NDLI: Modification of ELISA by Replacing Incubation of Microtiter Plates in an Incubator with Their Shaking in PVY, PVM and PLRV Detection

Content Provider	Springer Nature Link
Author	Wróbel, Sławomir
Copyright Year	2014
Abstract	ELISA (enzyme-linked immunosorbent assay) is a sensitive and reliable method of plant virus detection. It is commonly used in daily research carried out by scientific institutions and laboratories working on the certification of potato tubers. The key stage in this method is a 3–4-h-long incubation of microtiter plates with IgG and with a conjugate in an incubator at a temperature of 37 °C. The aim of the research was to replace this type of incubation process with a technique of mechanically shaking the plates using a shaker to induce a vibrating movement. Three durations of shaking, performed at room temperature, were adopted: 30, 60 and 90 min with two incubation periods at a temperature of 37 °C: 60 and 180 min which were applied at the stage of coating the IgG plates, following addition of the conjugate. The assessment was made for three dilutions of lyophilized sap from leaf of potatoes (1:10, 1:100, 1:1,000). Replacing the stages of plates incubation with IgG and conjugate at 37 °C with mechanical shaking allowed the whole process of DAS-ELISA to be reduced below 3–4 h without any significant impact on its quality. The process turned out to be equally efficient as the 3-h-long incubation in an incubator for PVY, PVM and PLRV detection by means of DAS ELISA. Applying the 90-min-long incubation on a shaker in comparison to a 3-h-long incubation in an incubator gave comparable or even slightly improved results. The reaction background, i.e. the value of absorbance for sap from healthy plants (negative control) was very low in all the combinations irrespective of the time of reading after the substrate was placed. No significant differences for this parameter were found between the combinations and times of reading. Only in the case of PLRV was a clearly visible decrease in test sensitivity found (no positive reactions) at diluted sap over 1:10. Moreover, it was observed that an increase in dilutions impacted the length of reaction. The dilution 1:10 seemed to be the most favorable (maximum 1:100 for PVY and PVM), wherein the sensitivity and pace of staining the substrate for each of the methods did not provoke any doubts regarding the reliability of the test.ELISA (ensayo de inmuno-absorción enzimática, serología con enzimas conjugadas), es un método sensible y confiable de detección de virus de plantas. Se usa comúnmente en la investigación diaria desarrollada por instituciones científicas y en laboratorios que trabajan en la certificación de los tubérculos de papa. La clave de este método es la incubación de 3 a 4 horas de las placas de microtitulación con IgG y con un conjugado en una incubadora a 37 º C de temperatura. El propósito de esta investigación fue reemplazar este tipo de proceso de incubación con una técnica de agitación mecánica de las placas mediante un agitador para inducir un movimiento de vibración. Se adoptaron tres duraciones de agitación a temperatura ambiente: 30, 60 y 90 minutos, condos períodos de incubación a 37 º C: 60 y 180 minutos aplicados en el paso de cubrimiento de las placas con IgG, siguiendo con la adición del conjugado. Se hizo el análisis para tres diluciones de savia liofilizada de hoja de papa (1:10, 1:100, 1:1,000). El reemplazo de los pasos de incubación de las placas con IgG y del conjugado a 37 º C con la agitación mecánica, permitió que se redujera el proceso completo de DAS ELISA por abajo de las 3–4 horas sin ningún impacto significativo en su calidad. El proceso resultó ser igualmente eficiente al de incubación de 3 horas en una incubadora para la detección de PVY, PVM y PLRV mediante la DAS ELISA. La aplicación de la incubación por 90 minutos en un agitador en comparación con la de 3 horas en la incubadora dio resultados comparables o aun ligeramente mejores. La reacción de impurezas, como el valor de la absorbancia de savia de planta sana (testigo negativo) fue muy bajo en todas las combinaciones, independiente del tiempo de lecturas después de que se aplicó el substrato. No se encontraron diferencias significativas para este parámetro entre las combinaciones y tiempos de lectura. Solo en el caso de PLRV hubo una disminución claramente visible en la sensibilidad de la prueba (no hubo reacciones positivas) con savia diluida a más de 1:10. Más aun, se observó que un aumento en las diluciones impactó la duración de la reacción. La dilución de 1:10 pareció ser la más favorable (máximo 1:100 para PVY y PVM), mientras que la sensibilidad y la coloración del sustrato para cada uno de los métodos no indujo ninguna duda en relación a la confiabilidad de la prueba.
Starting Page	354
Ending Page	362
Page Count	9
File Format	PDF
ISSN	1099209X
Journal	American Potato Journal
Volume Number	91
Issue Number	4
e-ISSN	18749380
Language	English
Publisher	Springer US
Publisher Date	2014-01-18
Publisher Institution	Potato Association of America
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	DAS-ELISA Shaker PVY PVM PLRV Incubation Potato Plant Sciences Agriculture Plant Genetics & Genomics Plant Breeding/Biotechnology Plant Pathology
Content Type	Text
Resource Type	Article
Subject	Plant Science Agronomy and Crop Science

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Assessment of Possibilities of Microtuber and in vitro Plantlet Seed Multiplication in Field Conditions. Part 1: PVY, PVM and PLRV Spreading

Seed Potato Production in Poland

Efficacy of Mineral Oil-Insecticide Mixtures for Protection of Potato Tubers Against PVY and PVM

Mechanical Transmission of Potato Virus Y (PVY) Through Seed Cutting and Plant Wounding

Improving Seed Health and Seed Performance by Positive Selection in Three Kenyan Potato Varieties

PVY: An Old Enemy and A Continuing Challenge

Comparison of Genome Sequence of PVY Isolates with Biological Properties

Sources and Effectiveness of Potato PVY Resistance in IHAR’s Breeding Research

The Adoption of Cv. Igorota in the Philippines and Vietnam

Modification of ELISA by Replacing Incubation of Microtiter Plates in an Incubator with Their Shaking in PVY, PVM and PLRV Detection

Similar Documents

Assessment of Possibilities of Microtuber and in vitro Plantlet Seed Multiplication in Field Conditions. Part 1: PVY, PVM and PLRV Spreading

Seed Potato Production in Poland

Efficacy of Mineral Oil-Insecticide Mixtures for Protection of Potato Tubers Against PVY and PVM

Mechanical Transmission of Potato Virus Y (PVY) Through Seed Cutting and Plant Wounding

Improving Seed Health and Seed Performance by Positive Selection in Three Kenyan Potato Varieties

PVY: An Old Enemy and A Continuing Challenge

Comparison of Genome Sequence of PVY Isolates with Biological Properties

Sources and Effectiveness of Potato PVY Resistance in IHAR’s Breeding Research

The Adoption of Cv. Igorota in the Philippines and Vietnam

Modification of ELISA by Replacing Incubation of Microtiter Plates in an Incubator with Their Shaking in PVY, PVM and PLRV Detection