Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan® real-time RT-PCR

Agindotan, Bright; Shiel, P. J.; Berger, P. H.

doi:10.1016/j.jviromet.2006.12.012

Cited by 113 publications

(57 citation statements)

References 32 publications

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“…The method, on the whole, had the following advantages over the two mentioned procedures and also the methods including RT-LAMP and RT-PCR: (1) fascinatingly, no need of RNA extraction (2), no requirement of expensive and sophisticated tools for amplification and detection; (3) no post-amplification treatment of the amplicons; and (4) a flexible and easy detection approach, that is visually detected by naked eyes using diverse visual dyes. On the other hand, among different visual systems HNB, SYBR Green I and GeneFinder TM were proved to be more powerful.…”

Section: Resultsmentioning

confidence: 99%

“…Potato plants, like other crops, are exposed frequently to plenty of infectious viruses, economically the most important of which are Potato leafroll virus (PLRV, genus Polerovirus), Potato virus Y (PVY, genus Potyvirus), Potato virus X (PVX, genus Potexvirus) and Potato virus S (PVS, genus Carlavirus) [2]. PVY, one species of the genus Potyvirus is a phloem-limited virus that infects potato leading to a significant reduction in potato annual yield as well as tuber quality worldwide [3,4].…”

Section: Introductionmentioning

confidence: 99%

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Colorimetric Immunocapture Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid Detection of the Potato virus Y

Almasi¹

2013

J Plant Pathol Microbiol

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Loop-Mediated Isothermal Amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. To diminish the time required for some diagnostic assays including Reverse Transcription PCR (RT-PCR), Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) and also DAS-ELISA into a minimum level, an innovative colorimetric IC-RT-LAMP and IC-RT-PCR protocol on the basis of Potato Virus Y (PVY) genome were used and optimized. Firstly, DAS-ELISA assay was performed to detect of the virus in a collection containing 95 suspicious samples. Lastly, five samples were detected as the positive samples. Then, the positive samples were verified by molecular methods. In this regard, all four RT-LAMP primers (i.e. F3, B3, FIP and BIP) together with RT-PCR primers (F and B) were selected on the basis of coat protein gene (CP) of PVY genome. Even though DAS-ELISA, RT-PCR and RT-LAMP assays could successfully detect positive infected plant samples, considering the time, safety, sensitivity, cost and simplicity, the last one was overall superior. Furthermore, the results demonstrated that the RT-LAMP assay was 100 times sensitive and 3 time faster compared to RT-PCR. LAMP assay was accomplished in the water bath either frees from any thermal cycler machine or sophisticated laboratories facility. Meanwhile, among six different visual dyes to accurately detect IC-RT-LAMP products, Hydroxynaphthol blue, GeneFinder TM and SYBR Green I could produce long stable colour change and brightness in a close tube-based approach to prevent cross-contamination risk, concluded eventually as the best ones. We accordingly propose this colorimetric assay as a highly reliable alternative viral recognition system regarding PVY recognition and probably other viral-based diseases.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Colorimetric Immunocapture Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid Detection of the Potato virus Y

Almasi¹

2013

J Plant Pathol Microbiol

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“…Reverse transcription PCR is much more sensitive and since 1990s has been adopted for detection of potato viruses directly in tuber extracts (Spiegel and Martin 1993;Singh and Singh 1996;Singh 1998;Singh et al 2002;Crosslin and Hamlin 2011). Many variations of RT-PCR have been developed focusing on (i) identification and elimination of factors negatively affecting virus detection in tubers (Singh and Singh 1996), (ii) development of standardized protocols (Singh 1998;Crosslin and Hamlin 2011), (iii) simplification of sample preparation (Nolasco et al 1993;Singh et al 2004), (iv) multiplexing (Nie and Singh 2000;Singh and Nie 2003;Du et al 2006;Li et al 2008;) and (v) real time detection (Knorr et al 1996;Boonham et al 2000;Agindotan et al 2007;Mortimer-Jones et al 2009;Singh et al 2012). However, reports comparing performance of RT-PCR (Barker et al 1993;Bolotova et al 2009), and real-time RT-PCR (Fox et al 2005) with grow-out test have shown that the reliability of the latter method is either equal or more.…”

Section: Introductionmentioning

confidence: 99%

The Adaptation of Silica Capture RT-PCR for the Detection of Potato Virus Y

2014

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In Poland, it is mandatory to index seed tubers for Potato virus Y (PVY), Potato virus M (PVM) and Potato leafroll virus (PLRV). Currently, the incidence of viral infection in seed tubers is determined by grow-out test. Direct testing of tubers after harvest by reverse transcription polymerase chain reaction (RT-PCR) can be beneficial but its application is so far hampered by the high cost of commercial kits for RNA isolation and poor usability of manual methods for routine diagnostics. In this work, we compare several commercial kits with the silica capture RT-PCR (SC-RT-PCR) for direct detection of PVY. The silica capture of RNA in conjunction with optimized PCR conditions facilitates quick and sensitive detection of PVY and offers cost effective and reliable alternative to commercial kits.Resumen En Polonia, los tubérculos-semilla son analizados obligatoriamente para el virus Y de la papa (PVY), virus M de la papa (PVM) y el enrollamiento de la hoja (PLRV). Actualmente, la incidencia de infección viral en tubérculo-semilla se determina por pruebas durante el crecimiento. Las pruebas directas de tubérculos después de la cosecha con la reverso-transcripción por la reacción en cadena de la polimerasa (RT-PCR) pudiera ser benéfica, pero su aplicación se limita por el alto costo de juegos comerciales para el aislamiento de ARN y por el pobre uso de métodos manuales para diagnósticos de rutina. En este trabajo comparamos varios juegos comerciales con la efectividad de costos de captura de sílice RT-PCR para detección directa de PVY. La sensibilidad en la detección del PVY en ARN preparado por captura de sílice fue igual o mayor que en ARN aislado usando juegos comerciales. El ARN por captura de sílice junto con condiciones óptimas de PCR facilita la detección rápida y sensible de PVY y ofrece una alternativa efectiva, en costos y confiabilidad, a los juegos comerciales.

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“…Despite the presence of a broad spectrum of diverse deleterious viruses, the most common and economic ones are Potato virus A, Potato virus Y (PVA, PVY, both from genus Potyviruses), Potato leafroll virus (PLRV, genus Polerovirus), Potato virus X (PVX, genus Potexvirus) and Potato virus S (PVS, genus Carlavirus) [1][2][3][4]. Viruses of the family Luteoviridae belongs to ssRNA plant viruses, divided into three genera: Enamovirus, Luteovirus and Polerovirus [5].…”

Section: Introductionmentioning

confidence: 99%

“…Typically, in the recent decades the molecular techniques have been applied for discrimination of viruses and other plant pathogens. In this regards, several molecular methods have been developed for detection of the PLRV, including nucleic acid sequence-based amplification (NASBA) [16], Northern blotting [7], immunecapture RT-PCR (IC-RT-PCR) [3], reverse transcription polymerase chain reaction (RT-PCR) [16][17][18][19][20][21][22][23][24][25][26][27], Multiplex AmpliDet RNA [18], and real-time RT-PCR [1,19]. Although those methods are sensitive and specific, they are either complicated or expensive.…”

Section: Introductionmentioning

confidence: 99%

Detection of Coat Protein Gene of the Potato Leafroll Virus by Reverse Transcription Loop-Mediated Isothermal Amplification

Almasi¹

2012

J Plant Pathol Microb

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Loop-mediated isothermal amplification assay amplifies DNA/RNA with high specificity and sensitivity. In this study, we describe an optimized reverse transcription-LAMP assay for detection of Potato Leafroll Virus. Firstly, DAS-ELISA assay was performed to detect of the virus in a collection containing 40 suspicious samples. Lastly, two samples were detected as the positive samples. Then, the positive samples were verified by RT-PCR and RT-LAMP methods. Furthermore, the results demonstrated that the RT-LAMP assay was 40 times sensitive and 4 time faster compared to RT-PCR. RT-LAMP assay was accomplished in the water bath either frees from any thermal cycler machine or sophisticated laboratories facility. Moreover, in RT-LAMP reaction the positive samples were detected through turbidity which produced by magnesium pyrophosphate. Interestingly, the application of CaCl 2 instead of MgSO 4 which create calcium pyrophosphate in reaction could significantly increase both stability and concentration of turbidity. Consequently, it could be an interesting alternative to MgSO4. Overall, the newly developed RT-LAMP assay can be a sensitive, specific and low-cost method for early detection of Potato Leafroll Virus and also other viral plant pathogens.

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Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan® real-time RT-PCR

Cited by 113 publications

References 32 publications

Colorimetric Immunocapture Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid Detection of the Potato virus Y

Colorimetric Immunocapture Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid Detection of the Potato virus Y

The Adaptation of Silica Capture RT-PCR for the Detection of Potato Virus Y

Detection of Coat Protein Gene of the Potato Leafroll Virus by Reverse Transcription Loop-Mediated Isothermal Amplification

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