A single tube, quantitative real-time RT-PCR assay that detects four potato viruses simultaneously

Mortimer-Jones, Sheila; Jones, M. G. K.; Jones, R. A. C.; Thomson, G.; Dwyer, G. I.

doi:10.1016/j.jviromet.2009.06.027

Cited by 56 publications

(30 citation statements)

References 24 publications

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“…Simultaneous multiplex detection systems like the MRLPM applied in this study are highly desirable, especially to analyze plant diseases caused by multiple viruses. In addition, potato crops are prone to contain several co-infecting viruses, demonstrating the value of having a simultaneous detection method for the analysis of field samples (Nie and Singh 2000;Boonham et al 2003;Mortimer-Jones et al 2009). To increase the confidence of data accuracy and reliability, several designed strategies and controls were included in the assay.…”

Section: Discussionmentioning

confidence: 99%

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Application of multiplex reverse transcription-ligase detection reaction-polymerase chain reaction (MRLP) mediated universal DNA microarray for detecting potato viruses in field samples

et al. 2011

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To facilitate the comprehensive and unbiased analysis of viral prevalence in a given biological setting, we have applied conventional PCR and multiplex reverse transcription-ligase detection reaction-polymerase chain reaction (MRLP) based universal microarray for the identification of potato virus infections in potato samples from tubers and leaves. With this MRLP based microarray (MRLPM) technique, total RNAs of 79 field samples were reverse transcribed by random primers, then subjected to a ligase detection reaction (LDR) and asymmetric labelling PCR as templates, finally, the MRLP amplicons were analyzed by zip-code microarray hybridization and subsequent scanning. The concordance rates between the MRLPM and the PCR results were 92.50 to 98.73% for ten potato viruses. In addition, the positive incidence obtained by the MRLPM was higher than that by PCR for corresponding viruses in field samples. In conclusion, the method was able to simultaneously detect ten potato viruses from samples with and without symptoms. Thus, this assay can be used as a rapid and sensitive technique for routine potato virus detection in field surveillance and in plant quarantine.

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Section: Discussionmentioning

confidence: 99%

“…Several multiplex assays for potato viruses have been developed in the last few years, such as multiplex PCR and DNA microarrays technology (Nie and Singh 2000;Boonham et al 2003;Rho et al 2004;Mortimer-Jones et al 2009). However, each method has its limitations.…”

Section: Introductionmentioning

confidence: 99%

Application of multiplex reverse transcription-ligase detection reaction-polymerase chain reaction (MRLP) mediated universal DNA microarray for detecting potato viruses in field samples

et al. 2011

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“…Multiplex PCR assays have proven to be great value in plant pathology Mumford et al 2000;Bertolini et al 2003;Uga and Tsuda 2005;Roy et al 2005;Lorenzen et al 2006;Mortimer-Jones et al 2009;Chikh Ali et al 2010, 2013. By using this multiplex protocol to detect these six important potato viruses in a single reaction, cost of testing is reduced, mixed infections of at least two viruses can be readily detected, and the time required for testing is reduced from days to hours.…”

Section: Resultsmentioning

confidence: 99%

A Multiplex Reverse Transcription (RT) High-Fidelity PCR Protocol for the Detection of Six Viruses that Cause Potato Tuber Necrosis

et al. 2015

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Viruses that cause necrotic symptoms in potato tubers can be difficult to distinguish based on symptoms and frequently require multiple molecular tests to identify the pathogen. In this study, a multiplex RT PCR high fidelity PCR protocol was developed using previously validated primers that could accurately detect six important potato viruses and detect multiple viruses in a single sample simultaneously. To test the protocol, 53 tubers previously tested using conventional PCR were retested using this multiplex protocol and blindly evaluated. In every case, the multiplex PCR results were consistent with previous findings. In addition, the multiplex PCR protocol also detected several mixed infections that were previously undetected. The results demonstrate that this technique could be a valuable assay not only for diagnostic laboratories, but also for seed certification and regulatory agency laboratories to diagnose the virus(es) that may be present in tubers with surface or internal necrotic symptoms.Resumen Los virus que causan síntomas necróticos en tubérculos de papa pueden ser difíciles de distinguir con base en los síntomas, y frecuentemente se requiere de múltiples pruebas moleculares para identificar al patógeno. En este estudio, se desarrolló un protocolo múltiple de RT PCR de alta fidelidad de PCR utilizando iniciadores previamente validados que pudieran detectar con precisión seis virus importantes de papa y múltiples virus en una única muestra simultáneamente. Para probar el protocolo, se analizaron nuevamente 53 tubérculos que habían sido probados previamente usando la PCR convencional, utilizando este protocolo múltiple con evaluación ciega. En cada caso, los resultados de PCR múltiple fueron consistentes con lo que se encontró previamente. Además, el protocolo de PCR múltiple también detectó algunas mezclas de infecciones que previamente no se detectaron. Los resultados demuestran que esta técnica pudiera ser un ensayo valioso no solo para laboratorios de diagnóstico, sino también para certificación de semilla y para laboratorios de agencias de regulación para diagnosticar el (los) virus que pudieran estar presentes en tubérculos con síntomas necróticos superficiales o internos.

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“…Moreover, the presence of inhibitors in the plant tissue could also pose a constraint on the detection of viruses by these techniques. The development of quantitative real-time PCR has revolutionized plant virus detection (Schoen et al, 1996;Eun et al, 2000;Mumford et al, 2000;Roberts et al, 2000;Boonham et al, 2002;Drosten et al, 2002;Korimbocus et al, 2002;Balaji et al, 2003;Lunello et al, 2004;Mortimer-Jones et al, 2009;Jarosova and Kundu, 2010). The technique is gaining widespread importance for its increased sensitivity (100-1000 fold more sensitive than end-point PCR/RT-PCR), accuracy, high-throughput virus detection and reduced risk of post-PCR contamination (Mackay et al, 2002).…”

Section: Nucleic Acid Based Methods Including End-point Polymerase Cmentioning

confidence: 99%

Development of a rapid, sensitive TaqMan real-time RT-PCR assay for the detection of Rose rosette virus using multiple gene targets

Babu

Jeyaprakash

Jones

et al. 2016

Journal of Virological Methods

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Rose rosette virus (RRV), belonging to the genus Emaravirus, is a highly destructive pathogen that causes rose rosette disease. The disease is a major concern for the rose industry in the U.S. due to the lack of highly sensitive methods for early detection of RRV. This is critical, as early identification of the infected plants and eradication is necessary in minimizing the risks associated with the spread of the disease. A highly reliable, specific and sensitive detection assay is thus required to test and confirm the presence of RRV in suspected plant samples. In this study a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of RRV from infected roses, utilizing multiple gene targets. Four pairs of primers and probes; two of them (RRV_2-1 and RRV_2-2) based on the consensus sequences of the glycoprotein gene (RNA2) and the other two (RRV_3-2 and RRV_3-5) based on the nucleocapsid gene (RNA3) were designed. The specificity of the primers and probes was evaluated against other representative viruses infecting roses, belonging to the genera Alfamovirus, Cucumovirus, Ilarvirus, Nepovirus, Tobamovirus, and Tospovirus and one Emaravirus (Wheat mosaic virus). Dilution assays using the in vitro transcripts (spiked with total RNA from healthy plants, and non-spiked) showed that all the primers and probes are highly sensitive in consistently detecting RRV with a detection limit of 1 fg. Testing of the infected plants over a period of time (three times in monthly intervals) indicated high reproducibility, with the primer/probe RRV_3-5 showing 100% positive detection, while RRV_2-1, RRV_2-2 and RRV_3-2 showed 90% positive detection. The developed real-time RT-PCR assay is reliable, highly sensitive, and can be easily used in diagnostic laboratories for testing and confirmation of RRV.

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A single tube, quantitative real-time RT-PCR assay that detects four potato viruses simultaneously

Cited by 56 publications

References 24 publications

Application of multiplex reverse transcription-ligase detection reaction-polymerase chain reaction (MRLP) mediated universal DNA microarray for detecting potato viruses in field samples

Application of multiplex reverse transcription-ligase detection reaction-polymerase chain reaction (MRLP) mediated universal DNA microarray for detecting potato viruses in field samples

A Multiplex Reverse Transcription (RT) High-Fidelity PCR Protocol for the Detection of Six Viruses that Cause Potato Tuber Necrosis

Development of a rapid, sensitive TaqMan real-time RT-PCR assay for the detection of Rose rosette virus using multiple gene targets

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