvi, R ., Syrjänen, K ., Syvänen, A . C . & Ranki, M . (1989) . Sandwich hybridization in solution . A rapid method to screen HPV 16 DNA in cervical scrapes . Molecular and Cellular Probes 3, 1-11 . 25 . Syvänen, A .-C., Bengtström, M ., Tenhunen, J . & Söderlund, H . (1988) . Quantification of polymerase chain reaction products by affinity-based hybrid collection . Nucleic Acids Research 16, 11327-38 . 26 . Ranki, M ., Leinonen, A . W ., Jalava, T . et al. (1990) . AffiProbe HPV test kit in type-specific detection of human papillomavirus DNA from genital scrapes . Journal of Clinical Microbiology (in press) . 27 . Flavell, R . A ., Birfelder, E. J ., Sanders, P . M . & Borst, P . (1974) . DNA-DNA hybridization on nitrocellulose filters . 1 . General considerations and non-ideal kinetics . European Journal of Biochemistry 47, 535-43 . 28 . Durst, M . (1987) . Physical state of papillomavirus DNA in tumors. In Papillomaviruses and Human Disease . (Syrjänen, K. J ., Gissmann, L., Koss, L . G ., eds) . pp . 393-408 . Berlin: Springer-Verlag . 29 . Rashtchian, A ., Eldredge, J ., Ottaviani, M . et al. (1987) . Immunological capture of nucleic acid hybrids and application to nonradioactive DNA probe assay . Clinical Chemistry 33, 1526-30 . 30. Urdea, M . S ., Kolberg, J ., Clyne, J . et al . (1989) . Application of a rapid non-radioisotopic nucleic acid
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