Detection Of Prunus Necrotic Ringspot Virus Research Articles

Dijagnostika virusa nekrotične prstenaste pegavosti koštičavog voća

Phytopathogenic viruses cause plant diseases that often lead to significant economic losses. Viruses, together with mycoses and bacterioses, are one of the limiting factors of agricultural production. Due to the multiple possibilities of infection, they have become widespread. Thereby, the main way to prevent and control virus infections is to produce and use healthy planting and sowing materials. As losses in fruit crops are much higher than in field crops and vegetables (high cost of planting material, raising and care of the orchard), it is very important to conduct plant material inspections on time and to apply detection methods for viruses. In this way, the harmful effects of viral infections can be avoided. Prunus necrotic ringspot virus (PNRSV) is one of the most significant stone fruit viruses. Due to the widespread of this virus and its devastating potential, various diagnostic methods are being used. In this paper, PNRSV detection methods based on the specificity of the protein cover (ELISA and DIBA) and nucleic acid-based virus detection methods (RT-PCR) are presented.

Rapid Detection of Prunus Necrotic Ringspot Virus by Reverse Transcription-cross-priming Amplification Coupled with Nucleic Acid Test Strip Cassette

Prunus necrotic ringspot virus (PNRSV) is one of the most devastating viruses to Prunus spp. In this study, we developed a diagnostic system RT-CPA-NATSC, wherein reverse transcription-cross-priming amplification (RT-CPA) is coupled with nucleic acid test strip cassette (NATSC), a vertical flow (VF) visualization, for PNRSV detection. The RT-CPA-NATSC assay targets the encoding gene of the PNRSV coat protein with a limit of detection of 72 copies per reaction and no cross-reaction with the known Prunus pathogenic viruses and viroids, demonstrating high sensitivity and specificity. The reaction is performed on 60 °C and can be completed less than 90 min with the prepared template RNA. Field sample test confirmed the reliability of RT-CPA-NATSC, indicating the potential application of this simple and rapid detection method in routine test of PNRSV.

A multiplex RT-PCR assay for simultaneous detection of four viruses from sweet cherry

A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed for simultaneous detection of four viruses: Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Little cherry virus-2 (LChV-2) and Cherry green ring mottle virus (CGRMV). Random hexamer primer was used for cDNA synthesis. Detection primers were designed against the genomic sequences of four viruses. The reaction conditions were firstly optimized by selecting primers combinations and standardizing the individual primer proportion and PCR protocols. Then, sensitivity of multiplex RT-PCR was evaluated. The assay could detect all four viruses in diluted cDNA (10−6, about 8.64×10−4ngμL−1) and RNA (10−1, about 0.2μg). The reliability of the assay was evaluated by cloning and sequence alignments. The developed multiplex RT-PCR method was then used to test virus infections from field samples of sweet cherry in this paper. It will be quite helpful for plant quarantine and certification programs. To our knowledge, it is the first report of the multiplex RT-PCR assay for simultaneous detection of PNRSV, PDV, LChV-2 and CGRMV.

Rapid detection of Prunus necrotic ringspot virus using magnetic nanoparticle-assisted reverse transcription loop-mediated isothermal amplification

Prunus necrotic ringspot virus (PNRSV) has seriously reduced the yield of Prunus species worldwide. In this study, a highly efficient and specific two-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect PNRSV. Total RNA was extracted from sweet cherry leaf samples using a commercial kit based on a magnetic nanoparticle technique. Transcripts were used as the templates for the assay. The results of this assay can be detected using agarose gel electrophoresis or by assessing in-tube fluorescence after adding SYBR Green I. The assay is highly specific for PNRSV, and it is more sensitive than reverse-transcription polymerase chain reaction (RT-PCR). Restriction enzyme digestion verified further the reliability of this RT-LAMP assay. To our knowledge, this is the first report of the application of RT-LAMP to PNRSV detection in Prunus species.

Preparation and Characterization of a Monoclonal Antibody to Prunus necrotic ringspot virus

AbstractA cell line named PVRSV1D11 secreting monoclonal antibody (McAb) against the prokaryotically expressed coat protein (CP) of Prunus necrotic ringspot virus (PNRSV) was developed using hybridoma technology including animal immunization, cell fusion, cell line culture and enzyme‐linked immunosorbent assay (ELISA)‐based for screening. The specificity, titre and detection sensitivity of the McAb were determined by indirect ELISA to establish optimal conditions. The antibody reacted strongly with PNRSV and showed no cross‐reactions with the proteins of Plum pox virus, Prunus dwarf virus, Apple stem pitting virus, Apple stem grooving virus, Apple mosaic virus or Apple chlorotic leafspot virus. The ascites developed with PNRSV1D11 cell line showed high absorbance until it was diluted to over 6.6 × 107 fold. The McAb belonged to IgG2a isotype and was diluted by 1.28 × 105 folds as an optimal detection concentration. The detection sensitivity of the monoclonal antibody was 11.7 ng/ml protein of PNRSV. The results indicated that the McAb against the CP of PNRSV is suitable for PNRSV detection in the plants and for monitoring the dynamics of the virus by using indirect ELISA.

Potential uses of in vitro expressed and purified recombinant Prunus necrotic ringspot virus coat protein gene

Prunus necrotic ring spot virus (PNRSV), an ilarvirus, has a wide host range especially on important commercial crops like almond, apple, apricot, hop, peach, and rose. PNRSV coat protein gene was cloned in expression vector pGEX-2Tk (Amersham Pharmecia, USA) using E. Coli BL 21 strain competent cells. Expression conditions were standardized for maximum recovery of soluble recombinant protein. The in vitro expressed protein was purified and used as an antigen for raising antisera. Both intramuscular and sub-cutaneous routes were used separately for antisera raising. A part of the raised and purified antisera was used in enzyme conjugate preparation. It was then formulated in the form of an ELISA-based diagnostic kit for PNRSV detection. It was also compared with the commercially available kit.

Development of Real-Time RT-PCR Assay for Detection of Prunus necrotic ringspot virus in Fruit Trees

A real-time fluorescent reverse-transcriptase polymerase chain reaction (RT-PCR) assay using a short fluorogenic 3' minor groove binder (MGB) DNA hydrolysis probe was developed for the detection of Prunus necrotic ringspot virus (PNRSV) in stone fruit trees. The covalent attachment of the minor groove binder moiety at the 3' end of the probe increased the probe target duplex stability and raised the melting temperature to a range suitable for real-time analysis. The real-time RT-PCR assay correlated well with conventional RT-PCR results for the detection of PNRSV. This assay reliably detects PNRSV in bark tissues of dormant cherry and plum trees. Furthermore, it is well adapted for the routine detection of PNRSV because it eliminates one risk of contamination by performing the whole test in a single closed tube. This system may replace the commonly used diagnostic techniques (e.g., woody indicators and immunological tests) to detect this virus.

Comparison of ELISA and RT-PCR for the detection of Prunus necrotic ring spot virus and prune dwarf virus in almond ( Prunus dulcis)

A technique based on the reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to detect the presence of Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) simultaneously in almond. This paper presents the results of a 3-year study comparing both enzyme-linked immunosorbent assay (ELISA) and RT-PCR for the detection of PNRSV and PDV using 175 almond leaf samples. Multiplex RT-PCR was found to be more sensitive than ELISA, especially when followed by nested PCR for the detection of PDV. The RT-PCR technique has the added advantage that plant material can be tested at any time throughout the growing season.

Seasonal variation of Prunus necrotic ringspot virus concentration in almond, peach, and plum cultivars.

Levels of Prunus necrotic ringspot virus (PNRSV) infection in almond, peach, and plum cultivars over the course of an entire year were determined by testing different plant parts of naturally infected trees, using the double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA). The data showed that spring was the best time of year for PNRSV detection in flowers, active growing buds, and young leaves. PNRSV detection was less reliable during the summer months. Young leaves of all cultivars were the most reliable source for distinguishing between healthy and infected plants, while flowers and buds yielded high values in some cultivars but not in others. Seasonal fluctuations in virus concentration did not follow the same pattern in all cultivars. It is therefore impossible to distinguish between infected and healthy trees on the basis of one single sampling time for all cultivars.

Detection of Prunus necrotic ringspot virus in almond: effect of sampling time on the efficiency of serological and biological indexing methodologies

The reliability of enzyme-linked immunosorbent assay (ELISA) and indexing methodologies for the detection of Prunus necrotic ringspot virus (PNRSV) in almond was assessed under local conditions. Thirteen field-grown almond trees were sampled fortnightly throughout the growing season. Petal and leaf homogenates were used for ELISA and to inoculate herbaceous indicator species, while buds collected from October onwards were budded to woody indicators. ELISA reliably detected PNRSV in petals and young leaves from bud-break until the cessation of stem elongation. While herbaceous indexing was not as reliable as ELISA, woody indicators could differentiate positive from negative samples reliably throughout the testing period. However, for mass screening of foundation plantings, nursery stock and elite germplasm, woody indexing is too costly and slow to give results. The use of ELISA can overcome these limitations but the timing of sample collection is critical. Petal or leaf tissue collected early in the season will yield the most reliable results.

Enzyme-Linked Immunosorbent Assay Testing of Shoots Grown In Vitro and the Use of Immunocapture-Reverse Transcription-Polymerase Chain Reaction Improve the Detection of Prunus necrotic ringspot virus in Rose

We developed and evaluated two different methods to improve the detection of the most prevalent virus of rose in Europe, Prunus necrotic ring-spot virus (PNRSV). Immunocapture-reverse transcription-polymerase chain reaction was estimated to be about 100 times more sensitive than double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and showed an equivalent specificity. Based on the observation that PNRSV multiplies actively in young growing tissues (axillary shoots and cuttings), an in vitro culture method allowing rapid (about 15 days) and homogeneous development of dormant axillary buds with high virus titers was standardized. ELISA tests of these young shoots showed, in some cases, a 10(4) to 10(5) increase in sensitivity in comparison to adjacent leaf tissues from the rose mother plants. Between 21 and 98% (depending on the season) more samples were identified as positive by using ELISA on samples from shoot tips grown in vitro rather than on leaves collected directly from the PNRSV-infected mother plants. This simple method of growing shoot tips in vitro improved the confidence in the detection of PNRSV and eliminated problems in sampling appropriate tissues.