MaataloustieteellinenAikakauskirja Vol. 59: 169—178, 1987
A simple kit for rapid field diagnosis of potato virus Y by latex serological test
AARNE KURPPA and MATTI VUENTO
Department
of
Plant Pathology, Agricultural Research Centre SF-31600 Jokioinen, FinlandDepartment
of
Biochemistry, Universityof
HelsinkiUnioninkatu 35, SF-00170Helsinki, Finland
Abstract. Asimplekit for rapid detection ofpotatovirus Ybylatex serological testwas developed.The test is carried outon a white cardboard sheet and the resultscanbe read by nakedeye intwominutes. Atestcard of 10x 6cm holdslatex sensitizedantibodies,buffers and othernecessaryingredientsasdryblue colored formulateonthe ringedareasof the card.
Atestcard includes spacefor six tests and positive and negative controls. The kit also includes disposable plasticsticks for mixing the samples with testreagentsand ahand presswith dis- posable plastic tips.
Fortesting,driedreagentsaredissolved indropsof sample and mixed. After gentle rota- tion,samples containingvirus appearclearly granulatedwhile samples from healthy plants remain unagglutinated.
The testing of undiluted extracts of evenly developed tubersproutsresulted inover91 % identitywith the results obtained with ELISA that wasusedas a control method. Testing of diluted leaf extracts reached thesamereliabilitybut undiluted leaf extracts from glasshouse grown potatoeswerenot well suitableastest samplesbecause of their darkgreencolor.No such problems occurred with fieldgrown material and acomplete identitywith the ELISA readingswas true when the samples included secondarily infectedpotatoplants.
No reaction to other potatoviruses thanPVY was obtained by the test kit.
Index words: virus detection,serology,latex test,ELISA, potatovirus Y, PVY
Introduction
Serological diagnosis of plant viruses has developed drastically during thepast decade, particularly with theintroductionoflabelled antibody techniques, especially the ELISA
(enzyme-linked immunosorbent assay, Clark and Adams 1977). Also, the latest research interest in developing more advantageous serological techniques has mainly focusedon labelled antibody techniques originating in medical immunology.However,interest in the
JOURNAL OF AGRICULTURAL SCIENCEIN FINLAND
study of essentially simpler but sufficiently reliable tests has remained limited.
The ELISA hasanumber of advantageous properties over the methods previously used forroutine detection of plant viruses, above all high sensitivity and specificity, and it is easy to be applied in any moderately equipped research laboratory. However, it cannot be used for field diagnosis, and it takesone to one and ahalf days to have the results.
Atpresent, the ELISA is the only relatively rapid test method for the detection ofpotato virus Y (PVY). Other moretraditional tech- niques such as the agglutination test, micro- precipitin test (vanSlogteren 1957), and ra- dial immunodiffusion test (Shepard 1972, Richter et al. 1979 a), are not sensitive enough for the routine detection of PVY in potato tubers or leaves.
The latex serological test (Bercks 1967, Abu Salihetal. 1968) offers potential sensi- tivity for the detection of barley yellow dwarf virus in plant extracts(Aapola and Rochow
1971). However, the test is too complicated with its preparatory steps, and thus has not aroused sufficient interest as aroutine tech- nique. The latestattemptsof developing latex serologicaltestkits for practical diagnosis of PVY (Talley et al. 1980, Wiedemann and Fuchs 1983, Fribourgand Nakashima 1984) have been promising, but thetestsare still to laborousorcomplicated for easy diagnosis in the field or on the farm.
The aim of this study was to develop a simple but highly sensitiveand reliabletestkit for the extremely rapid detection of potato virus Y by latex serological principle.
Materials and methods
Antibody production and characterization For the production of PVY specific anti- bodies for thetestkit, a Y" strain isolate F43
wasselected foran antigen. This isolate isse- rologically closely relatedtothe isolate YSFIS (Kurppa 1983,Kurppa and Korhonen 1984) and is thus suitable for raising up antibodies
to recognize all isolates of potato virus Y found in Finland.
The viruswas propagated in Nicotianata- bacum cv.Samsun and purified withaslightly modified method of Richter etal. (1979 b).
The final purification step was density gradient centrifugation in nuclease-freesucrosegradients (5 —35 %)prepared in 0.1 M borate bufferat pH 8.2. The purified material showedasingle polypeptide band having amol.wt. of33 000 when analyzed by polyacrylamide gel electro- phoresis in the presence of sodium dodecyl- sulphate (SDS-PAGE, Laemmli 1970). This value, although higher than that calculated from the aminoacid sequence (Shukla etal.
1986), agrees well with the values obtained by other investigators using SDS-PAGE (deßoxx 1981, Aruta 1983). The preparations had A26
0/A
2go-ratio
of 1.13—1.23,which is well in line with the value previously described for the purified virus (deßoxx 1981). Electron mi- croscopy of thepurified viruswas carriedout after negative staining with phosphomolyblic acid. It revealed long, thin flexiblestructures characteristic ofpotato virus Y (Fig. 1).Rabbits were immunized by subcutaneous injections of purified virus (100 ng) emulsified in Freund’s incomplete adjuvant. The injec- tions were given on days 0, 21 and 35, and bloodwas collectedon day44. Sera showing good litres (below) were pooled and im- munoglobulin fraction was precipitated with ammonium sulphate at a 50% saturation.
The precipitated immunoglobulins were dia- lyzed against 0.9% NaCl and stored at
—2O°C. For the characterization of antibo- dies in the ELISA, immunoglobulins were separated from the antisera using columns of protein A-Sepharose CL 4B and Sephadex G-25 (Pharmasia). Immunoglobulin fractions wereused atconcentrations of 0.25, 1 and 4 gg/m\ for coating the plates. Comparative di- lutions from enzyme conjugates of Ynand Y°
strain antibodies weremade for thetest.Test samples included known isolates of PYY in potatoleafextracts, and the double sandwich procedure of Clark and Adams (1977) was used.
Preparation
of
latexreagent and test cardFor preparation oftestlatexreagent, poly- styrenelatex particles (diameter 0.8 1 % final concentration)wereincubated with anti- potato virus Y antibodiesat varying concen- trations in 0.1 M-glycine buffer pH 8.2 for 2 h at37°C. After theincubation, bovine serum albumin was added to a concentration of 0.1 %. The latexreagent was sedimented by centrifugation and resuspended in the above buffer containing 0.1 %albumin. This wash- ing procedure was repeated twice. Control latexreagent was preparedas above but with immunoglobulins isolated from nonimmune rabbit serum.
The prepared latexreagents weretested for agglutination by purified potato virus Y and by plant sap frompotato sprouts andpotato leaves infected with the virus as well as by plant sap from healthy plants. Latex reagent (30
/il
1 % latex) was mixed with 30/d
ofsampleon awhitetestcardatroom tempera- ture.Thetest card was tilted back and forth
for exactly2 min,after which time the result wasread. Test latexreagent showing highsen- sitivity and aminimum of nonspecific reactiv- ity was selected for the preparation of test cards.
Preparation oftestcards that contained the testand control latexreagents in dry formwas carriedout by Ani BiotechLtd, Helsinki,Fin- land. The test cards also contained a dried positive controlreagentthat, when dissolved together with the dried test latex reagent,
caused anagglutination of the latexreagent.
Each card containedreagents for sixtests.In addition, one positive control (dried latex reagent together with dried positive control reagent) and onenegative control (dried con- trol latex)wereincluded in eachtestcard. The cardsweredelivered insets often cards sealed in air-tight bags together with a silica desic- cator.
Comparative virus testing
For the latextestkitasimple hand presswas Fig. 1. Electron micrographofpurified particlesof PVY" strainF43negativelystained with 1 % uranyl acetate.
Bar represents 200nm.
171
designed. With disposable plastic tipsastan- dard volume ofc 30
/d
of liquid samplecould be laidonthe test card. Dried latex-antibody dots on the card weredissolved in a sample drop and were mixed carefully but quickly witha disposable plastic stick. The test was ready tobe read with the naked eye aftertwo minutes of gentle rotation of thetest card. A PYY positive sample resulted in granulated accumulation of sensitized latex particles, similarto the positive test control. Negative samplesaswellasnegativetestcontrols stayed unagglutinated (Fig. 2).To characterize the practical properties and reliability of the latex test acomparison with the standard double sandwich ELISA (Clark and Adams 1977) was done. The same ex- tracts from potato sprouts and leaves were tested with both methods. In the ELISA antibodies and their enzyme conjugates to Y"
and Y° strain isolates were used.
Test groups of 18 sprouted tubers were formed. Each group included 6 to 12 tubers naturally infected withPYY, and the remain- ing tuberswerehealthyorinfected with other
potato viruses (Tables 1 and 2). The number ofpotatocultivars tested totalled eleven. The tubers withinthe groupsweremixed toavoid expected results in the latextest.After testing of sprouts, the tubers were planted in peat
substrate in a greenhouse. A few sample tubers from the preceding test hadto be re- placed becauseno emergingsprouts wereleft after sample taking.
The comparative tests from potato leaf sampleswere done threeto four weeks later.
At first undiluted leafextracts were used but because of the harmful effect of dark green coloron the visibility of latex agglutination reaction, thetestsampleswerelater diluted 1:5
in unbuffered saline(0.15 M NaCI).
For preliminary experience in testing of field grownpotatoplants, acomparativetest with 120 leaf samples was done in late July.
Thetestresults obtained with the latextest and with the ELISA were compared in each testgroupby counting identical and different recordings and tryingto findout thereason
for disagreement in each case.
Fig. 2. Atestcard of 10 x 6 cmwith clear granulated latex agglutination of virus positive samples (1 and 6) and the positive control. Sampleswerediluted 1:5 inunbuffered saline.
Table 1.Comparison of the latex test with the ELISA for the detection of PVY in eye sprouts of eleven potatocultivars.
Test PVYpositive samples
Latex ELISA Latex=
ELISA
1 12/18 12 10
2 11 9 9
3 10 10 10
42 4 10 4
52 5 8 5
6 10 10 10
7 9 9 9
8 8 11 8
9 9 10 9
10 8 9 8
11 11 10 10
122 6 10 6
13 8 10 8
14 8 8 8
15 10 10 10
16- 6 10 4
17 12 11 10
18 8 10 8
19 10 9 8
20 10 12 10
21 8 10 8
Totals 183/378 208 172
PVY-% 48.455.0
Identical recordings(%) 82.7 (all test groups) 91.1 (groups 4,5, 12 and
16rejected False positive recordings 6.0 (all test groups) inthe latex test (%)
1 Test groups I—91 —9 alsoincluded samples infected with PVM,PVSand PVX
: Poorly developed tiny sprouts, c0.5 cm long
Results
Potential and optimising
of
the latex method Antisera raised in the rabbits for thetestkit were highly virus specific. Some strain speci- ficity toPVY" was present, as expected, and there were also minor differences between antisera from different animals. Specific IgG fractions from the antisera, when used for coating the ELISA plates, resulted in the fol- lowing comparative absorbance readings from potato leaf samples (IgG, enzyme conjugate= 1 /ig/ml, substrate incubation 20 mins at 20°C):
a) PVY" conjugate, Ynsamples 1.553 (1.202—1.818), background 0.011 Y° samples 1.227 (0.939—1.419) b) PVY0conjugate, Y" samples 1.269
(0.880—1.373), background 0.016 Y° samples 1.416 (0.927—1.506) Immunoglobulins fromourhigh-titre anti- sera were effective in coating latex particles atconcentrationsas lowas 10—20/tg/ml. The resulting testlatexes wereagglutinated by sap from infected but not from healthy potato sprouts. However, usingverylow amountsof antibody for coating introduced a risk of getting false negative reactions by samples containing very high amounts of the virus (antigen excess). Therefore, wechose to coat the test latex with a slightly higher antibody concentration of 40pg/ml. Test latexes pre-
Table 2. Comparisonof the latex test with the ELISA for the detection ofPVY inpotatoleaf samples diluted in unbufferedsaline 1:5. The test in- cluded tenpotatocultivars.
Test PVY positive samples
grOUP
Latex ELISA Latex=
ELISA
1 13/18 13 13
2 13 12 12
3 8 8 8
4 5 6 5
5 8 7 7
6 8 8 8
7 8 8 8
8 6 6 6
9 9 9 9
10 9 8 8
11 12 12 12
12 9 9 9
13 11 10 9
14 14 12 12
15 12 9 9
16 16 16 16
17 13 11 10
18 12 12 11
Totals 186/324 176 171
PVY-% 57.454.3
Identical recordings (%) 91.9
False positive recordingsinthe latex test(%) 5.4
False negative » 2.7
' Thetestgroups werenot exactly identical with those presentedin Table 1.
pared by using this coating concentrationwere agglutinated by purified virus at concentra- tions of 1 /rg/ml or higher; we tested the reagent for agglutination by high viralcon- centrations and found thereagent tobe effec- tively agglutinated at the highest concentra-
tion tested (200 gg/m\). Control latexes prepared by using 40 /ig/ml of nonimmune rabbit immunoglobulis for coatingwere not agglutinated with purified virus.
Polystyrene latexes prepared by several manufacturers invarious colors were used in initial experiments. While the various latexes gave similar results with purified virus par- ticles, the initial results varied when plant materialwas usedas samples. The darkness ofpotato sap made it impossibletouse white or any light colored latex in the test. After someexperimentation, weselected dark blue latexto be used in thepresent test. Suitable materialwassupplied e.g. by Serva (Germany) or by Rhone-Poulanc (France).
The final driedreagent dots contain2 moles of glycine bufferedatpH 8.2. Inaddition, the dots contain reagents used in drying the latexes; however,they do not affect the ionic balance of dissolvedreagents.
Comparativetesting
of
sprout andleaf
extractsWith the latextest the agglutination reac- tion wasclear and easytoread if well devel- oped sproutsgrown in the darknessorunder weak non-continuous light wereused. There- action was normally completely developed aftertwo minutes’ gentle rotation of thetest card but in afew cases an extra minute of agitation increased the clearness of virus spe- cific agglutination. Rotation of the card for five minutes or longer may result in non- specific reactions. In testing of samples from well developed pale and juicy sprouts, the comparability with the ELISA recordingswas excellent, 91 per cent (Table 1).
Because of their violet colorsproutsamples from tubers kept under strong natural or artificial light caused somevisibility problems
of the virus specific reaction. The latex ag- glutination reaction on the white cardwas at
least as intensive as with the pale sproutsap but it became partially covered with colored material. The reaction remained reliably rea- dable, if the sample extract did not contain much crushed material with large plant cell particles. Coarce crushed opaque plantmate- rial mayaccumulate particularly atthe edges of the ringed test areaand completely cover the virus specific latex agglutination reaction.
Similarly negative reactions may remain unconfirmed.
Potato tubersat the early stage of sprout- ing were not suitable for testing PVY in the sproutswith the latextestorwith the ELISA.
No more thanc 50% of the virus infected samples could be detected with the latex test (Table 1) and false negativeswere also found with the ELISA fromsprouts ofc5 millimeter long or shorter. Almost no false positive recordings were made, which indicates low virus concentration being responsible for the unsatisfactory results.
Exceptionally high ELISA absorbances did notnecessarily indicate intensive agglutination in the latextest. In the range of absorbance values fromc0.3 to2.0 (30 mins of substrate incubation at 20°C) all comparative latex agglutination reactions could have almost the same intensity.
Intense non-transparent green color in undilutedpotato leaf sap disturbed the inter- pretation of the results in the latextest. Al- though virus positive reactions developed rapidly during card rotation, agglutinated latex particles were sometimes hardly visible through thick colored material. After ten test groups,testing wasdiscontinued because only c70 % of the positive recordings were true positives when compared with the ELISA. In- sufficient reliability in the latextest was due toboth false positive and negative recordings.
When the test comparisons were repeated using 1:5 diluted (in unbuffered saline) potato leaf extracts, the non-specificity problems al- most disappeared and a comparability of 92 % with the ELISA wasreached (Table 2,
Fig. 2). False positive recordings were more common than missing positive reactions, which indicatedat least sufficient sensitivity of the latextest in the detection of PVY in diluted potato leaf extracts. False positive readings weremostly caused by accumulation ofcoarse plant cell materialpresent in the sap samples.
Testing of leaf samples from field grown
potatoes gave completely identical results compared with the ELISA. Agglutination reaction was always clear ifa minimum suf- ficient volyme of undiluted sap(15—20 /d)to coverthetest area on the cardwas used. An increased sample volume may result in anti- body-antigen imbalance (antigen access), which appears in poor orprohibited aggluti- nation. The petioles of the leaveswerefound as exellent material for testing.
Potato virus Y could be detected reliably with the latextest in potato leafextracts di- luted 1:50 in unbuffered saline. A dilution of
1:100 causedanincreasing proportion of ques- tionable reactionstooccur. When dilutions of
1:3 or less were tested, false positive or masked reactions became increasingly com- mon.
The latextest didnot react toany otherpo- tato viruses than PVY.
Discussion
The results shown in Tables 1 and 2 indi- cate that our latex agglutination test was strictly specific for potato virus Y. This was nodoubt very much duetothe successful im- munization procedure producing high-titered antisera with good specificity. We feel thata high antibody titer is essential in developing thistypeofalatex since it allows the antibody to be usedatrelatively low concentrations in the preparation of the latexreagent. Although low titre antisera have been successfully used with the antibody sensitized protein, A-coated latex (PAL) in some cases as reported by Torrance (1980), it always introducesa new source of increased non-specificity.
The low concentration of antibody used in
the coating of the latex effectively dilutesout possible contaminating antibodies against plant tissue. The immunogen is never abso- lutely pure, and even minor impurities can give rise to significant production of conta- minating antibodies. Some ofourantisera did showtraces of antibodies against potato sap components. Also,using high immunoglobulin concentrations to coat latex particles, we found that normal potato sap components
(possibly lectins) agglutinated the latexeseven in the absence of viral antigens.
As compared to other latextests described for the detection ofpotato virusesX, S, and Y (Talley etal. 1980,Fribourgand Nakas-
hima 1984,Franc and Banttari 1986), our testis significantly faster. Incubation times of I—2 h have been used in previoustests, with our latextest, however,the result canbe ob- tained in two minutes. Prolonged incubation time could possibly increase sensitivity, but thisseemsunnecessaryin the light ofourpre- sentresults which show thatourlatextesthas apredictive value close tothat of the ELISA.
The buffersystem used in ourlatextestdid notgive maximum sensitivity for the test.In initial experiments with purified virus particles wenoted that lowering the pH of the assay systemtopH 6 considerably increased its sen- sitivity. However, those buffers were poorly compatible with the drying system used. We also tried Tween 20 and mercaptoethanol (Fribourg and Nakashima 1984) in our test buffer, but this ledtono improvement in the performance of the assay. However, the glycine buffer selected as the test buffer did give fair sensitivity, and itwascompatible with the reagent drying procedure used by Ani Biotech.
The properties of the antibodies produced for thetestkitwere well suitable for the pur- pose with exellentrecognition of heterologous isolates of Ynand Y° strains. Reliable detec- tion of various PVY isolates withasingle po- lyclonal antiserum agrees with the results of Torrance (1980) but disagrees with the data of Maat and deßoxx (1978), who have re- ported distinct serological relations between
thetwotypestrains of PVY. Perhaps the dif- ferent assays used can partially explain this discrepancy. In ourprevious studies (Kurppa 1983) significant serological differenceswere present between Yn and Y° strain isolates, but with careful selection of animmunogen, much of this diversity could be avoided.
Practical comparative tests clearly con- firmed the high potential of oursimple latex serological test for the detection of potato virus Y. The results obtained from naturally developedsproutswith the latextestdemons- trated exellent comparability with theELISA, which in the detection of PVY in potato sproutshas been found highly reliable bysev- eral researchers including Maat and deßoxx (1978), Gugerliand Gehringer(1980), and
Kurppa (1983). The low virus concentration together with the irregular distribution in pri- marily infectedtubers,reported by Beemster (1967) and Weidemann and Wigger (1984) maycauseinaccuracy in any testing procedure but thiscan be effectively avoided by breaking ofdormancy with Rindite treatmentasshown by Gugerliand Gehringer (1980) and Vet-
ten et ai. (1983). Low virus concentration was also clearly demonstrated in our experi-
ments insomegroups ofsamplesat the early stage of sprouting but a sufficiently high concentration was reached with prolonged sprouting. The drastic effect of Rindite treat- ment in raising up PVY concentration is
unquestionable, but for practical reasons it is an unwantedstep here. Our previous experi- ments have shown that PVY is relatively evenly distributed and it achieves asufficiently high concentration during tuber sproutingat a room temperaturewhen the sproutshavere- ached the length oftwo tothree centimeters.
The sufficient sensitivity of the latexsero- logical test for the detection ofpotato virus Y in potato leaves has been previously re- ported by Khan and Slack (1978), Talley et al. (1980), Torrance (1980) and Fribourg and Nakashima (1984). Thus the problem in the detectionisnotthe lack of sensitivity but non-specificity arising from chlorophyll, lectins and other cell materials.
Masking of specific latex agglutination re- actionsornon-specific accumulation of green cell materials could be almost completely avoided by dilution of the leaf extracts in unbuffered saline(1:5). However, this is an unwantedextra step for field diagnosis, and alternatives to sample dilution have to be studied. The use of leaf petioles instead of laminae or even stems as test samples may offer essentially less chlorophyll containing extracts. Our experience in testing field grown potatoplants islimited,but itseemsverylikely that no dilution of leaf sap is necessary for accuratevirus detection in secondarily infected leaf tissue. Potatostemsmay also be suitable material for testing and ata latestage of the growing season they may also serve as exel- lent indicators for primary tuber infection.
Atpresent ourlatextestkit offersaccurate detection of potato virus Y in sprouts, but it canalso be applied for virus detection in green plant samples, ifafew precautions aretaken.
Acknowledgements.This projectwassupportedinpart by Suomen Itsenäisyyden Juhlavuoden Teollisuusrahas- to (Sitra)and by Ani Biotech Ltd, Helsinki. We thank Mr.Anssi Mörttinen,Departmentof Virology, University ofHelsinki, for electron microscopy ofpotatovirus Y samples.
References
Aapola,A. 1. E.&Rochow, W. F. 1971.Relationships among three isolates of barley yellow dwarf virus.
Virology 46: 127—141.
Theuseof antibody-sensitised latex particles to detect plant viruses. J. Gen. Virol. 3: 299 —302.
Aruta,C. 1983.Untersuchungenzurelektrophoretischen und biologischen Differenzierungvon Stämmen des AbuSalih, H. S., Murant, A.F. &Daft, M. J. 1968.
176
PotatoVirusYSmith.Dissertation,UniversitätHan- nover, Germany.
Beemster, A. B. R. 1967.Partial infection with potato virus Y" of tubers from primarily infected potato plants, Neth. J.PL Path.73: 161—164.
Bercks, R. 1967.Metodische Untersuchungentiberden serologischenNachweis pflanzenpatogener Viren mit dem Bentonit-FlockungTest, dem Latextest unddem Bariumsulfat test. Phytopath.Z. 58: 1—l7.
Bokx, J.A. de& Huttinoa, H. 1981.Potato virus Y.
C.M.1./A.A.B. Descr. PI. VirusesNo 242.
Clark,M. F.&Adams, A.N. 1977.Characteristicsof themicroplatemethod of enzyme-linked immunosor- bent assayfor the detection of plant viruses. J.Gen.
Virol. 34: 475—483.
Franc, G.D. & Banttari, E. E. 1986.Comparisonof latex agglutination, enzyme-linked immunosorbent assay and indicator plants for detection ofpotato viruses S and X in potatoes. Am. Potato J. 63:
357—362.
Fribourg, C. E. &Nakashima, J. 1984. An improved latex agglutination test for routine detection ofpotato viruses. Potato Res 27: 237—249.
Gugerli,P.&Gehringer,W. 1980.Enzyme-linkedim- munosorbentassay (ELISA)for the detection ofpo- tatoleaf roll virus andpotatovirusYinpotatotubers after artificial break of dormancy. Potato Res. 23:
353—359.
Khan, M.A.&Slack, S.A. 1978.Studiesonthe sensi- tivityof latex agglutination test for the serologicalde- tection of potatovirus S and potatovirus XinWis- consin. Am. Potato J. 55: 627—637.
Kurppa, A. 1983. Potato viruses inFinland and their identification. J. Sci. Agric. Soc.Finl. 55: 183—301.
& Korhonen, K. 1984.ELISAreagents forpotato
virus Ystrains with signicantly low non-specificreac- tions. J.Agric. Sei.Finl. 56: 89—95.
Laemmli, U. K. 1970.Cleavage of structural proteins duringthe assembly of the head of the bacteriophage T4.Nature(London)227: 680 —685.
Maat,D. Z.& Bokx, J.A.de1978.Enzyme-linkedim- munosorbent assay(ELISA) forthe detection ofpotato virusesAand Yinpotato leaves and sprouts. Neth.
J. PI. Path. 84: 167—174.
Richter, J., Haack, I. & Eisenbrandt, K. 1979a.
Routineraässige Serodiagnoseder KartoffelvirenX,S, Mund Ymit dem Radialimmuno-diffusionstest. Arch.
Phytopath.Pfl.schutz. 15: 81—88.
—,Leiser, R.-M., Proll, E.& Döring,U. 1979b. Ver- suche zur Differenzierung von Stämmen der Kar- toffelvirenX, S, M undYan Hand ihrer Immuno- genität.Arch. Phytopath. Pfl.schutz 15: 13—20.
Shepard, J.F. 1972.Gel-diffusion methods forthese- rologicaldetection ofpotatovirusesX,SandM.Mont.
Agric. Exp. Sta. Bull.622.72p.
Shukla,D. D., Inolis, A.S.,McKern, N. M.&Gough, K. H. 1986.Coat proteinof potyviruses. 2.Amino acid sequenceof the coat protein ofpotatovirusY.Virol- ogy 152: 118—125.
Slooteren,D. H. Mvan 1957.Serologicalidentification of plant viruses and serological diagnosis of virus diseases of plants.Ann. Rev.Microbiol. II: 149—164.
Talley, J., Warren,F. H. J.,Torrance,L.&Jones,R.
A.C. 1980. Asimplekit for detection of plant viruses bythe latex serological test.PI. Path. 29; 77—79.
Torrance, L. 1980. Use of protein Ato improvesen- sitisation of latex particles with antibodies to plant viruses. Ann. Appi. Biol. 96: 45—50.
Vetten, H. J., Ehlers, U.&Paul, H. L. 1983.Detec- tion ofpotatovirusesYandAin tubers byenzyme- linked immunosorbentassayafter natural and artificial break of dormancy. Phytopath.Z. 108:41—53.
Weidemann, H.-L.von&Wigoer,E.-A. 1984.Thecon- centrations ofpotatovirusY(PVY)intubers and leaves ofpotatoplants.Abstr. Conf. Pap. 9th Triennial Conf.
EAPR. 1.—6. Juli 1984in Interlaken.
Wiedemann, W. &FUCHS,E. 1983.ZumNachweisvon Kartoffelviren mit dem Latextest bei Verwendungvon PVC-Tiefziehblistern. Arch. Phytopath. Pfl.schutz 19:
215—216.
177
SELOSTUS
Yksinkertainen nopea lateksiagglutinaatio- testi perunan Y-viruksen määrittämiseksi Aarne Kurppa
Kasvitautiosasto, Maatalouden tutkimuskeskus, 31600Jokioinen
Matti Vuento
Biokemianlaitos, Helsingin yliopisto, 00170 Helsinki
PerunanY-virus aiheuttaa jatkuvasti tuntuvia sadon- menetyksiä javähentäätätenperunanviljelynkannatta- vuutta.Virus rasittaa erityisesti Etelä-Suomen ruokape- runansiementuotantoa. Ainoa mahdollisuusestäävirus- tautivahingotonkäyttäätervettäsiemenperunaa. Tervey- den varmistamiseksi siemeneksi käytettäväperunaontes- tattava. Nykyinen testauslaboratoriokapasiteettiriittää vain valio- ja kauppasiemenen tutkimiseen. Ruokaperu- naatuotetaan yhä yleisesti tarkastamattomallasiemenellä, mikäonusein pahoin Y-viruksen tartuttamaa.Tilanteen korjaamiseksi tarvitaan lisää siemenperunan testauska- pasiteettia,mieluiten luotettavaa yksinkertaistatestiä,joka soveltuisi myösneuvonnanja perunatilojen käyttöön.
Tämätutkimus Y-viruksen pikatestin kehittämiseksi tehtiin Maatalouden tutkimuskeskuksen jaAniBiotech Oy;n(Helsinki) yhteistyönä. Alustavissa tutkimuksissa sel- vitettiin lateksiagglutinaatioperiaatteen sopivuus tarkoi- tukseen,minkä jälkeen valmistettiin tarkoitukseen erät vasta-aineseerumeita Y-viruksen Yn-rotua antigeeninä käyttäen.
Virusmääritystehdään valkealla pahvikortilla. Tähän muotoiltujenkohoumarenkaiden keskelle onkiinnitetty pieni sininen täplä, mikä sisältää lateksi-vasta-aine- kompleksinlisäaineineen. Kortilla (10 x 6cm)onkuusi testipaikkaasekä lisäksi paikat positiiviselle ja negatiivi- selle kontrollille. Testattaessa kuiva reagnessitäplä liuo- tetaannoin30 /d;nnäytepisaraan jasekoitetaan koko näy- teaineellelevittäen,minkä jälkeen näyte pidetään hitaassa liikkeessä korttia käännellen. Tulos luetaan kahden mi- nuutinkuluttuasilmävaraisesti. Y-virusta sisältävätnäyt- teetsekäpositiivinenkontrolli muuttuvat rakeisiksi kun taas viruksettomat näytteet ja negatiivinenkontrolli säi- lyvät hienojakoisensileinä. Testi ei reagoiperunanmui- hin viruksiin.
Lateksitestillä päästiin yli 91 °/o:n yhdenmukaisuuteen ELISA-testintulosten kanssa määritettäessä Y-viruspe- runan itujen puristemehusta. Virhe syntyi lähinnä joiden- kinheikkojen positiivisten reaktioiden havaitsematta jää- misestä. Näytteiksi soveltuvat parhaiten pimeässä tai hei- kossa valossa kasvaneet mehukkaat vaaleat idut. Valos-
sakehittyneiden itujenvioletti väri häiritsee tuloksenlu- kemista jonkinverran. Itämisen alussa olevia mukulanäyt- teitä ei pidä testata, koska viruskonsentraatio iduissaon tällöin vielä hyvin alhainen.
Kasvihuoneessa kasvatettujen perunantaimien laimen- netuista lehtimehunäytteistä saatiin92 %:sti oikeat virus- positiivisettulokset. Virus voitiin määrittää luotettavas- ti vielä 1:50laimennetusta näytteestä. Sopivaksi testilai- mennokseksi katsottiin kuitenkin 1:5,vaikka tällöinkin syntyi joitakinvoimakkaan vihreän värin aiheuttamiavir- hetulkintoja.Laimentamaton lehtimehu ei soveltunuton- gelmitta näytteeksi,sillä väri saattoi peittää tai naamioi- da vahvankin agglutinaatioreaktion tai saostua vähäisen- kin kuivumisen aikana. Lehtiruodeista tai varsistasaa- tava puristemehusoveltuu lehtimehua paremmin näyt- teeksi.
Normaalien kasvustonäytteiden virusmäärityksistätä- hän artikkeliin saatiin vasta alustavia tuloksia. Virusmää- ritysvoitiin tehdä aina täysin luotettavasti myös laimen- tamattomastalehtimehusta. Saostumareaktio oli erittäin selkeä,kun laimentamatonta mehua käytettiin minimi- määrä(15—20/il), mikä riitti peittämään tasaisesti testi- kortin näytetilan. Näytemäärän lisäännyttyä yli30/tl:n
reaktio heikkeni nopeasti, mikä aiheutui vasta-aine- konsentraation alenemisesta ja näin syntyvästä virus- ja vasta-ainekonsentraation epätasapainosta. Luotettavan määrityksenperustaon siten vakiotilavuuksinen näyte kortin kaikissa näytetiloissa.
Kehitetty testipakettisisältää testikorttien lisäksimuo- visia kertakäyttöisiä sekotustikkuja, pienen näytepuris- timen sekä puristimen kertakäyttökärjet. Testikortit säi- lyvät käyttökelpoisinakuivassa viileässä paikassa vähin- täänvuoden. Testi soveltuu nykyisellään iduistatehtäviin määrityksiin ja myös muista kasvinäytteistä voidaansaa- vuttaa riittävä luotettavuus. Testiä tulisi vielä kuitenkin parantaasiten,ettäkasvustosta ennennostoa otetustaver- sonäytteestä saataisiin luotettava tulos,jolloin viljelijävoi- si hyvissä ajoin varautua mahdolliseen siemenkannan uusimiseen.
