Detection Of Potato Leafroll Virus Research Articles

Rapid detection of Potato leafroll virus and Potato virus Y by reverse transcription loop-mediated isothermal amplification method in north-east India

Potato leafroll virus (PLRV) and Potato virus Y (PVY) are two important viruses causing serious potato yield losses in the North-east region and other planting areas in India. As a consequence, it is urgent to develop an efficient and quick method for the identification and diagnosis in the field. The results presented here showed that the reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was efficient and sensitive than reverse transcription-polymerase chain reaction (RT-PCR) for the detection of PLRV and PVY. The RT-LAMP primers specifically targeted PLRV and PVY (including PVYO, PVYN, and PVYNTN strains) and resulted in typical sigmoidal amplification curves. Ten-fold serial dilutions of PLRV and PVY total RNA indicated that RT-LAMP is faster and at least a hundred times more sensitive than RT-PCR in detecting both the viruses. Additionally, samples that RT-PCR could not detect at a diluted concentration of 10−3 and 10-4 ng/μl were identified by RT-LAMP. Thus, RT-LAMP offers many advantages over RT-PCR such as low cost and high accuracy, sensitivity, and specificity for the rapid diagnosis of plant virus diseases. In conclusion, the results highlighted the efficacy of the RT-LAMP method in quickly detecting PLRV and PVY in infected plants.

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

BackgroundCertification 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.ResultsWe 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.ConclusionThis 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.

Squash Print Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Detection of Potato leafroll virus in Single Aphid and in Potato

A squash print reverse transcription loop-mediated isothermal amplification amplification (SP-RT-LAMP) assay was developed for detection of Potato leafroll virus (PLRV) in a single aphid. Healthy aphids were given an acquisition feeding on potato plants infected with PLRV, and the acquired aphids were used for RNA isolation. The RT-LAMP assay was carried out using LAMP primers targeting the coat protein gene of PLRV. The amplified product was run on agarose gel; a typical ladder-like pattern in the acquired aphids indicated that the assay was able to detect PLRV from an individual aphid. The results of the RT-LAMP assay for the detection of PLRV in a single aphid were also visualized directly by adding fluorescent dye in LAMP amplified product. These results were confirmed by RT-PCR detection where an amplicon of expected size of 492 bp was observed in virus-acquired single aphids. The assay was optimized with respect to its isothermal amplification temperature and its incubation period. The technique was examined for its relative selectivity with other potato viruses transmitted by aphids where it did not show any cross-reactivity with the tested viruses indicating its specificity. With respect to its relative sensitivity, it was found to be equally sensitive as the existing RT-PCR assay. It was also found to be robust and highly reproducible as it was able to detect PLRV from known and unknown single aphid samples. The assay was successful in detection of PLRV in potato plants and tubers as well. Hence, we conclude that the assay is simple, sensitive and economical for detection of PLRV in single aphid and in potato plants and tubers.

Development of polyclonal antiserum against movement protein from Potato leafroll virus and its application for the virus detection

The serological method is one of the most important techniques extensively used in crop production to detect different pathogens, especially plant viruses. An antiserum is essential for serological tests. The 17 kDa movement protein (MP) of Potato leafroll virus (PLRV) is related to the membranous structures and localized to the plasmodesmata, but there is no report on preparation of PLRV-MP antiserum for detection of PLRV. To prepare PLRV-MP antiserum, reverse transcription polymerase chain reaction (RT-PCR) was carried out to amplify the PLRV-MP gene, which was constructed into a prokaryotic vector to express the protein in Escherichia coli for immunization of rabbits, after purification. Western blotting revealed that this developed antiserum could effectively detect PLRV, but with better results from the perspective of color development and economics by using antiserum at the ratio range of 1:10000 to 1:40000, presenting high sensitivity and specificity to PLRV. The serological detection results for PLRV of the field samples were identical to the RT-PCR detection. This is the first report on the development of PLRV-MP antiserum that has been successfully used for both laboratory and field detection of PLRV. The results provide a fundamental tool for further research on the function of PLRV-MP.

Silver-enhanced lateral flow immunoassay for highly-sensitive detection of potato leafroll virus

ABSTRACT Rapid non-laboratory screening of plants for pathogenic viruses crucially influences crop yields in modern agricultural technologies. The aim of this study was to develop a highly-sensitive lateral flow immunoassay (LFIA) for rapid detection of potato leafroll virus (PLRV), an infectious agent of one of the most widespread potato diseases. The proposed LFIA combines the formation of sandwich immune complexes with gold nanoparticles (GNP) as labels and silver enhancement. The enhancement stage was realized using mixture of silver lactate and hydroquinone and subsequent addition of chloride-containing buffer to stop silver reduction. LFIA with silver enhancement was 15 times more sensitive (detection limit 0.2 ng/mL; 15 min) compared with conventional LFIA (detection limit 3 ng/mL; 10 min). The enhanced LFIA detected PLRV in leaves’ extracts of infected potato in dilutions higher than enzyme-linked immunosorbent assay.

Field Evaluation and Enzyme-Linked Immunosorbent Assay Detection of Potato Leaf Roll Virus, Potato Virus X and Potato Virus Y in Potato Germplasm

Polerovirus: potato leaf roll virus (PLRV), Potyvirus: potato virus Y (PVY) and Potexvirus: potato virus X (PVX) is more destructive and well distributed throughout the Pakistan. Incidence has been reported to be as high as 90%, 25%, and ≥ 15%, respectively in the potato growing regions. To find out the source of resistance, twenty-nine virus free potato varieties were grown under field conditions with good agricultural practices. The disease severity of PLRV, PVY and PVX was recorded to determine the level of resistance of the potato varieties according to the disease rating scale. Infectivity and biological assay of all twenty-nine varieties were done in green house on potato, Datura stramonium, Nicotiana glutinosa and Physalis floridana. Non-inoculated plants were served as control. Leaf samples from potato varieties were collected for serological detection of PLRV, PVY and PVX by Double antibody sandwich Enzyme-Linked Immunosorbent Assay (DAS-ELISA). Out of twenty nine varieties, none of the variety was resistant to PLRV although three varieties; Mirrato, 394021-120 and Orla were moderately resistant. Only FD 48-4 and TPS 9813 showed resistance to PVX and PVY. While FD 3-10, FD 9616 and FD 37-13 were moderately to PVX and PVY. Rest of the varieties was found susceptible to all three viruses.

First Report of Five Viruses Infecting Potatoes in Tanzania.

Potato (Solanum tuberosum L.) is an increasingly important food and cash crop in Tanzania (3). Potato production is concentrated in the southern highlands and mainly carried out by smallholder farmers. A certification system for seed potatoes does not exist in the country. Currently, there is little information about viruses infecting potatoes in Tanzania. In October through December 2011, occurrence of the most common, globally distributed potato viruses, Potato leaf roll virus (PLRV), Potato virus A (PVA), M (PVM), S (PVS), Y (PVY), and X (PVX) (1), was determined in 219 potato plants in 16 fields ranging from 0.2 to 1 ha. Potato crops, 1 to 3 months old, consisted sometimes of mixtures of varieties identified as Arika, Chekundu, Kagiri, Kiazi, Kikondo, Sasamka, or Tigoni by farmers, but could not be independently confirmed. The fields were located in the regions of Mbeya (Kawetele, Kikondo, Umalia, Uyole) and Rungwe (Mwakaleli) ~100 km apart in the southern highlands. Virus-like symptoms observed in most fields included yellowish-green mosaic, leaf rolling, and veinal necrosis. Symptoms in tubers were not studied. Leaves from 10 symptomatic and three symptomless plants were sampled from each field and tested by double antibody sandwich (DAS)-ELISA (1) at ARI-Uyole. Virus-specific antibodies and negative and positive controls were used according to the supplier's instructions (Science and Advice for Scottish Agriculture, Edinburgh, United Kingdom). PVS and PLRV were detected in 55% and 39% of the samples, respectively, and in all fields sampled. PVX and PVM were found in most fields and in 14% and 5% of the samples, respectively. PVA and PVY were only detected in two localities. Co-infection with PVS and PLRV was detected in 14% of the tested plants. Mixed infections involving three or four viruses were detected in 5% of the plants. A total of 20 samples, which were collected from Uyole and Mwakeleli and found to be ELISA-positive for one or several viruses, were pressed on FTA cards (GE Healthcare, Buckinghamshire, United Kingdom), transported to University of Helsinki, and analyzed by reverse-transcription PCR (2) using virus-specific primers designed to amplify the coat protein (CP) encoding region. All ELISA-positive samples tested positive by reverse transcriptase (RT)-PCR. Four and five samples ELISA-negative for PVX or PVA, respectively, were positive when tested by RT-PCR, suggesting that the actual incidence of these viruses may be higher than detected by DAS-ELISA. The PCR products from three to five samples per virus were sequenced without cloning, which reconfirmed detection of PLRV, PVA, PVS, PVX, and PVM (GenBank Accession Nos. KC866618 through KC866622, respectively) and revealed few if any differences among isolates of the viruses. The CP sequences were compared with viruses from other countries and continents (4). CP similarities suggested that viruses might have been introduced to Tanzania through potato trade or through introducing new cultivars without adequate indexing for viruses. These results suggest the need for the development of virus control schemes in potato crops, including the nascent, domestic certified seed potato production in Mbeya.

Visual detection of Potato Leafroll virus by loop-mediated isothermal amplification of DNA with the GeneFinder™ dye

The most common virus affecting potatoes in the field worldwide is Potato Leafroll virus (PLRV), belonging to the family Luteoviridae, genius Plerovirus. There are several molecular methods to detect PLRV including polymerase chain reaction (PCR), Multiplex AmpliDet RNA and double antibody sandwich ELISA (DAS-ELISA). But these techniques take a long time for 3h to two days, requiring sophisticated tools. The aim of this study was to reduce the time required to detect PLRV, using a newly designed loop-mediated isothermal amplification (LAMP) technique requiring only an ordinary water bath or thermoblock.PLRV RNA was extracted from overall 80 infected naturally potato leaves. A set of six novel primers for the LAMP reaction was designed according to the highly conserved sequence of the viral coat protein (CP) gene. LAMP was carried out under isothermal conditions, applying the Bst DNA polymerase enzyme; the LAMP products were detected visually using the GeneFinder™ florescence dye.A positive result using the GeneFinder™ dye was a color change from the original orange to green. Results confirmed LAMP with GeneFinder™ provides a rapid and safe assay for detection of PLRV. Since with other molecular methods, equipping laboratories with a thermocycler or expensive detector systems is unavoidable, this assay was found to be a simple, cost-effective molecular method that has the potential to replace other diagnostic methods in primary laboratories without the need for expensive equipment or specialized techniques. It can also be considered as a reliable alternative viral detection system in further investigations.

Assessment of Performance Ability of Three Diagnostic Methods for Detection of Potato Leafroll Virus (PLRV) Using Different Visualizing Systems

To diminish the time required for some diagnostic assays including reverse transcription PCR (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP; due to mainly RNA extraction step) and also DAS-ELISA into a minimum level, an innovative immunocapture RT-LAMP (IC-RT-LAMP) and immunocapture reverse transcription (IC/RT-PCR) protocol on the basis of Potato Leafroll virus (PLRV) genome were used and optimized. In this regard, all six IC-RT-LAMP primers (i.e. F3, B3, FIP, BIP, LF and LB) together with IC/RT-PCR primers were designed on the basis of the highly conserved sequence (ORF3) of coat protein gene (GenBank accession number: U73777) of PLRV genome. Even though DAS-ELISA, IC/RT-PCR and IC-RT-LAMP assays could successfully detect positive infected plant samples, considering the time, safety, sensitivity, cost and simplicity, the last one was overall superior. Meanwhile, among five different visual dyes to accurately detect IC-RT-LAMP products, both hydroxynaphthol blue and GeneFinder™ 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. Altogether, as IC-RT-LAMP is sensitive, cost-effective, fairly user friendly and also can generate more accurate results than previous diagnostic procedures, we accordingly propose this colorimetric assay as a highly reliable alternative viral recognition system regarding PLRV recognition and probably other viral-based diseases.

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

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 CaCl2 instead of MgSO4 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.

Simple and Rapid Detection of Potato leafroll virus by Reverse Transcription Loop-mediated Isothermal Amplification

A new reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the Potato leafroll virus (PLRV) was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR) to address its advantages over RTPCR. RT-LAMP primers were designed from the open reading frame 3 (ORF3) sequence of PLRV. The RT-LAMP reactions were conducted without or with a set of loop primers. By real-time monitoring using Turbimeter, the RT-LAMP (with loop primers) detects PLRV in less than 30 min, compared to 120 min of RT-PCR. By adding fluorescent reagent during the reaction, final products of the RT-LAMP were fluorescently visualized under UV light or could be differentiated by naked-eye inspection under normal light. The RT-LAMP was extremely sensitive, about 2000-fold more sensitive than RT-PCR. This study presents great potential of the RT-LAMP for diagnosis and PLRV epidemiology because RT-LAMP method is speedy, sensitive, inexpensive, and convenient.

PCR-マイクロプレート・ハイブリダイゼーション法によるアブラムシからのジャガイモ葉巻ウイルスの検出

PCR-マイクロプレート・ハイブリダイゼーション法によるアブラムシからのジャガイモ葉巻ウイルスの検出

Development and validation of an analysis method for the detection of altered resistance in transgenic plants to herbivore-pathogen-complexes

AbstractGenetically modified plants (GMPs) have to pass several safety evaluations before they can be approved but identification of undesired changes in metabolism is hard to achieve due to its complexity. Such unexpected changes could be reflected by changes in virus resistance and feeding behaviour of herbivores such as aphids. Altered behaviour can be determined by so- called life table statistics where e. g. the lifetime and the reproduction rate are analysed. Such experiments are time consuming and costly. The behaviour alterations should also be detectable by changes in virus uptake und transmission. Thus, quantitative detection methods are needed in order to measure the absolute virus content in plants and in aphids. Potato leafroll virus (PLRV) and Potato virus Y (PVY) are efficiently transmitted by the green peach aphid Myzus persicae. PVY is transmitted non-persistently whereas PLRV is transmitted in a persistent and circulative manner. Consequently, PLRV is especially suited for experiments determining changes in the feeding behaviour.In recent years, several quantitative PCR-assays have been developed for the detection of PLRV in plants. Published assay conditions were not useful for the virus detection in aphids, since primers often bound non-specifically. Furthermore, particular extraction methods had to be developed, since reagents used for plant material were not suitable for aphids with their high content of inhibitory substances.However, RNA extraction is very costly and not suitable for high-throughput screening. Therefore, highly specific assays based on immunocapture-RT-qPCR were developed for the quantitative detection of both PLRV and PVY in aphids and plants. The efficiency of the assays is comparable for the detection of viruses in plants as well as in vectors.In the future, these methods will be applied to investigate the influence of differentgenetic modifications on virus content and feeding behaviour.

Detection of potato leafroll virus isolated from potato fields in Tehran province in aphids by immunocapture reverse transcription polymerase chain reaction

The surveys conducted during 2006 and 2007 revealed the infection of the virus in potato fields in Tehran province. Due to the important roll of aphids in transmission of the virus, immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) and reverse transcription polymerase chain reaction (RT-PCR) was developed using potato leafroll virus (PLRV) specific antibodies and specific primer pair (20 mer) located in the virus capsid gene to detect the virus in aphids. A 336-bp PCR product was detected from aphids (Myzus persicae) which had been fed on PLRV-infected plants. The PCR band was specific to PLRV as determined in PLRV-infected plants. This inquiry shows that this method is applicable to assess viroliferous nature of aphids in yellow -pan traps during season.

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

A high throughput, real-time multiplex, single tube RT-PCR assay was developed for simultaneous detection of Potato leafroll virus (PLRV), Potato virus X (PVX) and Potato virus S (PVS) in potato leaves and tubers, and Tomato spotted wilt virus (TSWV) in potato tubers and tomato leaves. The test uses four different fluorescently labelled TaqMan ® probes. Limits of detection sensitivity were established using a range of virus transcript copy numbers (8 × 10 1 to 8 × 10 9 copies of PVX and PVS, 1 × 10 2 to 1 × 10 10 copies of PLRV and 1 × 10 3 to 1 × 10 10 copies of TSWV). For each individual assay, the inter-assay reproducibility was high, with a coefficient of variation of the combined assays of <2%. Total RNA was extracted rapidly and efficiently from bulked samples equivalent to 300 dormant tubers to detect single infections of PLRV, PVX, PVS and TSWV simultaneously in a single assay. The multiplexed assay was validated in blind studies with leaves and tubers. This high-throughput test is accurate and sensitive, and provides seed potato industries with a cost-effective diagnostic tool to detect viruses reliably in bulked samples of dormant potato tubers.

A fully recombinant ELISA using in vivo biotinylated antibody fragments for the detection of potato leafroll virus

A recombinant antibody fusion protein, V3HCL, which was shown previously to have specific reactivity for potato leafroll virus (PLRV), was labeled with biotin using standard chemical coupling procedures and by an in vivo method. The in vivo method proved superior giving reproducible V3HCL-biotin preparations. A fully recombinant ELISA was devised incorporating V3HCL, V3HCL-biotin and streptavidin alkaline phosphatase conjugate. This assay gave comparable results for PLRV detection in potato to an assay based on immunoglobulins. The V3HCL-biotin preparations were stable and retained specific activity for more than 1 year when stored at 4 °C or −20 °C. The results demonstrate that scFv reagents derived from synthetic phage display platforms can provide effective alternatives to assays incorporating immune reagents.

Factors influencing detection of Potato Leafroll Virus and Potato Virus Y in potato tuber extracts

Factors influencing detection of Potato Leafroll Virus and Potato Virus Y in potato tuber extracts Detection of Potato leafroll virus (PLRV) and Potato virus Y (PVY) directly in potato tubers has been influenced by several factors. The most important were: the place of tuber sampling, preincubation of tuber sap before loading into wells of microplate and duration of tubers storage after collecting from field. The concentration of both viruses was highest in the heel part of tubers, whenever tested. Preincubation of tuber sap for several hours improved true/false signal ratio for dormant tubers and enabled reliable detection of both viruses. However after natural dormancy breaking it was necessary to change Cocktail-ELISA procedure to obtain reliable results, consistent with DAS-ELISA on leaves. The sap was not preincubated but loaded into wells directly after sample collecting and immuno-enzymatic reaction was developed overnight in refrigerator.

Beet necrotic yellow vein virus (benyvirus)

<i>Beet necrotic yellow vein virus</i> (<i>benyvirus</i>)

Annual Meeting, Québec, Quebec, 2006, The Canadian Phytopathological Society / Réunion annuelle, Québec, Québec, 2006, La Société canadienne de phytopathologie

Annual Meeting, Québec, Quebec, 2006, The Canadian Phytopathological Society / Réunion annuelle, Québec, Québec, 2006, La Société canadienne de phytopathologie

Development of a multiplex AmpliDet RNA for the simultaneous detection of Potato leafroll virus and Potato virus Y in potato tubers

A novel isothermal multiplex AmpliDet RNA system is described for the simultaneous amplification and detection of Potato leafroll virus (PLRV) and Potato virus Y (PVY) in seed potatoes. The risk of contamination by carry-over during diagnostic screening is eliminated by performing the reaction in a single closed tube. The viruses present in a sample are identified using differently coloured molecular beacons directed to a selected virus-specific sequence within the amplicon formed during amplification. With this system, as little as 10 fg of purified PLRV or PVY can be detected. The presence of both viruses in a sample is detected by the multiplex assay within a high range of virus concentrations. The reliability of the multiplex assay was compared with the enzyme-linked immunosorbent assay for detection of PLRV- or PVY-antigens in potato tubers. The multiplex assay detected clearly the viruses present originally in the potato tubers in all samples, demonstrating its potential for routine diagnostic work and high-throughput screening.