Evaluation of the enzyme-amplified ELISA for the detection of potato viruses A,M, S,X, Y,and leafroll

Various parameters,e.g. types of microtiter plate for DAS-ELISA (double antibody sandwich-enzyme-linked immunosorbent assay), use of fresh or frozen amplifier solutions for enzyme-amplified-ELISA, and use of sodium diethyldithiocarbamate (NaDIECA) in sample buffer in cocktail-ELISA were evaluated for the detection of potato viruses A, M, S, X, Y and leafroll from potato foliage. Dynatech Immulon immunoplates provided higher readings for all viruses. Fresh amplifier solution in amplifed-ELISA was superior to frozen solutions. Amplified ELISA gave only marginal improvement in the sensitivity over the standard DAS-ELISA. Addition of NaDIECA in sample buffer did not improve the detection of viruses in DAS-, amplified-, or cocktail-ELISA. Cocktail-ELISA can reduce antigen incubation time to as short as 15 min for PVA, PVM and PVX; 1 hr for PVY and PLRV; and 2–4 hr for PVS using pre-coated plates. Although amplified-ELISA is slightly more sensitive than DAS-ELISA for certain potato viruses, it is not suitable for large-scale indexing of potato viruses in Seed Certification Laboratories, in view of the additional steps needed in carrying out this procedure.     

A technique for rapid detection of leptine glycoalkaloids in potato foliage

A rapid screening procedure for detecting the presence of leptines in potato foliage was developed. The glycoalkaloids in expressed sap are separated by high performance thin-layer chromatography and detected with iodine vapor. About 80 foliage samples can be analyzed per day as compared to about 10 using other available methods, such as gas-liquid chromatography.     

Rapid detection of potato viruses by dot-ELISA

Summary The dot-ELISA, an immunosorbent assay on nitrocellulose (NC) membranes, was applied to potato viruses. The sensitivity of the test, as determined by visual assessment of the colour reactions in serial dilutions, allowed a reliable detection of the potato viruses M, S, X, Y, A and of potato leafroll virus in leaves of non-potato hosts and potato plants. Tuber extracts taken from the rose end resulted in higher dilution end points than those from the heel end. The test can be performed within two to three hours by using stored NC-membranes precoated with specific γ-globulin. The possibility of storing antigen treated NC-membranes for some days allows the assay to be quickly applied to samples taken in the field.     

Ultramicro-ELISA with a fluorogenic substrate for detection of potato viruses

Summary A double-antibody ELISA is described in which polystyrene-covered polyvinylchloride plates with 10 μl test volume in the wells are used as a solid phase. A fluorogenic substrate, 4-methylum-belliferyl phosphate is used instead of a colorigenic substrate. The plates are loaded with 10 μl samples by using a 50-channel micropipette and fluorescence units read at 0.16 mm thickness with a special measuring device. The practicability and sensitivity of the method are exemplified by the detection of potato viruses X, Y and M (secondary infections) and potato leafroll virus (primary infections), in leaf and tuber extracts. Ultramicro-ELISA is as reliable in detecting the viruses as the standard ELISA. Because of the very small volumes used in the wells, chemicals, antibodies and alkaline phosphatase are reduced by 95% as compared with the standard test.

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Zusammenfassung Es wird ein Ultramikro-ELISA in der Form eines Doppelantik?rper-ELISA (Clark & Adams, 1977) beschrieben, in dem als feste Phase polystyrolbeschichtete Polyvinylchlorid-Folien mit einem Füllvolumen von 10 μl verwendet wurden. Die Dosierung erfolgte in einem Raster von 5×10 mit einem speziellen 50-kanaligen Mikropipetter. Alle Inkubationsschritte wurden bei Raumtemperatur durchgeführt. Nach dem Ausspülen mit PBS-Tween wurden die Folien 20 Minuten mit Aqua dest. gewaschen. An Stelle des chromogenen Substratesp-Nitrophenylphosphat wurde 0,0005 mol/l 4-Methylumbelliferylphosphat in Diethanolaminpuffer verwendet. Die Reaktionszeit für das fluorogene Substrat betrug 20 Minuten. Ein Abstoppen der Substratreaktion entfiel, da die L?sung nach erfolgter Reaktion mit einem Mikropipetter auf eine im Raster 5×10 gekammerte Glasmessküvette überführt wurde. Die Messung der Fluoreszenzintensit?t erfolgte bei 0,16 mm Schichtdicke in einem speziellen Auswerteger?t. Dieser Ultramikro-ELISA, der zur quantitativen Bestimmung des α₁-Fetoproteins Anwendung findet (Horn et al., 1981; Hoffmann-Blume et al., 1983), wurde in seiner Nachweissicherheit mit einem üblichen Doppelantik?rper-ELISA verglichen (Richter et al., 1979). In die Untersuchungen wurden Blattpress?fte von Pflanzen einbezogen, die mit Kartoffelblattroll-Virus (PLRV), Kartoffel-Virus Y (PVY) und Kartoffel-Virus M (PVM) infiziert waren. Des weiteren wurden das Kartoffel-Virus X (PVX) sowie die Viren PVY und PVM in sekund?rinfizierten Knollen und Bl?ttern von Kartoffelzuchtst?mmen (spontane Freilandinfektionen) nachgewiesen. Zum Nachweis prim?rer Infektionen mit PLRV wurden gesunde Mutterpflanzen der Sorte Adretta an zwei Terminen mit Hilfe von Blattl?usen infiziert. Die mit beiden ELISA-Verfahren erhaltenen Ergebnisse stimmten überein. In Blattpresss?ften konnten die Viren bis zu einer Verdünnung von 10⁵ (PVM), 10³ (PVY) und 10² (PLRV) nachgewiesen werden. In 20 Individuen von Kartoffelzuchtst?mmen wurden mit beiden Verfahren übereinstimmend PVX und PVY in 5 Knollen- und Blattproben festgestellt; PVM wurde in 12 Blatt- und 13 Knollenproben nachgewiesen. Die in Blattproben ermittelten Extinktionen bzw. Fluoreszenzintensit?ten lagen um den Faktor 2–5 h?her als die Werte der Knollenextrakte. In 20 mit PLRV prim?rinfizierten Mutterpflanzen wurden im Rahmen der Blattuntersuchungen Ende Oktober mit beiden Verfahren 10 infizierte Stecklinge ermittelt. Die Ergebnisse wurden durch die Augenstecklingsprüfung, bei der 9 infizierte Pflanzen gefunden wurden, unterstützt. Bei der Untersuchung prim?rinfizierter Knollen wurde nach einer Zwischenlagerung von 2,5 Monaten in 17 von 18 dieser Knollen eine PLRV-Infektion best?tigt. Die anschliessende Augenstecklingsprüfung ergab nur 12 PLRV-infizierte Pflanzen. Eine Abh?ngigkeit vom Infektionszeitpunkt wurde nicht gefunden. Der vorgestellte Ultramikro-ELISA besitzt nach diesen Ergebnissen die gleiche Sensitivit?t wie ein üblicher Standard-ELISA. Bedingt durch die geringen Füllvolumina k?nnen jedoch Chemikalien, Antik?rper und alkalische Phosphatase um 95% der im Vergleich zum Standard ben?tigten Mengen reduziert werden.

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Résumé Dans ce travail est décrit une méthode Ultramicro-ELISA avec le principe du double anticorps-ELISA (Clark & Adams, 1977). La partie réceptrice solide se compose de feuilles en polyvinylchloride additionnée de couches en polystérole avec une capacité de 10 μl par alvéole. La sensibilisation des plaques qui correspondent à une grille de 5×10 alvéoles, est effectuée avec une micro-multipipette à 50 canaux. L'incubation a lieu à température ambiante. Après rin?age des plaques avec le PBS-Tween le lavage est effectué durant 20 minutes avec de l'eau distillée. A la place du substratp-nitrophénylphosphore on utilise une solution de 4-methylumbelliferylphosphore 0,0005 mol/l avec un tampon de diethanolamine. La durée de réaction du substrat fluorescent est de 20 minutes. Un stoppage de la réaction est superflu, étant donné qu'une fois la réaction obtenue, la solution est prélevée avec une micro pipette et versée sur une grille-cuvette en verre avec 5×10 champs. La mesure de l'intensité fluorescente est effectuée avec un appareil spécial sur une couche de 0,16 mm. Cet ELISA Ultramicro-test utilisé pour la détermination quantitative de la protéine α-1 Feto (Horn et al., 1981; Hoffmann-Blume et al., 1983) a été comparé sur sa fiabilité avec un double anticorps-ELISA courant (Richter et al., 1979). Les examens comprenaient des jus de feuilles de pommes de terre contaminées par PLRV, PVY, PVM. On a également détecté le PVX, PVY et PVM, infections secondaires de tubercules et feuilles de clones de pommes de terre. Pour la détection des infections primaires de PLRV, des plantes-mères saines de la variété Adretta ont été infectées à deux dates à l'aide de pucerons. Les résultats obtenus avec les deux méthodes ELISA concordent dans tous les essais. Dans le jus de feuilles, les virus ont été détectés jusqu'à une dilution 10⁵ (PVM), 10³ (PVY) et 10² (PLRV). Sur les 20 clones examinés on a décelé avec les deux méthodes 5 cas de PVX et PVY dans les échantillons de tubercules et feuilles. PVM a été détecté dans 12 cas sur les feuilles et 13 fois sur les tubercules. Les extinctions, c'est-à-dire les intensités fluorescentes, sont 2 à 5 fois plus élevées à partir de jus de feuilles que sur du jus de tubercules. Sur 20 plantes qui présentaient une infection primaire, on a obtenu par des analyses foliaires des boutures à fin octobre, 10 individus infectés avec chaque méthode. Ces résultats ont été confirmés par des tests d'indexage qui ont donné 9 individus infectés. Lors de la détection d'infections primaires, après entreposage des tubercules pendant 21/2 mois, on a obtenu 17 tubercules positifs sur 18 contaminés de PLRV. L'indexage d'oeilletons a permis de déceler 12 individus malades sur ce même matériel. On a pas observé de différence selon l'époque d'infection de la plante-mère. Cela avait cependant été démontré lors d'une étude plus conséquente (Richter et al., 1983). Selon les résultats obtenus avec les virus de la pomme de terre PVX, PVY et PVM d'infections secondaires, ainsi que du PLRV d'infections primaires l'ultramicro-test ELISA offre la même sensibilité que le test ELISA standard. En raison des petits volumes des plaques, les substances chimiques, anticorps et phosphatase alcaline, peuvent être réduites de 95% comparativement avec la méthode traditionnelle.

     

Oxygen and carbon dioxide treatment to break potato tuber dormancy for reliable detection of potato virus Y (PVY) by enzyme-linked immunosorbent assay (ELISA)

Summary Dormant tubers of the potato cv. Bintje were treated for 7 or 14 days in an atmosphere enriched to either 40% O₂ plus 1% CO₂, or 40% O₂ plus 20% CO₂, or they were stored in closed plastic bags for identical periods. Rindite-treated and untreated tubers served as references. Treatment with 40% O₂ plus 20% CO₂ for 7 days was nearly as efficient as rindite for inducing sprouting. Both the O₂ plus CO₂ treatments, for 7 and 14 days, considerably increased virus concentration in tubers and had an effect similar to that of rindite 40 days after treatment, but the plastic bag treatment was not as efficient. It is concluded that O₂ plus CO₂ enriched atmospheres could be used for breaking tuber dormancy in order to detect reliably PVY in tuber extracts.     

An improved latex agglutination test for routine detection of potato viruses

Summary A modified latex agglutination test with plastic Petri dishes and the dispersant Tween-20 in the extraction buffer proved to be a simple, reliable procedure for routine detection of potato viruses X, Y, S, Andean potato laten and Andean potato mottle in leaves both of singly infected indicator hosts and potato. The main advantages of the modified method are that the flocculates found are large and easy to see, that equal or less amounts of sensitized latex are used, and that non-specific reactions occur less often than with other methods. The method is equally sensitive for detecting more than one virus when polyvalent latex is used.

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Zusammenfassung Eine modifizierte Version des Latex-Agglutinationstests mit Plastikpetrischalen als Reaktionsgef?sse und 0,05% des Dispersionsmittels Tween-20 im Extraktionspuffer erwies sich sowohl bei Bl?ttern als auch Indikatorpflanzen und Kartoffeln als einfache und zuverl?ssige Prozedur zum routinem?ssigen Nachweis der Kartoffelviren X, Y, S, APLV und APMV (Tabelle 2). Gegenüber Mikropipettierung ergab Latex bei der Ermittlung der isometrischen Viren APLV und APMV eine 500- bis 1000-fach h?here, bei den st?bchenf?rmigen Viren PVX, PVS und PVY jedoch nur eine 16- bis 125-fach h?here Empfindlichkeit (Tabelle 3, Abbildung IB). Die Empfindlichkeiten von uni- und polyvalentem Latex zur Ermittlung sowohl isometrischer (APLV, APMV) wie st?bchenf?rmiger Viren (PVX, PVS, PVY) oder beider Typen waren gleich hoch. Simultan für beide Virustypen war polyvalentes Latex zur gleichzeitigen Ermittlung von vier Viren gleichermassen empfindlich (Tabelle 4). Alle fünf Viren liessen sich von einzelnen Kartoffelbl?ttern sowie von Mischungen von einem infizierten mit bis zu 80 gesunden Bl?ttern sicher nachweisen. APLV, PVS und PVX waren auch in Trieben und Knollengewebe sicher nachzuweisen, nicht jedoch PVY und APMV, wegen niedriger Lagertemperatur der Knollen vor der Testung (Tabelle 5). Hauptvorteil im Vergleich zu anderen Arten des Latextests sind gr?ssere und besser sichtbare Ausflockungen (Abbildung 1A); bei Verwendung von gleichem oder weniger sensitiviertem Latex werden nicht-spezifische Reaktionen gr?sstenteils verhindert, ein Nachweis von Viren wird über eine grosse Konzentrationsbreite m?glich. Ein Nachteil ist die Hemmung der Reaktion durch Antigen-überschuss bei denjenigen Viren, die in infiziertem Gewebe eine sehr hohe Konzentration erreichen.

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Résumé Une version modifiée du test d'agglutination à l'aide de latex en boites de Petri de plastique, utilisant pour l'extraction une solution tampon contenant 0,05% de Tween 20, s'est a vérée être une technique simple et s?re pour la détection de routine des virus X, Y, S, ainsi que des virus ‘latent’ et de la ‘marbrure’ des Andes de la pomme de terre (APLV et APMV), à partir de feuilles de plantes indicatrices et de pommes de terre (tableau 2); le latex rend ce test de 500 à 1000 fois plus sensible que la microprécipitation lors de la détection des virus de forme isométrique, APLV et APMV, mais seulement de 16 à 125 fois plus sensible pour les virus allongés tels que X, S et M (tableau 3, figure 1 B). La sensibilité des latex univalents et polyvalents est la même pour la détection des virus de forme isométrique ou allongée et pour les deux types de virus simultanément; le latex polyvalent s'est montré d'une bonne sensibilité pour la détection de 4 virus à la fois (tableau 4). Les cinq virus étudiés peuvent être détectés avec s?reté à partir d'une seule feuille de pomme de terre ou d'un mélange d'une feuille infectée et jusqu'à 80 feuilles saines. APLV et les virus X et S ont été décelés facilement dans les germes et la chair des tubercules; par contre, APMV et le virus Y n'ont pas été détectés en raison des basses températures auxquelles ont été conservés les tubercules avant le test (tableau 5). Les principaux avantages de cette méthode comparée aux autres utilisant le latex sont qu'elle donne des floculats plus gros, donc, plus visibles (figure 1 A), nécessite une quantité de latex égale ou moindre, préserve des réactions non spécifiques et permet la détection des virus sur une grande gamme de concentrations. L'inconvénient est une inhibition de la réaction par excès d'antigènes avec les virus dont la concentration est très élevée dans les tissus infectés.

     

Purification of potato virus A and its detection in potato by enzyme-linked immunosorbent assay (ELISA)

Potato virus A (PVA) was purified fromNicandra physaloides by a simple method that omitted organic solvent clarification and consisted of differential centrifugation followed by equilibrium centrifugation in CsCl. An antiserum was produced that specifically detected PVA in potato leaf sap using either the SDS-agar test or enzyme-linked immunosorbent assay (ELISA). No heterlogous reaction of the antiserum with potato virus Y was detected. Purified PVA was highly infectious; it had an A 258/280 nm absorbance ratio of 1.28. The particles had a normal intact appearance in the electron microscope. Detection of PVA in potato sprouts and foliage by ELISA was compared with the local lesion assay onPhysalis angulata L. plants. ELISA was superior over an indicator plant test when sprout tissue was used. PVA antiserum reacted similarly with mild and crinkle isolates.     

Treatments that improve serological detection of potato viruses X,S, M and Y

Summary It was found that addition of an equal volume of 0.2% sodium sulphite or a mixture of sodium sulphite and sodium azide (both 0.2%) to expressed leaf sap improved serological detection of viruses and prevented non-specific reactions. Incubations of the preparations at 20–24°C gave better serological detectability than incubating at 10–12°C. If the sap was centrifuged, better results were subsequently obtained if centrifuging had been done at 4200 or 5900 g than at lower values.     

Simplified extraction method for ELISA and PCR detection of potato leafroll luteovirus primary infection in dormant potato tubers

This report describes a simple, rapid and inexpensive procedure for sampling large numbers of dormant tubers for analysis of potato leafroll luteovirus (PLRV) infection. The procedure uses a common electric drill to simultaneously remove and macerate tuber-eye samples for detection of PLRV by the enzyme-linked immunosorbant assay (ELISA) and the polymerase chain reaction (PCR). By using these sampling and analysis approaches, 19 of 20 different PLRV isolates were detected in dormant tubers from plants with primary infections. Results from the dormant tuber analysis, were verified by planting the tubers and testing leaf tissue by ELISA and PCR. Similar sampling and testing done on healthy dormant tubers and sprouts from the tubers consistently gave negative results as expected.     

A rapid scoring technique for potato tuber disease assessment

Summary Three methods of scoring disease severity were compared on potato tubers infected with either common scab, gangrene or dry rot. For each disease, replicated samples of infected tubers were taken of 12–14 cultivars representing a range of reaction from resistant to susceptible, and disease severity assessed by 1) estimating the proportion of the surface area affected by the disease, 2) calculating the percentage of tubers infected to a pre-determined amount, 3) an overall ‘glance score’ on a 1–9 scale of decreasing infection. The third method gave scores which correlated closely with the others, and since it is direct and simple to use, it has become the preferred method in disease screening tests at the Scottish Crop Research Institute.     

Enzyme-linked immunosorbent assay (ELISA) for the detection of potato leafroll virus and potato virus Y in potato tubers after artificial break of dormancy

Summary Conditions necessary for the detection of potato leafroll virus (PLRV) and potato virus Y (PVY) in tubers from primary and secondary infected plants were investigated. Tubers were analysed before and after breaking dormancy by rindite treatment. PLRV was reliably detected indormant tubers whereas PVY was readily detected only when tubers had been rindite-treated and held for two to three weeks at 22°C and high humidity in the dark. PLRV occurred in higher concentration at the heel end than at the rose end of infected tubers and the concentration remained nearly unchanged during the experimental period of 35 days, whereas PVY was found to be more concentrated at the rose end and was rapidly accumulating in the tubers after the break of dormancy. In dormant tubers PVY concentration dropped during storage at 22°C. The use of ELISA for tuber indexing is discussed.     

A rapid method for determining blackspot susceptibility of potato clones

Two methods of determining susceptibility of potato clones to blackspot were compared: (1) bruising by weight dropping and (2) bruising by abrasive peeling. A highly significant positive correlation was obtained between the intensity of enzymatic discoloration following abrasive peeling and the amount of blackspot that developed by weight dropping (r=0.93). Abrasive peeling was more rapid than the weight-dropping method. Tuber samples were abraded 30 sec and the amount of enzymatic discoloration evaluated after 24 hr. The need for individually bruising and hand peeling of tubers was eliminated with this method. Because of the rapidity of the abrasive peeling method, it can be used effectively in potato breeding programs to screen large numbers of clones for blackspot susceptibility. Results indicate that tuber maturity affects enzymatic discoloration and blackspot susceptibility. Immature tubers, dug while the vines are still green, are more resistant to blackspot than mature tubers. Tuber maturity therefore must be considered when screening clones for susceptibility to blackspot.     

A reevaluation of bromoethane in comparison to rindite for the post-harvest detection of potato virus Y in tubers by ELISA

A reevaluation of breaking tuber dormancy with bromoethane to increase the concentration of potato virus Y in the tuber revealed a positive response, by ELISA testing, to the treatment. The degree of response increased with the maturity of the tuber. Response to treatment with rindite was generally stronger, although differences were slight.     

A rapid micro-starch quantitation method for potato callus and its application with potato tubers

The procedure described was developed as a micro-starch method for potato callus tissue (0.1-0.2% starch on a fresh weight basis) and then evaluated with macro-starch samples of potato tubers harvested during three physiological stages of development. This method consisted of the following steps: (a) a mild alkaline-sonic extraction of starch from lyophilized 80% aqueous ethanol-washed samples, (b) starch hydrolysis of the neutralized, filtered extracts by 1 N HC1, and (c) the determination of liberated glucose by the specific glucose oxidase system. Results with this procedure were as sensitive and accurate (SD ± 0.86%) as with other starch-specific methods with more extensive starch isolation and purification steps.     

A simple and rapid method for the detection of potato spindle tuber viroid (PSTVd) by RT-PCR

Summary A test procedure for PSTVd is described based on immobilisation of plant sap on filter paper, by dotting or tissue printing followed by RT-PCR. Tests were carried out using primarily and secondarily infected potato plants, primarily infected in vitro plants, and potato tubers. Print PCR was shown to be suitable for testing large samples of potato plants whereas dot PCR is recommended for in vitro plantlets and tuber tissue. Bulking one infected plant to 4 or 9 healthy plants gave reliable results with secondarily infected potato plants, but sometimes the test failed to detect PSTVd in primarily infected in vitro plants. Dotted and printed paper squares could be stored at 4°C for at least 2 weeks in Triton X-100 solution or under dry conditions. Storing at room temperature can lead to unreliable results.     

Comparison of penicillinase-based and alkaline phosphatase-based enzyme-linked immunosorbent assay for the detection of six potato viruses

Summary A penicillinase (PNC)-based enzyme-linked immunosorbent assay (ELISA) was compared with one based on alkaline phosphatase (ALP) to detect potato viruses A, S, V, X and Y, and potato leafroll virus. Tests were made with different ratios (w/w) of γ-globulin∶enzyme in the conjugation reaction. The γ-globulin fraction of antisera to the viruses was conjugated to ALP or to PNC by the single step glutaraldehyde bridge method using the ratio of 1∶1 (γ-globulin∶enzyme). The ALP conjugates worked well but PNC conjugates gave substantial non-specific reactions and in general were not suitable for ELISA. However, PNC conjgates made with a ratio of 1∶0.05 (γ-globulin∶enzyme) gave little or no non-specific reaction and were at least as sensitive as ALP conjugates. Both ALP and PNC conjugates were found to be useful in three variants of ELISA (double antibody sandwich; plate-trapped antigen and a simplified plate-trapped antigen form of ELISA) to detect the six potato viruses. Although PNC-based ELISA often required longer incubation with substrate to achieve the same sensitivity of detection as with ALP-based ELISA, this is only a minor disadvantage and PNC should prove to be more economical and safer to use than ALP.     

Comparison of latex agglutination,enzyme-linked immunosorbent assay,and indicator plants for detection of potato viruses S and X in potatoes

Potato virus S (PVS) was consistently detected by the enzyme-linked immunosorbent assay (ELISA) and the latex agglutination test (LAT) at dilutions (PVS sap: healthy sap) of 1:499 and 1:19, respectively. Mechanical inoculation ofNicotiana debneyi gave variable results with detection possible 38% of the time at dilutions of 1:499. Potato virus X (PVX) was consistently detected by ELISA and LAT at dilutions (PVX sap: healthy sap) of 1:199 and 1:49, respectively. Mechanical inoculation ofGomphrena globosa with PVX resulted in consistent detection of the virus at a dilution of 1:499.