scholarly journals Macroarray Detection of Plant RNA Viruses Using Randomly Primed and Amplified Complementary DNAs from Infected Plants

2007 ◽  
Vol 97 (1) ◽  
pp. 119-127 ◽  
Author(s):  
Bright Agindotan ◽  
Keith L. Perry
Keyword(s):  
Rna Viruses ◽  
Detection System ◽  
Leaf Roll ◽  
Simultaneous Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potato Leaf ◽  
Viral Rnas ◽  
Anchor Primer ◽  
Total Plant ◽  
Primer Sequence

Membrane-based macroarrays provide a relatively inexpensive technology with the potential to detect hundreds of pathogens in a single assay. For the simultaneous detection of a large number of pathogens, it is necessary to obtain sufficient nucleic acids for labeling, and any amplification reactions need to be performed using unbiased, pathogen-non-specific primers. A nonradioactive macroarray system is described to test for plant RNA viruses using 70-mer oligonucleotide probes immobilized on nylon membranes. Starting with a total plant RNA extract, complementary DNA (cDNA) and second-strand syntheses were carried out using an anchor primer sequence with random pentamers coupled at the 3′ end. Subsequent synthesis by polymerase chain reaction using the anchor primer alone resulted in a relatively unbiased amplification of plant and viral RNAs. These cDNAs were chemically labeled and the product used as a target in hybridization analyses. The system was validated using RNA extracts from plants infected with Cucumber mosaic virus, Potato virus Y, and Potato leaf roll virus (PLRV). Despite the relative excess of host-derived nonviral sequences, viral RNAs were amplified between 100- and 1,000-fold and were detected in single and mixed infections. The macroarray sensitivity was comparable to that of double-antibody sandwich enzyme-linked immunosorbent assay, with PLRV being detected in sap dilutions of 1:100. The potential for the development of a relatively inexpensive multipathogen detection system is discussed.

scholarly journals Presence and distribution of economically important potato viruses in Montenegro

2011 ◽  
Vol 26 (2) ◽  
pp. 117-127
Author(s):  
Jelena Zindovic
Keyword(s):  
Potato Virus ◽  
Leaf Roll ◽  
Potato Virus X ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potato Leaf ◽  
Potato Virus S ◽  
Potato Varieties ◽  
Potato Viruses ◽  
Das Elisa

The research was carried out, in the period 2002-2004 in order to determine the presence and distribution of potato viruses at 12 different locations and on 9 different potato varieties grown in Montenegro. The research included collecting of samples in seed potato crops and testing of six economically important potato viruses: Potato leaf roll virus (PLRV), Potato virus Y (PVY), Potato virus X (PVX), Potato virus S (PVS), Potato virus A (PVA) i Potato virus M (PVM). Using the direct enzyme-linked immunosorbent assay (DAS-ELISA) and commercial antisera specific for six potato viruses, it was found that PVY was the most frequent virus during the three-year research period. The second frequent virus was PVS, followed by PVA, PLRV, PVM and PVX. Single and mixed infections were detected, and the most prevalent were the single infections of PVY. Also, in the period 2002-2004, PVY had the highest distribution and the number of present viruses was different at different localities and on different potato varieties. Further investigations were related to detailed characterization of the most prevalent virus (PVY), which is at the same time economically the most important one. Serological characterization of PVY was performed utilizing DAS-ELISA kit with commercial monoclonal antibodies specific for detection of the three strain groups of PVY, and the two strain groups - necrotic (PVYN/PVYNTN) and common (PVYO), were identified. Necrotic strains were prevalent in 2002 and 2004, while in 2003 PVYO was the most frequent strain in virus population. The presence of stipple streak strain (PVYC) was not detected in any of the tested samples.

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

2017 ◽  
Vol 9 (7) ◽  
pp. 229 ◽  
Author(s):  
Romana Anjum ◽  
M. Aslam Khan ◽  
Kolawole Oluwaseun Olawale ◽  
Raheel Baber
Keyword(s):  
Immunosorbent Assay ◽  
Potato Virus ◽  
Potato Virus Y ◽  
Rating Scale ◽  
Leaf Roll ◽  
Potato Virus X ◽  
Potato Leaf Roll Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potato Leaf ◽  
Potato Varieties

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.

Utilization of Shoot Cuttings for Elimination of PLRV and PVY by Thermotherapy and Chemotherapy from Potato (Solanum tuberosum L.)

2007 ◽  
Vol 7 ◽  
pp. 1
Author(s):  
S. P. Dhital ◽  
B. M. Sakha ◽  
H. T. Lim
Keyword(s):  
Plant Height ◽  
Solanum Tuberosum L ◽  
Combined Treatment ◽  
Leaf Roll ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potato Leaf ◽  
Virus Elimination ◽  
Elisa Testing ◽  
Das Elisa

Heat and ribavirin treatments were applied for the elimination of potato leaf roll virus (PLRV) and potato virus Y (PVY) from the potato genotype F 9-99. The explants, rooted young plantlets, cultured on MS medium with and without ribavirin (20 mg l<sup>-1</sup>) were subjected to thermotherapy (35ºC/31ºC, 4 h alternating periods) and room temperature (25ºC) for 30 days. Double antibody sandwich - enzyme linked immunosorbent assay (DAS-ELISA) testing following the therapies revealed that ribavirin alone was enable to eliminate 10% each of PLRV and PVY, whereas along with thermotherapy its efficacy increased to 25% PLRV and 20% PVY elimination. In another experiment, three potato genotypes F 9-99 infected with PLRV and PVY, Gui Valley with PLRV infection and Rose Valley with PVY infection were evaluated. Ribavirin (20 mg l<sup>-1</sup>) and ASA (acetylsalicylic acid) (10<sup>-5</sup> M) were supplemented in liquid culture medium alone or in combination with thermotherapy for three successive cycles of 30-36 days interval. Heat and/or ribavirin suppressed survival and plant height whereas ASA promoted the survival as well as plant height even under heat treatment. After each cycle, the effect of treatment on virus elimination was evaluated by DAS-ELISA. The combined application of ribavirin and ASA with thermotherapy after three cycles of treatment showed up to 47.4% PLRV and 57.9% PVY elimination in F 9-99. In the case of single viral infection of PLRV or PVY the same combined treatment showed up to 58.8% PLRV and 61.1% PVY free plantlets. Virus elimination was also confirmed by transplanting in vitro grown plantlets in a net house and then retesting after 45 days of in vivo growth. This novel technique would be highly efficient for virus elimination within a short duration in diverse genotypes of potato. <i>Nepal Journal of Science and Technology </i> 7 2006

scholarly journals Identification, Characterization, and Molecular Detection of Alfalfa mosaic virus in Potato

2006 ◽  
Vol 96 (11) ◽  
pp. 1237-1242 ◽  
Author(s):  
H. Xu ◽  
J. Nie
Keyword(s):  
Mosaic Virus ◽  
Gene Sequence ◽  
Alfalfa Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Simple Method ◽  
Cp Gene ◽  
Nucleotide Sequence Alignment

Alfalfa mosaic virus (AMV) was detected in potato fields in several provinces in Canada and characterized by bioassay, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction (RT-PCR). The identity of eight Canadian potato AMV isolates was confirmed by sequence analysis of their coat protein (CP) gene. Sequence and phylogenetic analysis indicated that these eight AMV potato isolates fell into one strain group, whereas a slight difference between Ca175 and the other Canadian AMV isolates was revealed. The Canadian AMV isolates, except Ca175, clustered together among other strains based on alignment of the CP gene sequence. To detect the virus, a pair of primers, AMV-F and AMV-R, specific to the AMV CP gene, was designed based on the nucleotide sequence alignment of known AMV strains. Evaluations showed that RT-PCR using this primer set was specific and sensitive for detecting AMV in potato leaf and tuber samples. AMV RNAs were easily detected in composite samples of 400 to 800 potato leaves or 200 to 400 tubers. Restriction analysis of PCR amplicons with SacI was a simple method for the confirmation of PCR tests. Thus, RT-PCR followed by restriction fragment length polymorphism analysis may be a useful approach for screening potato samples on a large scale for the presence of AMV.

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