3 Maataloustieteellinen Aikakauskirja Vol. 59: 179—191, 1987 Diagnosis of plant viruses by nucleic acid hybridization REIJO KARJALAINEN Department of Plant Pathology, University of Helsinki SF-00710 Helsinki, Finland LEO ROUHIAINEN Department of Microbiology, University of Helsinki SF-00710 Helsinki, Finland HANS SÖDERLUND Orion Genetic Engineering Laboratory, Valimotie 7 SF-00380 Helsinki, Finland Abstract. Nucleic acid hybridization is a powerful technique for the diagnosis of many plant viruses not easily detected by serological techniques. It is particularly effective in the detection of viruses occurring in low amount in plant tissue, viruses that are poor immunogens or contain satellites. Molecular probes with desired specificities can be prepared by recombinant DNA tech- niques for large scale use. cDNA probes of potato virus X (PVX) RNA were made by mole- cular cloning, and the clones were 32 P labelled by nick translation. Hybridization of cDNA to PVX RNA revealed 1 ng of purified virus in 2 /d spots dried onto nitrocellulose filter. In- fected samples of crude leaf extracts were easily detected by hybridization, while probes did not react with healthy leaf samples. Nucleic acid hybridization research aims at replacing radiometric probes with non- radioactive methods involving enzymes which are directly or indirectly coupled to the probe and whose presence is observed with the aid of a colour changing substrate. Hybridization assay formats that can easily be automatized are under development. Sandwich hybridization is a simple test format developed for analyzing unpurified biological material, and it appears to be a powerful tool for microbial diagnostics. Sensitivity can be improved by using detection systems in which the specific activity of the probe is increased. Procedures such as ’polymerase chain reaction’, in which the amount of detectable nucleic acid sequences can be increased, are promising alternatives for increasing sensitivity. It is concluded that even if probe-based assays are in their infancy, they will no doubt develop towards such easy use as have immuno- logical test kits. Index words; virus detection, cDNA probes, potato virus X (PVX), spot hybridization, sandwich hybridization 179 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=DjZpFvVO8uY2ig7J.plBsf58FTrthtEGT3tutMA.xMVE7-ZscQ6WTZCbwV2S3-LEOftKqYJAy3-gYyszGgRNTOcQ0mq0LIz6XC8CwCo5TNvLanMwiSmZxjiKBO1byla6mcy7FZHNJrHHa25OzZsZrrk4AnFfZcELoZhN2nhk-eVJ-bN2aCWjsY168WcboboR0eLbmDXLUz3-Ik63rpyMk8cfAnPfkJOoZGRAN_Bg-oe2d8n3OZ87r7r-9navboEjw3o Introduction Crop losses caused by plant viruses are cur- rently controlled mainly by using virus-free plant material and disease resistant cultivars (Walkey 1985). The success of virus disease control is thus crucially dependent on the avai- lability of accurate, sensitive but simple diag- nostic techniques which enable the early detection of viral infections in plant materi- al. In many areas of plant production there is a great need for improved procedures for the rapid and sensitive detection of important plant viruses, particularly in laboratories res- ponsible for producing healthy horticultural and field crop plants, in plant quarantee la- boratories (Symons 1984), and plant breeding stations. Different virus diagnostic tools are needed for different purposes. For instance, extremely sensitive and accurate diagnostic techniques are required to detect some viru- ses in berry plants and other horticultural crops when producing virus-free material be- cause certain viruses occur in low concentra- tion in plant tissue. When producing virus-free potato cultivars or screening breeding mate- rial for virus resistance, diagnostic procedu- res should be rapid but reliable to detect various strains of the target virus because large numbers of samples are analyzed in a short time. Traditionally, plant virus diagnosis has mainly relied on electron microscopy, im- munological analyses, and symptom expres- sion on indicator plants. However, many of these methods are time-consuming and unre- liable and thus unsuitable for analyzing large numbers of samples (Symons 1984). Cur- rently, the most widely used serological tech- nique is the ELISA because it is simple and sensitive and appropriate even for small labo- ratories (review by Clark and Bar-Joseph 1984). However, the ELISA or related serolog- ical techniques are not always reliable enough. For instance, some viruses which occur in low concentration in plant tissue (e.g. barley yel- low dwarf virus, potato leafroll virus), are poor immunogens or difficult to purify, are not easily detected by serological techniques (Symons 1984, Hull 1986). For some viruses which have a wide host range (e.g. cucumber mosaic virus) and are unstable or contain satellite RNA encapsidated by the coat pro- tein of the associated virus, serological diag- nosis is also impossible (Harrison et al. 1983, Palukaitis et al. 1985). Viroids, the smallest known pathogenic agents of plants, have no protein coat, and are thus not detectable by immunological methods (Owens and Diener 1984). Nucleic acid hybridization is a new, power- ful diagnostic alternative for the detection of viral infections in plants (Maule et al. 1983). Hybridization techniques are based on the ability of complementary single-stranded nucleic acid sequences to hybridize under appropriate conditions. Because of the unique base pairing tendency, hybridization is an attractive method since it is highly specif- ic, accurate and able to detect very low con- centrations of viral nucleic acids in plant extract. Nucleic acid hybridization has beco- me a realistic diagnostic technique in the past few years, when recombinant DNA techniques made it possible to produce complementary nucleic acid probes (cDNA) with desired spec- ificity for large scale use. Nucleic acid hybridization has been used in the detection of various human viruses and other microbes of clinical importance for several years (review by Viscidi and Yolken 1987), as well as in the detection of some food contaminating microbes (Fitts et al. 1983). It was first shown by Owens and Diener (1981) that plant viroid infections can be effectively detected by nucleic acid hybridization using cloned cDNA probes. Since then nucleic acid hybridization has been widely tested as a means of diagnosing various plant virus dis- eases (Hull 1986). However, there are still many problems limiting its wide diagnostic application both in medical and agricultural fields. One serious limitation is the use of ra- dioactively labelled recombinant DNA probes, which are still more reliable than the non- radioactive ones available. However, non- 180 radioactive labelling techniques are rapidly being developed (Syvänen et al. 1986, Li et al. 1987). Progress has also been made in developing hybridization test formats. Sandwich hybrid- ization technique has been developed for the detection of nucleic acids in crude clinical samples (Ranki et al. 1983). In this hybrid- ization method, the specimen is kept in solu- tion, and thus sample pretreatments are easy and background problems can be reduced. Sandwich hybridization has been used to diagnose various viral and bacterial infections during the past five years (Ranki et al. 1987), and its value in the detection of plant viral in- fections is being evaluated. This paper is a review of recent develop- ments in nucleic acid hybridization and its applications for plant virus diagnostics. The use of hybridization techniques is illustrated using potato virus X (PVX) as a test virus. Methodological aspects In principle, the use of hybridization tech- niques for the detection of plant viruses is rather simple. The first step is to prepare la- belled complementary DNA (cDNA) to the target viral nucleic acid. In the case of dot- blot (sap spot) hybridization, a small amount of plant extract is immobilized on a solid sup- port, usually nitrocellulose or nylon filter, and the labelled cDNA probe is added and incu- bated with it. As both samples are rendered single-stranded, the probe will hybridize with homologous sequences in the plant extract on the support. The extent of hybrid formation is a measure of the concentration of viral sequences in the plant extract, which can be visualized on the filter by autoradiography. Although most plant viruses contain RNA genomes, RNA probes are relatively little used in nucleic acid hybridization diagnostics. Viral RNA to be used as end-labelled RNA probes can be prepared from ssRNA isolated from purified virions or from dsRNA replicative forms (Garger and Turpen 1986). RNA probes can also be conveniently made in vitro using RNA polymerase and cloned cDNA in plasmid vectors with RNA polymerase pro- moter sites (Melton et al. 1984). The use of synthetic oligonucleotides is a rapid hybridization method. Several synthetic probes have been used to diagnose various viral and bacterial infections (Hill et al. 1985, Lin et al. 1987). However, synthetic oligonucleotide probes have not been much used in plant virus diagnostics because of the lack of published sequence information and partly because of their poor sensitivity (Bar- Joseph et al. 1986). Complementary DNA (cDNA) to viral genomic RNA is the most widely used type of probe in nucleic acid hybridization. There are several techniques for its preparation. In general, these techniques involve four steps (Palukaitis 1986): 1. the cDNA synthesis re- action, 2. the separation of the cDNA from the template and the enzyme, 3. the separa- tion of the cDNA from the unincorporated radioisotope and other components of the reaction mixture, and 4. the concentration of the cDNA probe. The appropriate method de- pends on the properties of the virus, its molec- ular weight, structure, and 3’ polyadenylate sequences. The majority of plant viruses con- tain single-stranded RNA genomes, and only a small percentage contains DNA (Hull and Davies 1983). In this paper, potato virus X (PVX) was used as a test virus to prepare a cDNA probe and to use it for detecting plant viral infec- tions. Cloning strategies and preparation of cDNA probes of PVX RNA Potato virus X (PVX) is world-wide distri- buted in potato growing countries, and it is estimated that tuber yields of infected plants can be reduced by 5— 15 % (Torrance et al. 1986). The virus causes mild mosaic on po- tato leaves, but foliage symptoms are not re- liable indications of infection (Torrance et al. 1986). Several strains of PVX can be distinguished, but in some cases they are not 181 easily detected by serological assays based on polyclonal antibodies (Moreira et al. 1980). In order to reveal their serological relation- ships in more detail, monoclonal antibodies have been produced from two strains ofPVX (Koenig and Torrance 1986, Torrance et al. 1986). PVX is a potexvirus containing single- stranded RNA. Its molecular weight is 2.1 x 106 , and its coding capacity is sufficient for three polypeptides (Morozov et al. 1983). Recently, Morozov et al. (1983) showed that the RNA of PVX has a poly-A tail of about 50—200 bases at its 3’ end, and there is a cap m 7 GpppG at the 5’ end. Part of PVX RNA has been sequenced, and its amino acid se- quence has been deduced from nucleotide se- quences (Morozov et al. 1983). Virus purification and RNA extraction The PVX isolate used in this work was orig- inally isolated by Dr. A. Kurppa. The virus was purified from infected Nicotiana glutinosa leaves, mainly according to Shephard (1972), but further purification was made by centri- fugation into CsCl gradient. The viral RNA was treated with 0.5 °/o SDS and phenol ex- tracted, then precipitated withethanol. Purity of the RNA was analyzed spectrophotometri- cally and by agarose gel electrophoresis. cDNA synthesis and molecular cloning Complementary DNA (cDNA) to the ge- nomic RNA of PVX was synthesized by the method of Gubler and Hoffman (1983). In general, first-strand cDNA was synthesized by AMV reverse transcriptase (Promega Biotech) using oligo-dT as a primer and the poly- adenylated RNA of PVX as a template. The second strand of cDNA was synthesized with DNA polymerase I. Double-stranded cDNA was digested with Sau3 and cloned into the plasmid p8R322 at the BamH\ site. Recombinant clones were identified on the basis of their sensitivity or resistance to tetracycline and ampicillin. The clones were screened for the size of cDNA in- sert by agarose gel electrophoresis, and those containing inserts larger than 500 bases were selected for hybridization studies. The cDNA probes were labelled by nick-translation to a specific activity of approximately 108 cpm/pg. Sample preparation and hybridization For hybridization, 2 pi of crude leaf sap ex- tract or purified virus was spotted onto nitrocellulose filters, which were first soaked in water and then in 20 x SSC. The filters were baked at 80°C for 2 h in a vacuum oven. The filters were prehybridized in a water- bath at 45°C for 4—5 h using sealed plastic bags and then hybridized at 50°C for about 16 h. The hybridization buffer contained denatured 32P labelled cDNA at a concentra- tion of approximately 30 ng/ml. After hybrid- ization, the filters were washed four times at room temperature for 5 min and twice at 50°C for 15 min in 0.1 SSC + 0.2 % SDS. Then filters were autoradiographed at 80°C for 24 h. The cDNA clone 19 of PVX RNA used as the reference probe was a kind gift of Dr. D. Baulcombe, Plant Breeding Institute, Cam- bridge. Preparation of reagents for sandwich hybridization The sandwich hybridization method is based on two separate nucleic acid reagents, which are derived from two non-overlapping but adjacent regions of the target microbial genome (Ranki et ai. 1983). One of the frag- ments is immobilized on a nitrocellulose filter in single-stranded form (filter-DNA), and the other fragment is radioactively labelled (probe- DNA). In the reaction any nucleic acid se- quence homologous to the DNA reagents will hybridize both to the filter-DNA and to the probe-DNA, thus binding the probe to the filter (Ranki et ai. 1983). In this system, the reagents have no common sequences, there- 182 fore no hybrids are formed with incorrect sample nucleic acids. In general, the preparation of nucleic acid fragments for sandwich hybridization involves various molecular biological techniques (Fig. I). Various restriction enzymes are usually first used to map the target DNA fragment, and then two adjacent restriction fragments are subcloned into two different vectors. DNA fragments for filter-DNA are subcloned into the plasmid vector p8R322 or its derivative pATIS3 (Ranki et ai. 1983). Single-stranded probe-DNA is cloned in the bacteriophage Ml 3. In the case of subcloning of reagent pairs of starter cDNA of PVX for sandwich hybridization, the size of filter-DNA was 150 bp, and for the probe-DNA 700 bp was used. As in the spot hybridization described pre- viously, double-strandedrecombinant plasmid DNA is denatured in 0.2 M NaOH at 100°C for 5 min, cooled at O°C, and applied to the nitrocellulose filter in ice-cold 6 X SSC under slight pressure, then fixed onto the filter by baking under vacuum at 80°C for 2 h (Ran- ki et al. 1983). In sandwich hybridization re- action, each hybridization contains one filter with microbe-specific DNA and one or two control filters with calf thymus or no DNA, respectively (Ranki et ai. 1983). Incubation is usually allowed to proceed overnight (16 — 20 h) at 65 °C, after which the filters are carefully washed. Hybrid formation is quan- titatively measured by a radioactivity counter (Ranki et ai. 1983). Applications for virus detection cDNA cloning ofPVX RNA revealed three distinct types of inserts after Sau3 digestion (Fig. 2). Of these inserts, the one of 850 bp was selected as the test clone, called clone PVX 59, to demonstrate the use of cloned cDNA probes for detecting plant viral infec- tions. After mass production of the plasmid in E. coli cells, it was labelled with 32P by nick-translation. In the first test, the 32P-labelled cDNA probe was used to detect purified PVX virus. It was found (Fig. 3) that this probe of 850 bp complementary to PVX RNA easily re- vealed as little as 1 ng of purified virus con- taining 50 pg of RNA by hybridization to 2 /d spots dried onto nitrocellulose filters. In the second test, dilution series of crude extract from infected tobacco leaves indicated that dilutions with water of up to 500 times were readily detectable by hybridization auto- radiographed for 24 h (Fig. 4). In addition, an experiment was carried out where 36 sap samples (2 /d), including random samples, in- fected and healthy potato leaves, were spotted onto filter. The results showed (Fig. 5) that infected samples were easily detected by cDNA probe. The results presented here showed that cDNA hybridization is a reliable way of de- tecting PVX infections in crude plant sap. The sensitivity of our cDNA probes appears to be Fig. I. Principle of sandwich hybridization assay. 183 Fig. 2. Agarose gel electrophoresis of fragments of the pPVX 59 and pPVX 19 clones. Clone 59 (lane 2) was cut with Sau 3A, clone 19(lane 2) with Pst I, and the DNA markers (lane 1) with Hindlll + Ecoßl. The arrow indicates the position of PVX cDNA inserts. Fig. 3. Dot blot hybridization of purified PVX with 52P-labelled, nick-translated probes of pPVX 19 and pPVX 59. 24 h exposure. Fig. 4. Dot blot from crude sap dilutions of N. glutinosa leaves infected with PVX. The clones were J2P- labelled by nick-translation. 24 h exposure. Fig. 5. Detection of PVX in sap extracts. 2 pi samples of sap from infected, healthy, and randomly taken leaves were spotted onto nitrocellulose filter. After 24 h exposure, samples containing viral RNA (lanes 1,4, 5, spots 2C, 3C, 2F and 3F) were easily detected. 184 of the same order as detected by Baulcombe et al. (1984). In general, cDNA hybridization seems to be as sensitive as the ELISA or even more sensitive to detect PVX infections in plants (Boulton et al. 1984). Hybridization has been found to be an efficient way of screening large numbers of potato clones for resistance to PVX. Boulton et al. (1984, 1986) have pointed out that cDNA probes require less sap and fewer manipulations than the ELISA, and they are more rapid for screening large numbers of clones for PVX infections in a few days than the ELISA. However, PVX is highly immunogenic and occurs abundantly in plant tissue, and it is in most cases easily detectable by standard ELISA techniques (Goodwin and Banttari 1984). It has been suggested that antisera against certain strains of PVX cannot always detect all strains (Mo- reira et al. 1980). In these cases, alternative strategies using either monoclonal antibodies (Torrance et al. 1986) or cDNA probes carefully prepared from certain regions of the PVX genome (Baulcombe et al. 1984) can be useful diagnostic tools and also reveal the strain diversity of PVX. Within the past three years nucleic acid hybridization has been increasingly used for the detection of plant virus infections. For example, cDNA probes have been produced from various virus groups (Table 1), including large potyviruses, potexviruses, closteroviruses, luteoviruses, and the Fiji disease virus be- longing to the small group of reoviruses. cDNA probes are not only sensitive for de- tecting some viruses which occur in high amounts in plant tissue, e.g. TMV and PVX, but also a powerful way of diagnosing viruses such as tobacco rattle (Flarrison et al. 1983, Fluub et al. 1986), which infect large numbers of different agricultural and horticultural crops and which are not reliably detected by serological techniques. In addition, it has been recently shown that cDNA techniques are effective in diagnosing viruses which occur in low concentration in plant tissue, such as the economically very important barley yellow dwarf (Flabili et al. 1987) and the Fiji disease Table 1. Some of the cDNA probes prepared from different plant viruses as reported by various authors. Virus group Virus Viral Reference nucleic acid Luteoviruses Barley yellow dwarf virus (BYDV) ssRNA Waterhouse et at. 1986 Subterranean clover red leaf virus (CRLV) ssRNA Jayasena et al. 1984 Potato leafroll virus (PLRV) ssRNA Baulcombe et al. 1984 Potyviruses Potato virus Y (PVY) ssRNA » Bean yellow mosaic virus (BYMV) ssRNA Hammond and Hammond 1985 Rosni r et al. 1986 Dlßokx and Cuperus 1987 Potexviruses Potato virus X (PVX) ssRNA Baulcombe et al. 1984 Tobamoviruses Tobacco mosaic virus (TMV) ssRNA Sila et al. 1984 Bar-Joseph et al. 1986 Tobraviruses Tobacco rattle virus (TRV) ssRNA Harrison et al. 1983 (2 div.) Hum et al. 1986 Tombusviruses Tomato bushy stunt virus (TBSV) ssRNA Gai i itelli and Hull 1985 Closteroviruses Citrus tristeza virus (CTV) ssRNA Rosner et al. 1983 Reoviruses Fiji disease virus (FDV) dsRNA Skuinicki et al. 1986 Comoviruses Cowpea mosaic virus (CpMV) dsRNA Maui.e et al. 1983 Geminiviruses African cassava mosaic virus (ACMV) ssDNA Robinson et al. 1984 Caulimoviruses Cauliflower mosaic virus (CaMV) dsDNA Maui.e et al. 1983 Figwort mosaic virus (FMV) dsDNA » Carnation etched ring virus (CERV) dsDNA » 185 of sugarcane (Skutnicki et al. 1986). The pre- sent status of the sensitivity of cDNA probes compared with other diagnostic methods is not well demonstrated. Only a few direct com- parisons are available between the ELISA and cDNA probes, and in general they suggest that nucleic acid hybridization is at least as sensi- tive as the ELISA (Maule et al. 1983) or even more sensitive for the detection of, for example, tobacco mosaic virus (TMV) and potato virus Y (PYY) (Sela et al. 1984, Deßokx and Cu- perus 1987). The actual limit of sensitivity for the detection of plant viruses using cDNA probes is poorly known. Maule et al. (1983) showed using 32P-labelled probes that the li- mit of sensitivity for several plant viruses was about 5—20 pg of purified RNA. Baulcombe et al. (1984) were able to detect 1 ng of PYX (50 pg RNA) in a 1 /j! spot. Sensitivity limits for the detection of viroid infection in plant tissue are somewhat better known than those of plant viruses. For example, 80 pg of PSTV (30 ng/g tuber) has been detected (Palukai- tis et al. 1985). About 300 pg of avocado sunblotch viroid (ASBV) was detected without any purification (Rosner et al. 1983), and with partial purification and concentration about 5 pg in 3—5 jd spots, which means about 20 pg ASBV/g fresh weight leaf (Bar- ker et al. 1985). Sandwich hybridization has been used to diagnose various animal viruses as well as bacterial pathogens (Ranki et ai. 1987). Ad- vantages of sandwich hybridization over spot hybridization are that sample pretreatments can be kept simple and crude samples can be tested without causing unspecific hybridiza- tion background. Sandwich hybridization has been shown to be as sensitive in adenovirus detection as radioimmunoassay (review by Ranki et ai. 1987). So far, however, this tech- nique is just beginning to be applied for the diagnosis of plant viruses. Prospects for improving nucleic acid hybridization as a diagnostic tool Any diagnostic procedures which are likely to be used on a large scale in routine plant virus testing should fulfill a number of criteria. The main requirements are a) specificity, b) sensitivity, c) simplicity to perform, and d) they should not contain decaying reagents. In nucleic acid hybridization the specificity is an intrinsic advantage. A gene region spe- cific for the organism or group of organisms to be detected can always be found by using recombinant DNA techniques. The cloning of specific viral nucleic acid sequences and the preparation of probes for desired specificity provide powerful tools for the detection of various isolates of the target virus and for the characterization of strain variation. The sensitivity of nucleic acid based tests appears, in chemical terms, very good. Today the best sensitivity is obtained using 32P- labelled probes, in which case down to 2 X 10~20 moles of target nucleic acid can be found. This corresponds to about 10 000 molecules of DNA or RNA (Syvänen 1986). With non-radiometric methods the detection limit is usually reduced several hundred fold. There is, however, usually only one genome per micro-organism and in many applications a test in which e.g. 100 000 bacteria per 100 /d is the detection limit, is simply not satis- factory. A lot of work is done on improving the sen- sitivity of probe-based tests. One simple way is to assay for a nucleic acid present in many copies per micro-organism. Such multicopy sequences which can be assayed for include ribosomal RNA (Göbel and Standbridoe 1986), multicopy plasmids (Totten et al. 1983), and repetitive DNA sequences in the genome (Gonzales et al. 1984). Another direct way of improving the tests is to use detection systems in which the speci- fic activity of the probe (i.e. signals generated per mass unit of DNA) is increased over those used presently. This is at least theoretically ac- hievable using time-resolved fluorescence and Europium label (Soini and Kujala 1983, Sy- vänen et ai. 1986). Bioluminescent systems can in principle give extremely high sensitivity (Tanaka and Ishi- 186 kawa 1986), as can coupled multienzyme re- actions in which the final detectable product is amplified over the primary one (Self 1985). The major breakthrough in improving the sensitivity of probe-based tests is, how- ever, found in one of the unique properties of nucleic acids. The very basis of heredity is that DNA is duplicated in dividing cells and can be multiplicated in propagating orga- nisms. Specific regions of DNA can by the same principle be enzymatically amplified in vitro. In a reaction called ’polymerase chain reaction’ (Saiki et al. 1985) a given DNA- sequence can be duplicated many times giving an exponential increase in the copies of the target DNA which is then easy to detect by hybridization. Even a few copies of DNA can be detected in this way. The polymerase chain reaction has the potential to solve the sen- sitivity problem of hybridization-based tests. Extremely high sensitivity is only seldom re- quired in plant virus diagnostics, perhaps most often in the detection of viruses of berry or woody plants whenproducing virus-freeplant material. When comparing the published data on sensitivity values in detecting plant viruses and those of thepotential detection sensitivi- ty provided by nucleic acid hybridization (Sy- vänen 1986), it is clear that the present probe-based tests are not nearly as sensitive as they could be. This is probably due to the fact that sample treatments are not optimal for obtaining high sensitivity. Using simple pretreatments for plant material and effecti- ve extraction buffers, detection sensitivity can be improved (Palukaitis et al. 1985). In ad- dition, slight modifications of procedures, changes in hybridization buffers, for instan- ce, can in some cases considerably improve the sensitivity compared to the original buffer (Palukaitis 1986). However, extra steps complicate procedures, and multistep pretre- atments are justified only in cases where vi- rus amounts in plants are low and high sensitivity is necessarily required. Reaction times in minutes rather than hours or days are often important in diagnostics. Due to low concentrations, hybridization reactions are relatively slow. However, the situation has improved recently. Very high probe concentrations (Leary et al. 1983), the use of oligonucleotides rather than large pro- bes (Jablonski et al. 1986), and volume ex- cluders like dextran sulfate and polyethylene glycol (Amasino 1986) have all been impor- tant steps in increasing the reaction rate sig- nificantly. As hybridization time can be shortened to a few hours without any signifi- cant loss of sensitivity by using oligonucleo- tide probes (Lin et al. 1987), their use in plant viral diagnostics might be useful in ca- ses where speed is more important than high sensitivity. Synthetic probes may be an attrac- tive alternative for the diagnosis of dangerous viroid diseases because they can be prepared without the need of propagating the target or- ganism (Bar-Joseph et al. 1985). Many plant viroids have been sequenced and oligonucle- otides could be easily constructed (Riesner and Gross 1985). The first generation of probe-based tests is now becoming available for the diagnostics of some micro-organisms. These tests have their roots in the methodology used in research laboratories and their use is still dependent on laboratory surroundings. The development of more convenient assay formats and simple tools and kits is, however, in progress. One important aspect is the development of non-radioactive probes which do not self- decay. Several different approaches have been tried to replace 32P or 125 1 as detectors. One useful method involves enzymes, directly or indirectly coupled to the probe (Leary et al. 1983, Renz and Kurz 1984, Tchen et al. 1984, Jablonsky et al. 1986, Li et al. 1987), the presence of which is observed with the aid of a colour changing substrate. This approach will lead to tests with certain ELISA-like features. Another possibility is to use probes labelled with fluorecent or luminescent markers (Matthews et al. 1985, Syvänen et al. 1986). 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L., Falkow, S. 1983. DNA hybridization technique for the detection of Neisseria gonorrhoeae in men with urethritis. J. Inf. Dis. 148: 462—471. Viscidi, R. P. & Yolken, R. G. 1987. Molecular diag- nosis of infectious diseases by nucleic acid hybridiza- tion. Mol. Cell. Probes I: 3—14. Waterhouse, P. M., Gerlach, W. L. & Miller, W. A. 1986. Serotypic-specific and general luteovirus probes from clones cDNA sequences of barley yellow dwarf virus. J. Gen. Virol. 67; 1273—1281. SELOSTUS Kasvivirusten tunnistaminen nukleiinihappohybridisaatiolla Reijo Karjalainen Kasvipatologian laitos, Helsingin yliopisto, 00710 Helsinki Leo Rouhiainen Mikrobiologian taitos, Helsingin yliopisto, 00710 Helsinki Hans Söderlund Geeniteknologian laboratorio, Orion-yhtymä Oy, Valimotie 7, 00380 Helsinki Geeniteknologia tarjoaa uuden keinon tunnistaa kasvi- viruksia niiden perintöaineksen perusteella. Tätä pika- diagnostista menetelmää kutsutaan nukleiinihappohyb- ridisaatioksi, koska se perustuu nukleiinihappomolekyy- lien puolikkaiden pariutumiseen. DNA-molekyyli koos- tuu kahdesta toisiaan tarkasti vastaavasta osasta, juos- teesta, jotka toisistaan erotettuina pyrkivät pariutumaan uudelleen. Nukleiinihappohybridisaatiossa tunnistin eli koetin on tunnistettavan patogeenin nukleiinihappomo- lekyylin keinotekoinen puolikas, toinen juoste.Kasvinäyt- teessä olevien virusten nukleiinihapot ’halkaistaan’ yksi- juosteisiksikuumentamalla jakiinnitetään erikoissuodat- timelle, nitroselluloosafiltterille. Tähän lisätään koetin- juosteet, jotkapariutuvat vastaavanpuolikkaansa kans- sa, mikäli niitä on näytteessä, eli mikäli kasvi on viruk- sen infektoima. Pariutuminen havaitaan esimerkiksi au- toradiografisesti, tuikelaskimella tai entsymaattisten vä- rireaktioiden perusteella. Tässä kirjoituksessa tarkastel- laan nukleiinihappohybridisaation kehitysnäkymiä kas- vivirusten tunnistamisessa sekä kuvataan sen työvaiheet käyttäen perunan X-virusta (PVX) testiviruksena. Nukleiinihappohybridisaatiossa tarvittavien koetinmo- lekyylien valmistamiseksi puhdistettiin ensin perunan X- virus ja eristettiin sen RNA. Tämän jälkeen syntetoitiin toista juostetta vastaava eli komplementaarinen DNA- juoste (cDNA) spesifisten entsyymien avulla. Kaksisäikei- set komplementaariset DNA-molekyylit pilkottiin tä- män jälkeen SaM3-restriktioentsyymillä ja kloonattiin p8R322-plasmidiin SamHl-alueelle. Yhdistelmä-DNA -molekyylejä eli PVX:n nukleiinihappoa sisältävät bak- teeripesäkkeet tunnistettiin antibioottimarkkerien avul- la, ja valitut koetinmolekyylit leimattiin radioaktiivisel- la 32P:11ä nick-translaation avulla. Hybridisaatiota var- ten 2/il puhdistamatontaperunan tai tupakan mehua tai puhdistettua virusta pipetoitiin nitroselluloosafiltterille, joka oli ensin käsitelty 20 x SSC -puskurissa. Tämän jäl- keen nukleiinihapot kiinnitettiin filtterille kuumentamalla sitä 80°C:ssa kaksi tuntia. Tämän jälkeen filtterit esihyb- ridisoitiin muovipusseissa 4—5 tuntia, minkä jälkeenkoe- tin lisättiin varsinaiseen hybridisaatioliuokseen jahybri- disaation annettiin jatkua 50°C:ssa 16 tuntia. Hybridi- soinnin jälkeen suodattimet pestiin useaan kertaan pus- kurissa, jolloinhybridisoitumaton leima huuhtoutui pois. Tulokset osoittivat, että PVX:n RNA:sta kloonatuil- la koettimilla pystyttiin tunnistamaan Ing puhdasta vi- rusta 2 p\ pisarassa. Koettimien avulla voitiin myös no- 190 peasti ja luotettavasti tunnistaa PVX:n infektoimat me- hunäytteet suodattimelta, sillä koettimet reagoivat vain virusta sisältävien näytteiden kanssa. Nämä hybridisaa- tiotulokset tukevat viimeaikaisia DNA-diagnostiikka- tulkimuksia, joiden mukaan tämän tekniikan avulla voidaan tarkasti ja luotettavasti tunnistaa monia viruk- sia. Monien virusten tunnistuksessa DNA-tekniikka on ELISAa herkempi ja nopeampi, joskin ELISA on tois- taiseksi paljon yksinkertaisempi jahelpompi. Nopeuden ja tarkkuuden vuoksi nukleiinihappohybridisaatiotakui- tenkin käytetään jo laajalti perunan jalostuksessa seulo- malla sillä nopeasti virusta kestävät kloonit jalostusaineis- toista. Nukleiinihappohybridisaatio soveltuu parhaiten sel- laisten virusten tunnistamiseen, joihin ELISA ja muut se- rologiset menetelmät eivät sovellu, kuten viruksiin, jot- ka ovat kasvissa pieninä pitoisuuksina, joilla on huonot antigeeniset ominaisuudet, jotkasisältävät satelliitteja ja joidengenomi on moniosainen. Tällaisia viruksia on hy- vin paljon, jane ovat hyvin haitallisia sekä maa- että puu- tarhataloudessa. Nukleiinihappohybridisaation käyttöä rajoittaa toistai- seksi eniten se, että tunnistuksessa tarvittavat koettimet on leimattava radioaktiivisesti, mikä on kallista ja edel- lyttää erikoistiloja. Nukleiinihappohybridisaatio on kui- tenkin hyvin uusi menetelmä, ja ei-radioaktiivisista koettimista on jo saatu lupaavia tuloksia. On myös ke- hitetty uusia hybridisaatiomenetelmiä, jotka soveltuvat entistä paremmin automatisoitaviksi. Kerroshybridisaa- tio on Suomessa kehitetty menetelmä (Orion-yhtymä Oy), jolla mikrobeja voidaan tunnistaa helposti puhdistamat- ta näytettä. Nukleiinihappohybridisaatiomenetelmätovat nopeasti kehittymässä siihen suuntaan, että niillä voidaan tehostaa monien tällä hetkellä vaikeasti tunnistettavien kasvivirusten, viroidien ja kasvipatogeenistenbakteerien diagnostiikkaa. Tämä uusi menetelmä tulee lähivuosina helpottamaan esimerkiksi kasvintarkastustoimintaa, ter- veen kasvimateriaalin tuotantoa ja taudinkestävyysjalos- tusta. 191