Immunology Today, vol. 6, No. 9, 1985
268
Nucleic acid hybridization-an alternative tool in diagnostic microbiology Ulf Pettersson and Timo Hyypi~i* The use of radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs) has revolutionized diagnost# microbiology. Their high specificity and sensitivity make them versatile, they are simple to carry out eitherfor direct detection of microorganisms in specimens orfor serological diagnosis, and they can easily and reliably be standardized. Monoclonal antibodies have further improved these immunoassays. However, the development of simple and highly sensitive detection methods for nucleic acids has neverthelesspromoted an interest also in diagnostic methods based on nucleicacid hybridization. Here Ulf Pettersson and Timo Hyypi/i discuss methods which are likely to becomea usfful complement to the immunoassays in the nearfuture. Duplex D N A is composed of two complementary polynucleotide chains. The D N A molecule can easily be denatured by heat or by increasing the pH. Denaturation leads to a reversible separation of the two complementary strands. The latter will under the appropriate conditions gradually reunite and reform a duplex molecule, identical to the original one. The reassociation reaction is highly specific: two separated polynudeotide chains will only reassociate if they are complementary. If a foreign polynucleotide chain is introduced in a solution of denatured D N A it will form a duplex with one of the strands provided that it shares sequences with the denatured DNA. This reaction, designated nucleic acid hybridization, occurs both for R N A and DNA. The requirement for complementarity, however, is not absolute. A certain amount of mismatch is tolerated, still allowing duplex formation. This means that non-identical but related nucleic acids can participate in the hybridization reaction. The resulting hybrids are, however, less stable when subjected to heat in buffers of low ionic-strength. This property provides the basis for a method to discriminate between precisely and imprecisely matched duplexes. The sensitivity and the specificity of the hybridization method is perhaps best illustrated by its use for detection of mammalian genes; employing the Southern hybridization procedure it is possible to detect the presence of a single-copy gene using a probe that is less than 20 nucleotides long. The probe represents in this case less than 10 -8 of the sequences in the total D N A sample. P r a c t i c a l considerations For a detailed description of the nudeic acid hybridization methods, the review by Meinkoth and WahP is recommended. The principle behind the practical use of the hybridization technique is simple; two complementary polynudeotide chains will form a duplex molecule (hybrid) at the appropriate temperature and at the appropriate cation concentration. Oligonucleotides consisting of as few as 12 nucleotides are able to form specific and
Department of Medical Genetics, Uppsala University, Biomedical Center, Box 589, S-75123 Uppsala~ Sweden. *Permanent address: Department of Virology, University of Turku, sF-20520 Turku, Finland. © 1985, Elsevier Science Publishers B.V., Amsterdam 0167 - 4919/85/$02.00
stable hybrids, albeit at a reduced temperature. R N A can also form a hybrid with a complementary D N A or R N A strand and it is in fact possible to arrange the hybridization conditions in such a way that R N A / D N A hybrids are formed in the absence of D N A / D N A duplex formation 2. A practical problem with the hybridization reaction is that two competing reactions take place; the sample D N A will reassociate with itself while reacting with the nucleic acid that is used for detection, the so called probe. This obviously reduces the efficiency of the hybridization reaction. The problem is usually circumvented by immobilization of the D N A on a solid matrix, most commonly a nitrocellulose membrane. In this way renaturation is prevented while the probe can react with the exposed strands on the filter. A key problem in the practical use of hybridization reactions concerns the detection of hybrid formation. Several properties ofnudeic acids can be exploited for this purpose; the absorbance of ultraviolet light differs between single and double-stranded nucleic acids ('hyperchromicity'). The reaction can hence be followed in the spectrophotometer. This requires, however, pure D N A samples and is thus not applicable for detection of nucleic acids in crude specimens. Physical separation methods can also be used to discriminate between single and double-stranded nucleic acids. One of the most powerful methods is hydroxylapatite chromatography, which very efficiently separates single- and doublestranded DNAs. The differential sensitivity of duplex and singlestranded nucleic acids to enzymatic degradation can also be exploited. The single-strand specific nudeases are thus widely used in basic research to detect the formation of nuclease-resistant hybrids. For R N A , ribonucleases can be used to discriminate between single stranded R N A and hybrids. By far the most common way to detect hybrid formation is to use nucleic acids immobilized on nitrocellulose membranes. This method, originally described more than 20 years ago 3'4'5, is one of the most widely used in molecular biology. The principle is simple: the nucleic acid to be analysed is denatured and filtered through a nitrocellulose membrane. T h e single strands bind to the filter in such a way that they are able to interact with nucleic acids in a solution that is in contact with the filter. A radioactively labeled probe is usually added to detect
Immunology Today, vol. 6, No. 9, 1985
269 Fig. 1. Detection of adenovirus in stool specimens by hybridization.
32P-labelecladenovirus-2DNA was used as the probe. Upper row (I). The indicated amounts of adenovirus-2 (left) and HSV-1 (right) DNA were immobilizedon the filter. Lowerrow (II). Analysisof8 stoolspecimens. Specimens 1,3,6 and 7 were positive according to an adenovirus-detectingRIA, whereas specimens 2,4,5 and 8 were negative in the same assay.
the presence of a specific nucleic acid in the immobilized sample. If complementarity exists between the probe and the immobilized nucleic acid, hybridization will occur which can be measured by scintillation counting or by subjecting the filter to autoradiography. Recombinant D N A technology has in many ways facilitated nucleic acid hybridization methods. The use of genomes isolated from microorganisms as probes has several disadvantages; it is often laborious, expensive, and sometimes even impossible to prepare a nucleic acid sample of sufficient purity and homogeneity~ for use in a practical test. It is moreover desirable in many cases to utilize probes which represent restricted parts o f a genome to discriminate between closely related microorganisms. Gene technology has circumvented many of these shortcomings. Essentially any nucleic acid can be cloned in a bacterial vector and in this way can be produced in large quantities. It is also possible to derive segments from the original clone by subcloning fragments. In this way probes can be selected which represent unique parts of a genome or parts which are shared by other members of e.g. a virus family. Recent advances in gene technology provide further practical advantages. By the use of certain phage vectors it is possible to manufacture single stranded probes that are labeled to very high specific radioactivity6. It is also possible to clone fragments in vectors which allow high level transcription of the cloned sequence, thus providing a method to synthesize very highly labeled R N A probes 7. A n additional important technical improvement concerns novel methods for oligonucleotide synthesis. Commercially available synthesizers have the capacity to rapidly manufacture oligonucleotides which exceed 50 nucleotides in length. This technology opens possibilities to design very specific hybridization probes based on established nucleotide sequences. For sensitive detection the probes need to be labeled to a high specific activity. Probes can be radioactively labeled both in vivo and in vitro. In-vitro methods are, for practical reasons, much more common and the so-called nick translation method is very convenient for labeling of double-stranded D N A probes, yielding specific activities >10 a cpm//~g. 32P-phosphorous is the most common label,
although it has a short half life. 3H-labeled nucleotide precursors are usually unsatisfactory because of their unfavourable detection limit. More recently 3aS-labeled nucleotides with a half life of about 80 days have been introduced as an alternative to 3~p. Also I25I-labeled nucleic acids can be obtained both by chemical labeling and by enzymatic synthesis. Non-radioactive labeling methods are needed to convert hybridization into a practically useful diagnostic method. Several non-radioactive labeling procedures have been describedS-11, the most widely used being based on biotinylated nucleotides. Biotinylated D N A can be synthesized enzymatically and is detected by an avidinenzyme conjugate combined with a color-producing substrate. This method appears promising, although in most studies reported so far the sensitivity is less than that noted with 32P-labeled probes. Methods for direct labeling of D N A or hybrids have also been described although none has so far been evaluated in practical diagnostic tests ~0-~2.
Spot h y b r i d i z a t i o n ; a u n i v e r s a l t o o l for d e t e c t i o n of microbial DNA
A simple way to detect the microbial D N A in a specimen is to extract the nucleic acids, immobilize them on a nitrocellulose fiker and then to reveal their presence by a labeled hybridization probe. Probes consisting of D N A or R N A extracted from a cultivated microorganism, cloned D N A sequences, in vitro synthesized R N A or a synthetic oligonucleotide can be used. The method, commonly known as spot hybridization (Fig, 1), is very straightforward and has been used for the detection of a variety of microorganisms. Brandsma and Miller 13 were the first to describe a practical application of the method, i.e. the detection of Epstein-Barr virus (EBV) sequences in lymphoblastoid cells. They designed a simple protocol according to which the cells to be tested were immobilized on a nitrocellulose membrane. The D N A in the immobilized sample was denatured in situ and hybridized to a probe consisting of radioactively labeled EBV DNA. Variations of the
270 method have subsequently been reported which makes it possible to detect EBV directly in nasopharyngeleal aspirates 14. St~lhandske and Pettersson ~5used spot hybridization to detect herpes simplex virus (HSV) in infected cells. They used a cloned restriction enzyme fragment of HSV-1 D N A as the probe and were able to discriminate between HSV-1 and HSV-2 infected cells. The method exploits the fact that imprecisely matched hybrids, as obtained after hybridization to the heterologous serotype, are less stable after washing under stringent conditions (see Fig. 2 for details). The test can thus be used for HSVtyping. Subsequently, the assay has also been used for direct detection of H S V in clinical specimens16. Several investigators have used spot hybridization for detection of human cytomegalovirus (CMV) in urine specimens 17-19and here the results look very promising. The detection level is approximately 5 pg of viral DNA. An interesting and important application of the spot hybridization test is in diagnosis of hepatitis B virus (HBV). The test has been used by several investigators for direct detection of HBV-specific D N A sequences in blood 2°'21. The blood can be applied directly to the filter without purification of the D N A and the test is hence very simple to perform. The sensitivity is comparable to that obtained with currently used immunoassays. An important application of the spot hybridization test concerns detection of papillomaviruses. Here hybridization offers several advantages. Firstly the different human papillomavirus serotypes can only be discriminated by hybridization or their typical restriction-enzyme cleavage patterns 22. Secondly, it is impossible to cultivate these viruses in vitro and therefore difficult to obtain sufficient amounts of antigen for an immunoassay. This is particularly true for the important serotypes 16 and 18 which are commonly associated with human genital tumors 2~'24. Many applications in diagnostic bacteriology have also been described. Spot hybridization has, for instance, been used to detect enterotoxigenic E. coli where a particular advantage of hybridization is exploited, namely the specific detection of genes which predetermine pathogenicity 2a'26. More recently, tests based on hybridization have also been used for diagnosis of sexually transmitted diseases, like those caused by Chlamydia trachomatis 27a8and Neisseria gonorrhoeae29. A useful modification of spot hybridization, so-called sandwich hybridization, has also been described 3°. An advantage with this method is its low background, which is particularly useful when impure samples are analysed. The method has been used for detection of adenovirus in nasopharyngeal specimens s0 and C M V in urine 19.
Detection of RNA viruses The use of the spot hybridization method for the detection of RNA-containing microorganisms is somewhat more complicated since R N A binds less well to nitrocellulose and is more sensitive to high pH and to nucleases than DNA. The construction of the probes is also more tedious since cDNA copies have to be cloned rather than DNA fragments. The first practical application was the detection of the potato spindle tuber
Immunology Today, voL 6, No. 9, 1985
viroid 31. In this case the immunoassays provide no akernative to spot hybridization since viroids lack protein components. Hyypi~ et aL used a cloned probe dervied from the replicase gene of coxsackievirus B3 for detection of enteroviruses 32. The probe was capable of identifying enteroviruses in tissue cultures that had been inoculated with specimens but was not sensitive enough to detect reproducibly the virus directly in stool specimens. An interesting property of the probe is that it is broadly reacting, i.e. hybridizes to different serotypes of poliovirus, echoviruses, and all tested coxsackievirus B serotypes. This is a valuable characteristic since there is no group-reacting immunological reagent capable of detecting the entire enterovirus group. Flores et al. 33used spot hybridization to detect rotavirus R N A in stool specimens. An R N A probe produced by in-vitro transcription was used and the test was found to be at least as sensitive as the currently used ELISA for rotavirus detection. Other modifications of the spot hybridization assay The sensitivity of the hybridization test can obviously be increased if the sequence used as a probe is repeated several times in the microorganism. This property has been exploited by some investigators. Franzen et al. 34 cloned a highly reiterated sequence from the genome of Plasmodium falciparum. This sequence, which represents approximately 1% of the P. falciparum genome, was used for parasite detection in malaria patients. The test is simple to perform; D N A is extracted from a small blood sample, immobilized on a nitrocellulose membrane and hybridized to a radioactive probe containing many copies of the reiterated sequence (Fig. 3). By this method it was possible to detect approximately 25 pg of P. falciparum DNA, and the test had a sensitivity comparable to that of other tests currently in use for parasite detection, including light microscopy of stained blood smears and antigen detection by ELISA. The probe seems to recognize no other plasmodium species than P.falciparurn. A similar method has been designed for detection of the parasite Trypanosorna cruzi 35. Cloned minichromosomal repetitive DNA is used as the probe and 30 parasites are sufficient to give a positive signal in the test. Also for diagnosis ofLeishmania, a hybridization test has been described which utilizes cloned kinetoplast D N A as the probe. The test has been used to detect Leishmania in cutaneous lesions 36. Recently, a commercial assay has been introduced for detection of mycoplasmas in tissue cultures ('Geneprobe'). This test utilizes cloned sequences complementary to ribosomal RNA. Since a large number of ribosomal R N A copies exist in the microorganism, the test becomes obviously much more sensitive than tests based on probes representing single-copy genes. Are nucleic acid hybridization assays feasible alternatives to the immunoassays? Although a versatile tool, spot hybridization has not yet been introduced in the diagnostic laboratories to any significant extent. There are several reasons for this: the
Immunology Today, vol. 6, No. 9, 1985
Fig. 2. (from Ref. 15) Detection of HSV DNA by hybridization, DNA was extracted from uninfectedcells and cells infectedwith HSV-1 or HSV-2 and immobilizedon nitrocellulosefilters. A cloned fragmentfromthe HSV-1 genomewas used as the hybridizationprobe (its preciselocationin the HSV-genomeis unknown). A: DNA fromuninfectedcells. B: DNA from HSV-1 (upper spot) and HSV-2 (lowerspot) infected cells. The filter was washed at 65°C in 2 x SSC (standard citrate saline). C: The same filteras in B after washingat 65°C in 0.1 x SSC. sensitivity obtained is at best comparable to that obtained with immunoassays, the test usually requires more than 24 h to provide an answer, it requires short-lived isotopes, and is based on methodologies which are not used in a routine diagnostic laboratory. The immunoassays, with which the clinical microbiologist is already so familiar have moreover been improved dramatically thanks to hybridoma technology. However, in some circumstances the hybridization test is the only diagnostic alternative or it has certain significant advantages. Immunoassays cannot be applied for detection of viroids, since they lack any protein component and hybridization is thus the only diagnostic alternative. In the diagnosis of HBV, convenient immunoassays are available and the hybridization test offers no advantages, with regard to sensitivity or convenience. The immunoassays have, however, one serious disadvantage: they score for the presence of viral proteins rather than for infectious particles. This is a drawback because some carriers excrete viral antigens in the absence of infectious particles. The detection of the viral DNA by hybridization is thus already an important complement to the antigen-detecting assays 37. For the diagnosis of CMV, current isolation procedures are tedious and require often more than 2-3 weeks to give a definite result. The direct detection of C M V D N A in the urine specimens is an important contribution to diagnostic virology. Hybridization methods for C M V detection in urine specimens have already been developed but the sensitivity obtained is not yet sufficient to make them superior to the conventional isolation procedures. For viruses which are difficult or impossible to propagate in vitro, hybridization is a valid diagnostic alternative. This includes tests for EBV DNA in nasopharyngeal specimens. The h u m a n papillomaviruses, particularly the very important types 16 and 18, cannot be propagated
271 at all in tissue culture and there is no means yet known for enriching virus particles. Hence nucleic acid hybridization is the only existing diagnostic alternative. For the enteroviruses hybridization offers some significant advantages. Probes can, at least in theory, be designed which react with group-specific as well as typespecific parts of enteroviral genomes. Since enteroviruses are one of the major obstacles in clinical virology, hybridization methods are likely to become an important alternative to the existing immunoassays in the near future. In the analysis of stool specimens the hybridization assay again may be a useful complement, particularly for detection of rotaviruses and adenoviruses (Fig. 1) and in clinical bacteriology the specific recognition of genes in bacteria which are linked to pathogenicity is a property which ultimately should make hybridization tests attractive alternatives to currently used diagnostic procedures. Examination of microscopic slides currently dominates test procedures for parasites, which is very time-consuming and, in the case of diagnosis of malaria, subjective. Hybridization may here become an alternative for mass screening of samples, as for instance screening of blood banks. In situ hybridization is a useful variant of the hybridization method which also may find important applications in diagnostic microbiology, since it allows the identification of foreign nucleic acids in specific parts of a tissue section. Future prospects Methods detecting nucleic acids have several advantages which can be exploited in diagnostic tests: (1) They are versatile and can be used for detection of microorganisms which are unable to replicate in vitro, microorganisms which lack protein components, etc.
Fig. 3. (from Ref. 34) Detection of Plasmodium falclparum by hybridization. Row A. 250ng, 25 ng, 2.5 ng, 250pg and 25 pg ofP.falciparum DNA. Rows B-C. Samplesof patientswith parasitemiaas determinedby microscopy. B1, B2, C1, C4, C5 P. vivax. All the others P. falciparum.
272
(2) They can be used to identify essentially any specific gene in a microorganism. Immunoassays preferentially detect surface proteins or other antigens which are synthesized in abundance during replication of the microorganism. These proteins do not necessarily correlate with the pathogenic properties of the microorganism. Specific identification of a gene or part of a gene that is responsible for the pathogenicity can be achieved with nucleic acid hybridization. There is, as outlined above, already today a need for hybridization methods in clinical microbiology. Will the hybridization tests then replace the immunoassays? Probably not, unless the technique is improved dramatically. The major obstacle concerns the label of the probe. Currently the short lived isotope 32p is the most commonly used, which is a disadvantage for several reasons. Alternative labeling methods must be found before hybridization can be introduced as a routine method in the diagnostic laboratory. Non-radioactively labeled probes have been designed and the biotin-labeled probes seem to be particularly promising 3s. Also the hybridization method as such might be improved in several ways. So far most investigators have used a standard protocol according to which the specimen D N A or R N A is immobilized on a filter and cloned fragments are used for detection. Simple methods, like the use ofhydroxylapatite chromatography, single-strand specific nucleases etc., need to be further exploited. Also the availability of single-stranded DNA and R N A probes offers new possibilities for the design of more sensitive and convenient assays. Finally, the synthetic oligonucleotides may turn out to be crucial instruments for specific discrimination between closely related microorganisms. The time required to complete the test, currently 1-2 days, could also be reduced by altering the hybridization conditions. It should be kept in mind that nucleic acid hybridization methods also are applicable to other important areas of diagnostic medicine. The genetic diseases, for instance, can now be studied due to restriction enzyme fragment length polymorphism. The combined forces of investigators from many fields are likely to result in solutions to some of the practical problems which presently prevent hybridization from becoming a widely used routine method in clinical microbiology. [=~
Acknowledgements The authors are grateful to Drs Z. Dinter, H. Diderholm and G. Magnusson for valuable discussions during preparation of the manuscript.
Immunology Today, vol. 6, No. 9, 1985
References 1 Meinkoth, J. and Wahl, G. (1984) Analyt. Biochem. 138,267-284 2 Casey, J. and Davidson, N. (1977) Nud. Acids Res. 4, 1539-1552 3 Nygaard, A. P. and Hall, B. D. (1963) Biochem. Biaphys. Res. Commun. 12, 98-104 4 Nygaard, A. P. and Hall, B. D. (1964)Ji MoL Biol. 9, 125-142 5 Gillespie, D. and Spiegelman, S. (1965).]'. Mol. BioL 12, 829-842 6 Messing, J. (1983), in Methods in Enzymology (Wu, R., Grossman, L. and Moldave, K., eds). Vol. 101, pp. 20-78, Academic Press, New York 7 Green, M. R., Maniatis, T. and Melton, D. A. (1983) Cell 32,681-694 8 Langer, P. R., Waldrop, A. A. and Ward, D. C. (1981) ProcNatlAcad. Sci. USA 78, 6633-6637 9 Leafy, J. J., Bfigati, D.J. and Ward, D. C. (1983) Proc. NatlAead. Sci. USA 80, 4045-4049 10 Renz, M. and Kurz, C. (1984) Nucl. Acids Res. 12, 3435-3444 11 Tehen, P., Fuehs, R. P. P., Sage, E. and Leng, M. (1984) Proe. Natl Acad. Sci. USA 81, 3466-3470 12 Forster, A. C., McInnes, J. L., Skingle, D. C. and Symons, R. H. (1985) Nucl. Acids Res. 13, 745-761 13 Brandsma, J. and Miller, G. (1980) Proc. Natl Acad. Sci. USA 77, 6851-6855 14 Bornkamm, G. W., Desgranges, C. and Gissmann, L. (1983) Curt. Top. in Microbiol. Immunol. 104, 287-298 15 Stiilhandske, P. and Pettersson, U. (1982)J. Clin. Microbial. 15,744--747 16 Redfield, D. C., Riehman, D. D., Albanil, S., Oxman, M. N. and Wahl, G. M. (1983) Diag. Microbial Infect. Dis. 1, 117-128 17 Chou, S. and Merigan, T. C. (1983) N. Engl...L Med. 308, 921-925 18 Spector, S. A., Rua, J. A:, Speetor, D. H. and MeMillan, R. (1984) J. Infect. Dis. 150, 121-126 19 Virtanen, M., Syv/inen, A-C., Oram, J., S6derlund, H. and Ranki, M. (1984)J. Clin. Microbiol. 20, 1083-1088 20 Berninger, M., Hammer, M., Hoyer, B. and Gerin,J. L. (1982)J. Med. Virol. 9, 57-68 21 Br~chot, C., Degos, F., Lugassy, C., Thiers, V., Zafrani, S. Franco, D., Bismuth, H., Tr~po, C., Benhamou, J. P., Wands, J., Isselbaeher, K., Tiollais, P. and Berthelot, P. (1985) Ni Engl. J. Med. 312, 270-276 22 Pfister, H. (1984) Reo. Physiol. Bioehem. Pharmacol. 99, 111-178 23 Diirst, M., Gissmann, L., Ikenberg, H. and zurhausen, H. (1983) Proc. Natl Acad. SoL USA 80, 3812-3815 24 Crum, C. P., Ikenberg, H., Riehart, R. M. and Gissman, L. (1984) iV. Engl. J. Med. 310, 880-883 25 Moseley, S. L., Echeverria, P., Seriwatana, J., Tirapat, C., Chaieumpa, W., Sakuldaipeara, T. and Falkow, S. (1982)J. Infect. Dis. 145, 863-869 26 Echeverria, P., Seriwatana, J., Leksoboon, U., Tirapat, C., Chaieumpa, W. and Rowe, B. (1984) Lancet i, 63-66 27 Hyypi~i, T., Jalava, A., Larsen, S. H., Terho, P. and Hukkanen, V. (1985),]. Gen. Microbial. 131,975-978 28 Palva, A., Jousimies-Somer, H.,Saikku, P., V~iS~n~aen,P., SSderlund, H. and Ranki, M.(1984) FEMS Microbial. Lett. 23, 83-89 29 Totten, P. A., Holmes, K. K., Handsfield, H. H., Knapp, J. S., Perine, P. L. and Falkow, S. (1983)J. Infect. Dis. 148, 462-471 30 Virtanen, M., Palva, A., Halonen, P., Laaksonen, M., S~lerlund, H. and Ranki, M. (1983) Lancet i, 381-383 31 Owens, R. A. and Diener, T. O. (1981) Science 213, 670-672 32 Hyypi~i, T., St~lhandske, P., Vainionpii~i, R. and Pettersson, U. (1984) J. Clin. Microbial. 19, 436-438 33 Flores,J., Boeggeman, E., Purcell, R. H., Sereno, M., Percz, I., White, L., Wyatt, R. G., Chanock, R. M. and Kapikian, A. Z. (1983) Lancet i, 555-559 34 Franzen, L., Westin, G., Shabo, R., ~slund, L., Perlman, H., Persson, T., Wigzell, H. and Pettersson, U. (1984) Lancet i, 525-528 35 Gonzales, A., Prediger, E., Hueeas, M. E., Nogueira, N. and Lizardi, P. M . (1984) Pmc. NatlAcad. Sci, USA 81, 3356-3360 36 Wirth, D. F. and MeMahon Pratt, D. (1982)Proc. NatlAcad. Sci. USA 79, 6999-7003 37 Harrison, T.J., Wheeler, E. G., Meaeock, T.J., Harrison, J. F. and Zuekerman, A. J. (1985) Brit. Med. J. 290, 663-664 38 Hyypi[i, T. (1985)J. Clin. Microbial. 21, 730-733
268
Nucleic acid hybridization-an alternative tool in diagnostic microbiology Ulf Pettersson and Timo Hyypi~i* The use of radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs) has revolutionized diagnost# microbiology. Their high specificity and sensitivity make them versatile, they are simple to carry out eitherfor direct detection of microorganisms in specimens orfor serological diagnosis, and they can easily and reliably be standardized. Monoclonal antibodies have further improved these immunoassays. However, the development of simple and highly sensitive detection methods for nucleic acids has neverthelesspromoted an interest also in diagnostic methods based on nucleicacid hybridization. Here Ulf Pettersson and Timo Hyypi/i discuss methods which are likely to becomea usfful complement to the immunoassays in the nearfuture. Duplex D N A is composed of two complementary polynucleotide chains. The D N A molecule can easily be denatured by heat or by increasing the pH. Denaturation leads to a reversible separation of the two complementary strands. The latter will under the appropriate conditions gradually reunite and reform a duplex molecule, identical to the original one. The reassociation reaction is highly specific: two separated polynudeotide chains will only reassociate if they are complementary. If a foreign polynucleotide chain is introduced in a solution of denatured D N A it will form a duplex with one of the strands provided that it shares sequences with the denatured DNA. This reaction, designated nucleic acid hybridization, occurs both for R N A and DNA. The requirement for complementarity, however, is not absolute. A certain amount of mismatch is tolerated, still allowing duplex formation. This means that non-identical but related nucleic acids can participate in the hybridization reaction. The resulting hybrids are, however, less stable when subjected to heat in buffers of low ionic-strength. This property provides the basis for a method to discriminate between precisely and imprecisely matched duplexes. The sensitivity and the specificity of the hybridization method is perhaps best illustrated by its use for detection of mammalian genes; employing the Southern hybridization procedure it is possible to detect the presence of a single-copy gene using a probe that is less than 20 nucleotides long. The probe represents in this case less than 10 -8 of the sequences in the total D N A sample. P r a c t i c a l considerations For a detailed description of the nudeic acid hybridization methods, the review by Meinkoth and WahP is recommended. The principle behind the practical use of the hybridization technique is simple; two complementary polynudeotide chains will form a duplex molecule (hybrid) at the appropriate temperature and at the appropriate cation concentration. Oligonucleotides consisting of as few as 12 nucleotides are able to form specific and
Department of Medical Genetics, Uppsala University, Biomedical Center, Box 589, S-75123 Uppsala~ Sweden. *Permanent address: Department of Virology, University of Turku, sF-20520 Turku, Finland. © 1985, Elsevier Science Publishers B.V., Amsterdam 0167 - 4919/85/$02.00
stable hybrids, albeit at a reduced temperature. R N A can also form a hybrid with a complementary D N A or R N A strand and it is in fact possible to arrange the hybridization conditions in such a way that R N A / D N A hybrids are formed in the absence of D N A / D N A duplex formation 2. A practical problem with the hybridization reaction is that two competing reactions take place; the sample D N A will reassociate with itself while reacting with the nucleic acid that is used for detection, the so called probe. This obviously reduces the efficiency of the hybridization reaction. The problem is usually circumvented by immobilization of the D N A on a solid matrix, most commonly a nitrocellulose membrane. In this way renaturation is prevented while the probe can react with the exposed strands on the filter. A key problem in the practical use of hybridization reactions concerns the detection of hybrid formation. Several properties ofnudeic acids can be exploited for this purpose; the absorbance of ultraviolet light differs between single and double-stranded nucleic acids ('hyperchromicity'). The reaction can hence be followed in the spectrophotometer. This requires, however, pure D N A samples and is thus not applicable for detection of nucleic acids in crude specimens. Physical separation methods can also be used to discriminate between single and double-stranded nucleic acids. One of the most powerful methods is hydroxylapatite chromatography, which very efficiently separates single- and doublestranded DNAs. The differential sensitivity of duplex and singlestranded nucleic acids to enzymatic degradation can also be exploited. The single-strand specific nudeases are thus widely used in basic research to detect the formation of nuclease-resistant hybrids. For R N A , ribonucleases can be used to discriminate between single stranded R N A and hybrids. By far the most common way to detect hybrid formation is to use nucleic acids immobilized on nitrocellulose membranes. This method, originally described more than 20 years ago 3'4'5, is one of the most widely used in molecular biology. The principle is simple: the nucleic acid to be analysed is denatured and filtered through a nitrocellulose membrane. T h e single strands bind to the filter in such a way that they are able to interact with nucleic acids in a solution that is in contact with the filter. A radioactively labeled probe is usually added to detect
Immunology Today, vol. 6, No. 9, 1985
269 Fig. 1. Detection of adenovirus in stool specimens by hybridization.
32P-labelecladenovirus-2DNA was used as the probe. Upper row (I). The indicated amounts of adenovirus-2 (left) and HSV-1 (right) DNA were immobilizedon the filter. Lowerrow (II). Analysisof8 stoolspecimens. Specimens 1,3,6 and 7 were positive according to an adenovirus-detectingRIA, whereas specimens 2,4,5 and 8 were negative in the same assay.
the presence of a specific nucleic acid in the immobilized sample. If complementarity exists between the probe and the immobilized nucleic acid, hybridization will occur which can be measured by scintillation counting or by subjecting the filter to autoradiography. Recombinant D N A technology has in many ways facilitated nucleic acid hybridization methods. The use of genomes isolated from microorganisms as probes has several disadvantages; it is often laborious, expensive, and sometimes even impossible to prepare a nucleic acid sample of sufficient purity and homogeneity~ for use in a practical test. It is moreover desirable in many cases to utilize probes which represent restricted parts o f a genome to discriminate between closely related microorganisms. Gene technology has circumvented many of these shortcomings. Essentially any nucleic acid can be cloned in a bacterial vector and in this way can be produced in large quantities. It is also possible to derive segments from the original clone by subcloning fragments. In this way probes can be selected which represent unique parts of a genome or parts which are shared by other members of e.g. a virus family. Recent advances in gene technology provide further practical advantages. By the use of certain phage vectors it is possible to manufacture single stranded probes that are labeled to very high specific radioactivity6. It is also possible to clone fragments in vectors which allow high level transcription of the cloned sequence, thus providing a method to synthesize very highly labeled R N A probes 7. A n additional important technical improvement concerns novel methods for oligonucleotide synthesis. Commercially available synthesizers have the capacity to rapidly manufacture oligonucleotides which exceed 50 nucleotides in length. This technology opens possibilities to design very specific hybridization probes based on established nucleotide sequences. For sensitive detection the probes need to be labeled to a high specific activity. Probes can be radioactively labeled both in vivo and in vitro. In-vitro methods are, for practical reasons, much more common and the so-called nick translation method is very convenient for labeling of double-stranded D N A probes, yielding specific activities >10 a cpm//~g. 32P-phosphorous is the most common label,
although it has a short half life. 3H-labeled nucleotide precursors are usually unsatisfactory because of their unfavourable detection limit. More recently 3aS-labeled nucleotides with a half life of about 80 days have been introduced as an alternative to 3~p. Also I25I-labeled nucleic acids can be obtained both by chemical labeling and by enzymatic synthesis. Non-radioactive labeling methods are needed to convert hybridization into a practically useful diagnostic method. Several non-radioactive labeling procedures have been describedS-11, the most widely used being based on biotinylated nucleotides. Biotinylated D N A can be synthesized enzymatically and is detected by an avidinenzyme conjugate combined with a color-producing substrate. This method appears promising, although in most studies reported so far the sensitivity is less than that noted with 32P-labeled probes. Methods for direct labeling of D N A or hybrids have also been described although none has so far been evaluated in practical diagnostic tests ~0-~2.
Spot h y b r i d i z a t i o n ; a u n i v e r s a l t o o l for d e t e c t i o n of microbial DNA
A simple way to detect the microbial D N A in a specimen is to extract the nucleic acids, immobilize them on a nitrocellulose fiker and then to reveal their presence by a labeled hybridization probe. Probes consisting of D N A or R N A extracted from a cultivated microorganism, cloned D N A sequences, in vitro synthesized R N A or a synthetic oligonucleotide can be used. The method, commonly known as spot hybridization (Fig, 1), is very straightforward and has been used for the detection of a variety of microorganisms. Brandsma and Miller 13 were the first to describe a practical application of the method, i.e. the detection of Epstein-Barr virus (EBV) sequences in lymphoblastoid cells. They designed a simple protocol according to which the cells to be tested were immobilized on a nitrocellulose membrane. The D N A in the immobilized sample was denatured in situ and hybridized to a probe consisting of radioactively labeled EBV DNA. Variations of the
270 method have subsequently been reported which makes it possible to detect EBV directly in nasopharyngeleal aspirates 14. St~lhandske and Pettersson ~5used spot hybridization to detect herpes simplex virus (HSV) in infected cells. They used a cloned restriction enzyme fragment of HSV-1 D N A as the probe and were able to discriminate between HSV-1 and HSV-2 infected cells. The method exploits the fact that imprecisely matched hybrids, as obtained after hybridization to the heterologous serotype, are less stable after washing under stringent conditions (see Fig. 2 for details). The test can thus be used for HSVtyping. Subsequently, the assay has also been used for direct detection of H S V in clinical specimens16. Several investigators have used spot hybridization for detection of human cytomegalovirus (CMV) in urine specimens 17-19and here the results look very promising. The detection level is approximately 5 pg of viral DNA. An interesting and important application of the spot hybridization test is in diagnosis of hepatitis B virus (HBV). The test has been used by several investigators for direct detection of HBV-specific D N A sequences in blood 2°'21. The blood can be applied directly to the filter without purification of the D N A and the test is hence very simple to perform. The sensitivity is comparable to that obtained with currently used immunoassays. An important application of the spot hybridization test concerns detection of papillomaviruses. Here hybridization offers several advantages. Firstly the different human papillomavirus serotypes can only be discriminated by hybridization or their typical restriction-enzyme cleavage patterns 22. Secondly, it is impossible to cultivate these viruses in vitro and therefore difficult to obtain sufficient amounts of antigen for an immunoassay. This is particularly true for the important serotypes 16 and 18 which are commonly associated with human genital tumors 2~'24. Many applications in diagnostic bacteriology have also been described. Spot hybridization has, for instance, been used to detect enterotoxigenic E. coli where a particular advantage of hybridization is exploited, namely the specific detection of genes which predetermine pathogenicity 2a'26. More recently, tests based on hybridization have also been used for diagnosis of sexually transmitted diseases, like those caused by Chlamydia trachomatis 27a8and Neisseria gonorrhoeae29. A useful modification of spot hybridization, so-called sandwich hybridization, has also been described 3°. An advantage with this method is its low background, which is particularly useful when impure samples are analysed. The method has been used for detection of adenovirus in nasopharyngeal specimens s0 and C M V in urine 19.
Detection of RNA viruses The use of the spot hybridization method for the detection of RNA-containing microorganisms is somewhat more complicated since R N A binds less well to nitrocellulose and is more sensitive to high pH and to nucleases than DNA. The construction of the probes is also more tedious since cDNA copies have to be cloned rather than DNA fragments. The first practical application was the detection of the potato spindle tuber
Immunology Today, voL 6, No. 9, 1985
viroid 31. In this case the immunoassays provide no akernative to spot hybridization since viroids lack protein components. Hyypi~ et aL used a cloned probe dervied from the replicase gene of coxsackievirus B3 for detection of enteroviruses 32. The probe was capable of identifying enteroviruses in tissue cultures that had been inoculated with specimens but was not sensitive enough to detect reproducibly the virus directly in stool specimens. An interesting property of the probe is that it is broadly reacting, i.e. hybridizes to different serotypes of poliovirus, echoviruses, and all tested coxsackievirus B serotypes. This is a valuable characteristic since there is no group-reacting immunological reagent capable of detecting the entire enterovirus group. Flores et al. 33used spot hybridization to detect rotavirus R N A in stool specimens. An R N A probe produced by in-vitro transcription was used and the test was found to be at least as sensitive as the currently used ELISA for rotavirus detection. Other modifications of the spot hybridization assay The sensitivity of the hybridization test can obviously be increased if the sequence used as a probe is repeated several times in the microorganism. This property has been exploited by some investigators. Franzen et al. 34 cloned a highly reiterated sequence from the genome of Plasmodium falciparum. This sequence, which represents approximately 1% of the P. falciparum genome, was used for parasite detection in malaria patients. The test is simple to perform; D N A is extracted from a small blood sample, immobilized on a nitrocellulose membrane and hybridized to a radioactive probe containing many copies of the reiterated sequence (Fig. 3). By this method it was possible to detect approximately 25 pg of P. falciparum DNA, and the test had a sensitivity comparable to that of other tests currently in use for parasite detection, including light microscopy of stained blood smears and antigen detection by ELISA. The probe seems to recognize no other plasmodium species than P.falciparurn. A similar method has been designed for detection of the parasite Trypanosorna cruzi 35. Cloned minichromosomal repetitive DNA is used as the probe and 30 parasites are sufficient to give a positive signal in the test. Also for diagnosis ofLeishmania, a hybridization test has been described which utilizes cloned kinetoplast D N A as the probe. The test has been used to detect Leishmania in cutaneous lesions 36. Recently, a commercial assay has been introduced for detection of mycoplasmas in tissue cultures ('Geneprobe'). This test utilizes cloned sequences complementary to ribosomal RNA. Since a large number of ribosomal R N A copies exist in the microorganism, the test becomes obviously much more sensitive than tests based on probes representing single-copy genes. Are nucleic acid hybridization assays feasible alternatives to the immunoassays? Although a versatile tool, spot hybridization has not yet been introduced in the diagnostic laboratories to any significant extent. There are several reasons for this: the
Immunology Today, vol. 6, No. 9, 1985
Fig. 2. (from Ref. 15) Detection of HSV DNA by hybridization, DNA was extracted from uninfectedcells and cells infectedwith HSV-1 or HSV-2 and immobilizedon nitrocellulosefilters. A cloned fragmentfromthe HSV-1 genomewas used as the hybridizationprobe (its preciselocationin the HSV-genomeis unknown). A: DNA fromuninfectedcells. B: DNA from HSV-1 (upper spot) and HSV-2 (lowerspot) infected cells. The filter was washed at 65°C in 2 x SSC (standard citrate saline). C: The same filteras in B after washingat 65°C in 0.1 x SSC. sensitivity obtained is at best comparable to that obtained with immunoassays, the test usually requires more than 24 h to provide an answer, it requires short-lived isotopes, and is based on methodologies which are not used in a routine diagnostic laboratory. The immunoassays, with which the clinical microbiologist is already so familiar have moreover been improved dramatically thanks to hybridoma technology. However, in some circumstances the hybridization test is the only diagnostic alternative or it has certain significant advantages. Immunoassays cannot be applied for detection of viroids, since they lack any protein component and hybridization is thus the only diagnostic alternative. In the diagnosis of HBV, convenient immunoassays are available and the hybridization test offers no advantages, with regard to sensitivity or convenience. The immunoassays have, however, one serious disadvantage: they score for the presence of viral proteins rather than for infectious particles. This is a drawback because some carriers excrete viral antigens in the absence of infectious particles. The detection of the viral DNA by hybridization is thus already an important complement to the antigen-detecting assays 37. For the diagnosis of CMV, current isolation procedures are tedious and require often more than 2-3 weeks to give a definite result. The direct detection of C M V D N A in the urine specimens is an important contribution to diagnostic virology. Hybridization methods for C M V detection in urine specimens have already been developed but the sensitivity obtained is not yet sufficient to make them superior to the conventional isolation procedures. For viruses which are difficult or impossible to propagate in vitro, hybridization is a valid diagnostic alternative. This includes tests for EBV DNA in nasopharyngeal specimens. The h u m a n papillomaviruses, particularly the very important types 16 and 18, cannot be propagated
271 at all in tissue culture and there is no means yet known for enriching virus particles. Hence nucleic acid hybridization is the only existing diagnostic alternative. For the enteroviruses hybridization offers some significant advantages. Probes can, at least in theory, be designed which react with group-specific as well as typespecific parts of enteroviral genomes. Since enteroviruses are one of the major obstacles in clinical virology, hybridization methods are likely to become an important alternative to the existing immunoassays in the near future. In the analysis of stool specimens the hybridization assay again may be a useful complement, particularly for detection of rotaviruses and adenoviruses (Fig. 1) and in clinical bacteriology the specific recognition of genes in bacteria which are linked to pathogenicity is a property which ultimately should make hybridization tests attractive alternatives to currently used diagnostic procedures. Examination of microscopic slides currently dominates test procedures for parasites, which is very time-consuming and, in the case of diagnosis of malaria, subjective. Hybridization may here become an alternative for mass screening of samples, as for instance screening of blood banks. In situ hybridization is a useful variant of the hybridization method which also may find important applications in diagnostic microbiology, since it allows the identification of foreign nucleic acids in specific parts of a tissue section. Future prospects Methods detecting nucleic acids have several advantages which can be exploited in diagnostic tests: (1) They are versatile and can be used for detection of microorganisms which are unable to replicate in vitro, microorganisms which lack protein components, etc.
Fig. 3. (from Ref. 34) Detection of Plasmodium falclparum by hybridization. Row A. 250ng, 25 ng, 2.5 ng, 250pg and 25 pg ofP.falciparum DNA. Rows B-C. Samplesof patientswith parasitemiaas determinedby microscopy. B1, B2, C1, C4, C5 P. vivax. All the others P. falciparum.
272
(2) They can be used to identify essentially any specific gene in a microorganism. Immunoassays preferentially detect surface proteins or other antigens which are synthesized in abundance during replication of the microorganism. These proteins do not necessarily correlate with the pathogenic properties of the microorganism. Specific identification of a gene or part of a gene that is responsible for the pathogenicity can be achieved with nucleic acid hybridization. There is, as outlined above, already today a need for hybridization methods in clinical microbiology. Will the hybridization tests then replace the immunoassays? Probably not, unless the technique is improved dramatically. The major obstacle concerns the label of the probe. Currently the short lived isotope 32p is the most commonly used, which is a disadvantage for several reasons. Alternative labeling methods must be found before hybridization can be introduced as a routine method in the diagnostic laboratory. Non-radioactively labeled probes have been designed and the biotin-labeled probes seem to be particularly promising 3s. Also the hybridization method as such might be improved in several ways. So far most investigators have used a standard protocol according to which the specimen D N A or R N A is immobilized on a filter and cloned fragments are used for detection. Simple methods, like the use ofhydroxylapatite chromatography, single-strand specific nucleases etc., need to be further exploited. Also the availability of single-stranded DNA and R N A probes offers new possibilities for the design of more sensitive and convenient assays. Finally, the synthetic oligonucleotides may turn out to be crucial instruments for specific discrimination between closely related microorganisms. The time required to complete the test, currently 1-2 days, could also be reduced by altering the hybridization conditions. It should be kept in mind that nucleic acid hybridization methods also are applicable to other important areas of diagnostic medicine. The genetic diseases, for instance, can now be studied due to restriction enzyme fragment length polymorphism. The combined forces of investigators from many fields are likely to result in solutions to some of the practical problems which presently prevent hybridization from becoming a widely used routine method in clinical microbiology. [=~
Acknowledgements The authors are grateful to Drs Z. Dinter, H. Diderholm and G. Magnusson for valuable discussions during preparation of the manuscript.
Immunology Today, vol. 6, No. 9, 1985
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