Optimization of an isothermal recombinase polymerase amplification method for real-time detection of Potato virus Y O and N types in potato

https://doi.org/10.1016/j.jviromet.2019.02.006Get rights and content

Highlights

  • Primers targeting the highly conserved pipo region specifically recognized different Potato virus Y isolates by RPA.

  • The RPA assay efficiently detects viral RNA in low viral titer samples, including the peridermal tissues of potato tubers.

  • The RPA procedure was streamlined by using crude extract from infected-potato tissues.

  • The real-time detection of Potato virus Y using fluorescent probe picked up the viral RNA within 6 min.

Abstract

Potato virus Y (PVY) is a global challenge for potato production and the leading cause of seed crop downgrading and rejection for certification. Accurate and timely diagnosis is key to effective control of PVY. Here we optimized the isothermal recombinase polymerase amplification (RPA) for accurate detection of different PVY O and N types that were tested, present in different tissues of potato plants including tubers with a primer set that specifically targets the highly conserved pipo region within the viral genome. Combined with a simplified preparation of the template by tissue homogenization, we established a rapid RPA procedure, which can allow real time detection in less than 10 min with a fluorescent probe. Specificity of the reaction was determined by the lack of cross-reactivity with other common potato viruses. Although RPA reagents remain more expensive than PCR reagents, RPA technology is equivalent in that results can be visualized by gel electrophoresis or with a fluorescent probe with greater sensitivity; and it is superior to the common PCR-based assay in its versatility, speed, and lack of need for a highly purified RNA template.

Introduction

Potato is the most important dicot food crop worldwide and about $3.4 billion in potato is produced in the United States annually. The potyvirus Potato virus Y (PVY) is the most important potato virus in North America and the most common reason for rejection of seed potato lots from certification (Frost et al., 2013), which is one of the reasons that PVY is considered to be among the 10 most important viruses in the world (Scholthof et al., 2011). PVY has become increasingly important in the United States since recent regulatory changes require that seed must be certified for low levels of PVY prior to crossing state borders or being planted in fields greater than 1–10 acres, depending on the state. As a result, the need for rapid, inexpensive, and accurate PVY diagnosis has escalated.

PVY causes tuber yield reduction of up to 80% depending on the varieties and time of infection (Nolte et al., 2003; Whitworth et al., 2006; Bantarri et al., 1993; Hane, 1999). Several major changes have contributed to the re-emergence of PVY as a serious disease threat in the United States. An important component is the popularity of several russet varieties (Hane and Hamm, 1999; Rykbost et al., 1999) that show mild or no PVY foliar symptoms but serve as large reservoirs for PVY. One of the challenges in the control of PVY is the emergence of new recombinants of the virus, which are derived from the ordinary PVYO strain and the necrotic PVYN strain (Green et al., 2017, 2018; Karasev and Gray, 2013). These recombinant strains cause mild foliar symptoms on potato, may cause necrosis on tubers, and unlike PVYO, are not self-limiting in varieties that encode N-gene mediated resistance (Quenouille et al., 2013).

Potato virus Y is the type member of the genus Potyvirus, family Potyviridae. It is a positive sense RNA virus of about 9700 nts, which encodes a single large polypeptide, which is then processed into nine multifunctional proteins. A second small open reading frame (ORF) termed pipo, predicted to encode a 7 kDa protein, was discovered to overlap with the P3 coding region as fusion to the N-terminal part of P3 protein in all members of the Potyviridae family (Chung et al., 2008). This highly conserved ORF is an ideal target for development of detection assays.

Rapid and accurate diagnosis of PVY is key for effective disease control. While the double sandwich enzyme-linked immunosorbent assay (DAS-ELISA) is routinely used to test seed potato crops as part of seed certification, many nucleotide-based procedures with high specificity and automation are also available including RT-PCR and isothermal-detection based assays (Nie, 2005; Przewodowska et al., 2015; Singh et al., 2013; Singh, 1999; Treder et al., 2018; Zacharzewska et al., 2014; Glais and Jacquot, 2015). Such methods have not yet been adapted for routine diagnostic schemes. They are hindered largely by the existing challenges in isolating high quality RNA from potato tubers required for PCR-based amplification, the presence of inhibitory compounds and/or the low virus titer in dormant tubers and the time-consuming sample preparation for the assays (Boonham et al., 2008).

Here we describe the optimization of the isothermal-based reverse transcription recombinase polymerase amplification assay (RPA) for robust and rapid detection of PVY from crude extracts of potato leaves and tubers. Isothermal DNA amplification methods such as the loop-mediated isothermal amplification (LAMP) and RPA represent alternatives to PCR-based assays (Magrina Lobato and O’Sullivan, 2018). Their reactions are set in one single temperature without the need of sophisticated equipment. While the LAMP approach is based on six different primers that recognizes distinct regions of the target sequence and relies on a DNA polymerase with strand displacement activity at 60–65 C, the RPA reaction involves a specific combination of two core enzymes and a protein for simple amplification of molecular targets at a single temperature (Magrina Lobato and O’Sullivan, 2018). This includes the recombinase, which pairs a single set of primers to the homologous sequences; the single stranded DNA binding protein, which displaces the strands of the DNA; and the strand-displacing polymerase, which initiates DNA synthesis from the bound primers. For RNA samples, the reverse transcription can be synchronistically performed in the same reaction just by adding the reverse transcriptase enzyme. Here we designed specific PVY primers that target a consensus region among different PVY strains within the highly conserved pipo coding sequence present in all members of the Potyviridae family (Chung et al., 2008). The specificity of the assay for PVY was validated by detection of different strains of PVY and the lack of cross-reactivity with other potato-infecting viruses, including other potyviruses. The power of the assay is based on the fluorescent probe system that allowed real time detection of the PVY amplicon up to 10000-fold dilution of the total RNA sample, in less than 10 min. In addition, the assay was sensitive enough to allow the detection of PVY when using crude extract from peridermal tuber tissues. The present study offers an alternative procedure to conventional nucleic acid-based assays for successful and rapid detection in low viral titer samples, and an optimization of a previously reported RPA approach (Glais and Jacquot, 2015), which now eliminates the need of additional purification step of the products prior visualization and works with low quality RNA materials.

Section snippets

Materials and methods

Frozen PVYO (isolate NY090031), PVYN:O (isolate NY090029) and PVYNTN (isolate NY090004) were maintained in tobacco Nicotiana benthamiana leaves at – 80 °C Potato tubers infected with the known strains of PVY (PVYO, PVYN:O, PVYWi PVYNTN) were kindly provided by Stewart Gray (Cornell University) to Amy Charkowski.

Frozen PVY-infected Nicotiana benthamiana leaves were used for extracting total RNA for the optimization of the designed primers, and as source of inoculum for rub-inoculation of potato

Detection of the different PVY strains with PIPO-targeting specific primers

The RPA assay is based on an isothermal single stranded displacement of DNA. The high performance of the method heavily relies on the efficacy of the recombinase enzyme to successfully pair the designed primers to the target sequence. We thus designed three sets of primers, which targeted a consensus sequence among the PVY strains available in the NCBI database within the pipo open reading frame (Green et al., 2017) (Fig. 1). We tested different forward and reverse primer combinations in the

Discussion and conclusion

We optimized the RPA assay for PVY detection in potato leaves and tubers. We validated a set of primers that specifically amplified all PVY strains tested with no cross-reactivity with other potato viruses including potyviruses, Here we revealed that the PVY amplicon can be directly and easily visualized on a gel electrophoresis. This is to contrast with a previous report of PVY detection by RPA, which required an additional purification step (Glais and Jacquot, 2015). The real-time detection

Acknowledgments

We thank the Wisconsin Seed Potato Certification Agency for their assistance with this work. This work was supported by grant number 73999-10921 from SCRI-USDA National Institute of Food and Agriculture to AOC, by Wisconsin Potato Industry grant (MSN 180863) and an Hatch Formula Act grant (MSN 179998) to AMR, by a Obihiro University of Agriculture and Veterinary Medicine, President leadership grant for young scientists to DA.

References (23)

  • K.J. Green et al.

    Phylogenetic study of recombinant strains of Potato virus Y

    Virology

    (2017)
  • E.E. Bantarri et al.

    Management of diseases caused by viruses and virus-like pathogens

  • N. Boonham et al.

    Exploiting generic platform technologies for the detection and identification of plant pathogens

    Eur. J. Plant Pathol.

    (2008)
  • B.Y. Chung et al.

    An overlapping essential gene in the Potyviridae

    Proc. Natl. Acad. Sci. U. S. A.

    (2008)
  • S.L. Dellaporta et al.

    A plant DNA minipreparation: version II

    Plant Mol. Biol. Rep.

    (1983)
  • K.E. Frost et al.

    Integrated control of potato pathogens through seed potato certification and provision of clean seed potatoes

    Plant Dis.

    (2013)
  • L. Glais et al.

    Detection and characterization of viral species/subspecies using isothermal recombinase polymerase amplification (RPA) assays

  • K.J. Green et al.

    Genetic diversity of potato virus Y (PVY): sequence analyses reveal ten novel PVY recombinant structures

    Arch. Virol.

    (2018)
  • D.C. Hane

    Effects of seedborne potato virus Y infection in two potato cultivars expressing mild disease symptoms

    Plant Dis.

    (1999)
  • D.C. Hane et al.

    Effects of seedborne potato virus Y infection in two potato cultivars expressing mild disease symptoms

    Plant Dis.

    (1999)
  • A.V. Karasev et al.

    Continuous and emerging challenges of Potato virus Y in potato

    Annu. Rev. Phytopathol.

    (2013)
  • Cited by (0)

    View full text