Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR

Huo, P; Shen, Wei; Yan, Peiguang; Tuo, Decai; Li, X Y; Zhou, Peng

doi:10.4149/av_2015_04_380

Cited by 8 publications

(7 citation statements)

References 29 publications

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“…Real-time PCR has been widely used for the detection and quantification of the low titre pathogens in a given plant sample [36][37][38]. However, in spite of its robustness, very few studies have adopted the real-time PCR as a multiplex detection assay.…”

Section: Discussionmentioning

confidence: 99%

Targeted Genome Sequencing (TG-Seq) Approaches to Detect Plant Viruses

Maina

Zheng

Rodoni

2021

Viruses

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Globally, high-throughput sequencing (HTS) has been used for virus detection in germplasm certification programs. However, sequencing costs have impeded its implementation as a routine diagnostic certification tool. In this study, the targeted genome sequencing (TG-Seq) approach was developed to simultaneously detect multiple (four) viral species of; Pea early browning virus (PEBV), Cucumber mosaic virus (CMV), Bean yellow mosaic virus (BYMV) and Pea seedborne mosaic virus (PSbMV). TG-Seq detected all the expected viral amplicons within multiplex PCR (mPCR) reactions. In contrast, the expected PCR amplicons were not detected by gel electrophoresis (GE). For example, for CMV, GE only detected RNA1 and RNA2 while TG-Seq detected all the three RNA components of CMV. In an mPCR to amplify all four viruses, TG-Seq readily detected each virus with more than 732,277 sequence reads mapping to each amplicon. In addition, TG-Seq also detected all four amplicons within a 10−8 serial dilution that were not detectable by GE. Our current findings reveal that the TG-Seq approach offers significant potential and is a highly sensitive targeted approach for detecting multiple plant viruses within a given biological sample. This is the first study describing direct HTS of plant virus mPCR products. These findings have major implications for grain germplasm healthy certification programs and biosecurity management in relation to pathogen entry into Australia and elsewhere.

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Section: Discussionmentioning

confidence: 99%

Targeted Genome Sequencing (TG-Seq) Approaches to Detect Plant Viruses

Maina

Zheng

Rodoni

2021

Viruses

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“…Our assay could detect the four viruses simultaneously from the mixture of four homogenates of singly-virus-infected potato leaves using newly designed primers. A few real-time mRT-PCR assays with melt curve analysis for plant viruses were reported [ 12 , 17 – 19 ], and all of them separately performed reverse transcription reaction and real-time multiplex PCR. According to our knowledge, this is the first report of a one-step real-time mRT-PCR assay with melt curve analysis for plant viruses performed.…”

Section: Discussionmentioning

confidence: 99%

“…The two criteria also affect the evaluation of detection sensitivity. The detection sensitivity of real-time RT-PCR assays has been confirmed using purified genome RNA [ 11 ] and plasmid DNA [ 12 , 17 , 19 ]. Our purpose is the development of high-throughput detection protocol for seed potato quarantine.…”

Section: Discussionmentioning

confidence: 99%

One-step real-time multiplex reverse transcription-polymerase chain reaction assay with melt curve analysis for detection of potato leafroll virus, potato virus S, potato virus X, and potato virus Y

Onozuka

Ohki

Oka

et al. 2021

Virol J

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Background Certification of seed potato as free of viruses is essential for stable potato production. Among more than 30 virus species infecting potato, potato leafroll virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY) predominate worldwide and should be the targets of a high-throughput detection protocol for seed potato quarantine. Results We developed an assay based on one-step real-time multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with melt curve analysis for the four viruses and one internal control, potato elongation factor 1 alpha gene (EF1α). Virus-specific primers were derived from conserved regions among randomly selected representatives considering viral genomic diversity. Our assay simultaneously detected representative Japanese isolates of PLRV, O lineage of PVS, PVX, and NTN strain of PVY. The variability of melting temperature (Tm) values for each virus was confirmed using Japanese isolates, and virus species could be identified by the values of 87.6 for PLRV, 85.9 for PVX, 82.2 (Ordinary lineage) to 83.1 (Andean lineage) for PVS, and 79.4 (NA-N strain) to 80.5 (O strain and NTN strain) for PVY on average. The reliability of calculation was validated by comparing the calculated Tm values and measured Tm values and the values had a strong linear correlation (correlation of determination: R2 = 0.9875). Based on the calculated Tm values, representative non-Japanese isolates could also be identified by our assay. For removing false positives, two criteria were set for the evaluation of result; successful amplification was considered as 30.0 ≥ threshold cycle value, and the virus-specific peak higher than the EF1α-specific peak was considered as positive. According to these criteria, our assay could detect PLRV and PVS from 100-fold dilution of potato leaf homogenate and PVX and PVY from 1000-fold in a model assay. Conclusion This new high-throughput detection protocol using one-step real-time mRT-PCR was sensitive enough to detect viruses in a 100-fold dilution of singly-virus contaminated homogenate in a model assay. This protocol can detect the four viruses in one assay and yield faster results for a vast number of samples, and greatly save the labor for seed potato quarantine and field surveys.

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“…Another alternative to the analysis of the amplicons by agarose gel-based detection, is the multiplex real-time PCR in which, the amplified fragments are detected directly during the reaction by using non-probe based fluorescent dyes such as SYBRGreen or EvaGreen ( Zipper et al, 2004 ; Mao et al, 2007 ) or specific fluorescent probes such as TaqMan ® probes ( Livak et al, 1995 ), molecular beacons ( Tyagi and Kramer, 1996 ), or the minor groove binding (MGB) probes ( de Kok et al, 2002 ). Real-time PCR allows quantification of the pathogen and reduced significantly the detection limit to as little as a few molecules ( Torres et al, 2005 ; Mortimer-Jones et al, 2009 ; Tuo et al, 2014 ; Huo et al, 2015 ). However, despite the clear advantages of the real-time procedure, the references of multiplex detection assays based on this technique, either using non-probe fluorescent days or fluorescent probes, represent only 28.1% of such test since 2005 ( Figure 1 ).…”

Section: Multiplex Pcrmentioning

confidence: 99%

Recent Advances on the Multiplex Molecular Detection of Plant Viruses and Viroids

2018

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Plant viruses are still one of the main contributors to economic losses in agriculture. It has been estimated that plant viruses can cause as much as 50 billion euros loss worldwide, per year. This situation may be worsened by recent climate change events and the associated changes in disease epidemiology. Reliable and early detection methods are still one of the main and most effective actions to develop control strategies for plant viral diseases. During the last years, considerable progress has been made to develop tools with high specificity and low detection limits for use in the detection of these plant pathogens. Time and cost reductions have been some of the main objectives pursued during the last few years as these increase their feasibility for routine use. Among other strategies, these objectives can be achieved by the simultaneous detection and (or) identification of several viruses in a single assay. Nucleic acid-based detection techniques are especially suitable for this purpose. Polyvalent detection has allowed the detection of multiple plant viruses at the genus level. Multiplexing RT polymerase chain reaction (PCR) has been optimized for the simultaneous detection of more than 10 plant viruses/viroids. In this short review, we provide an update on the progress made during the last decade on techniques such as multiplex PCR, polyvalent PCR, non-isotopic molecular hybridization techniques, real-time PCR, and array technologies to allow simultaneous detection of multiple plant viruses. Also, the potential and benefits of the powerful new technique of deep sequencing/next-generation sequencing are described.

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Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR

Cited by 8 publications

References 29 publications

Targeted Genome Sequencing (TG-Seq) Approaches to Detect Plant Viruses

Targeted Genome Sequencing (TG-Seq) Approaches to Detect Plant Viruses

One-step real-time multiplex reverse transcription-polymerase chain reaction assay with melt curve analysis for detection of potato leafroll virus, potato virus S, potato virus X, and potato virus Y

Recent Advances on the Multiplex Molecular Detection of Plant Viruses and Viroids

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