scholarly journals Optimization and Application of a Multiplex RT-PCR System for Simultaneous Detection of Five Potato Viruses Using 18S rRNA as an Internal Control

Plant Disease ◽  
2006 ◽  
Vol 90 (2) ◽  
pp. 185-189 ◽  
Author(s):  
ZhiYou Du ◽  
JiShuang Chen ◽  
Chuji Hiruki
Keyword(s):  
Potato Virus ◽  
Internal Control ◽  
18S Rrna ◽  
Degradation Kinetics ◽  
Simultaneous Detection ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Potato Viruses ◽  
Das Elisa

Search for a host RNA molecule appropriate as an internal control for reverse transcription-polymerase chain reaction (RT-PCR) detection of viruses in potato (Solanum tuberosum) was conducted. The 18S ribosomal RNA (rRNA) was compared with the commonly used nad2 mRNA in terms of detection sensitivity and degradation kinetics. Detection of 18S rRNA was 5 magnitudes more sensitive than that of nad2 mRNA. The 18S rRNA also displayed degradation kinetics more similar to that of Potato virus X (PVX). Based on this result, reaction components and cycling parameters were optimized for a multiplex RT-PCR protocol for simultaneous detection of five potato viruses using 18S rRNA as an internal control. The protocol simultaneously amplified cDNAs from Potato virus A, PVX, Potato virus Y, Potato leaf roll virus, Potato virus S, and 18S rRNA. The multiplex RT-PCR protocol was able to detect all viruses in different combinations. The technique was 100-fold greater for detection of PVX than that of commercial double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA), and also could detect viruses in some samples that DAS-ELISA failed to detect. This multiplex RT-PCR technique demonstrates a higher sensitivity of virus detection than DAS-ELISA.

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 Application of cDNA Macroarray for Simultaneous Detection of 12 Potato Viruses

Plant Disease ◽  
2010 ◽  
Vol 94 (10) ◽  
pp. 1248-1254 ◽  
Author(s):  
T. Maoka ◽  
S. Sugiyama ◽  
Y. Maruta ◽  
T. Hataya
Keyword(s):  
Mosaic Virus ◽  
Potato Virus ◽  
Colorimetric Detection ◽  
Simultaneous Detection ◽  
Alfalfa Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potato Virus S ◽  
Cdna Macroarray ◽  
Potato Viruses ◽  
Homologous Virus

A complementary DNA (cDNA) macroarray was developed for simultaneous detection of 12 different potato viruses. A suitable region in the viral genome for each was selected for Alfalfa mosaic virus, Cucumber mosaic virus, Potato aucuba mosaic virus, Potato leafroll virus, Potato mop-top virus, Potato virus A, Potato virus M, Potato virus S, Potato virus X, Potato virus Y, Tomato ringspot virus, and Tomato spotted wilt virus, and their respective cDNAs were cloned into plasmid vectors. Capture probes for each virus ranging from 290 to 577 bp were generated by polymerase chain reaction (PCR) and immobilized on a nylon membrane. Total RNAs were extracted from each of these virus infected-plants, and cDNAs were synthesized from the RNA extracts using a random 9-mer primer. Subsequently, PCR reactions were performed using one primer pair for each of the 12 viruses. During PCR, amplified cDNAs were labeled with biotin and used as a target for hybridization analyses on a macroarray membrane. Hybridization signals between capture probes for the 12 viruses and their respective target cDNAs were observed using chemiluminescent or colorimetric detection. In all viruses, hybridization signals with capture probes were detected only when homologous virus targets were examined, and no hybridization to healthy plant extract was observed, facilitating identification of each virus. The results by colorimetric detection agreed with those obtained using chemiluminescence. The macroarray method developed was 5 × 102 to 4 × 106 times more sensitive than enzyme-linked immunosorbent assay and 5 to 5 × 104 times more sensitive than reverse-transcription PCR, except for Alfalfa mosaic virus. Colorimetric detection and substantial reduction in cross-hybridization signals much improved the method compared with other array-based detection methods for practical use.

scholarly journals Monitoring Current-Season Spread of Potato virus Y in Potato Fields Using ELISA and Real-Time RT-PCR

Plant Disease ◽  
2013 ◽  
Vol 97 (5) ◽  
pp. 641-644 ◽  
Author(s):  
Manphool S. Fageria ◽  
Mathuresh Singh ◽  
Upeksha Nanayakkara ◽  
Yvan Pelletier ◽  
Xianzhou Nie ◽  
...  
Keyword(s):  
Real Time ◽  
Potato Virus ◽  
Potato Virus Y ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Current Season ◽  
Seed Market ◽  
Polymerase Chain ◽  
Time Of Harvest

The current-season spread of Potato virus Y (PVY) was investigated in New Brunswick, Canada, in 11 potato fields planted with six different cultivars in 2009 and 2010. In all, 100 plants selected from each field were monitored for current-season PVY infections using enzyme-linked immunosorbent assay (ELISA) and real-time reverse-transcription polymerase chain reaction (RT-PCR) assay. Average PVY incidence in fields increased from 0.6% in 2009 and 2% in 2010 in the leaves to 20.3% in 2009 and 21.9% in 2010 in the tubers at the time of harvest. In individual fields, PVY incidence in tubers reached as high as 37% in 2009 and 39% in 2010 at the time of harvest. Real-time RT-PCR assay detected more samples with PVY from leaves than did ELISA. A higher number of positive samples was also detected with real-time RT-PCR from growing tubers compared with the leaves collected from the same plant at the same sampling time. PVY incidence determined from the growing tubers showed a significant positive correlation with the PVY incidence of tubers after harvest. Preharvest testing provides another option to growers to either top-kill the crop immediately to secure the seed market when the PVY incidence is low or leave the tubers to develop further for table or processing purposes when incidence of PVY is high.

scholarly journals First Report of Cucurbit aphid-borne yellows virus in Iran Causing Yellows on Four Cucurbit Crops

Plant Disease ◽  
2006 ◽  
Vol 90 (4) ◽  
pp. 526-526 ◽  
Author(s):  
K. Bananej ◽  
C. Desbiez ◽  
C. Wipf-Scheibel ◽  
I. Vahdat ◽  
A. Kheyr-Pour ◽  
...  
Keyword(s):  
Citrullus Lanatus ◽  
Healthy Plant ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
First Report ◽  
Chain Reactions ◽  
Reference Isolate ◽  
Sigma Chemical ◽  
International Mycological Institute ◽  
Das Elisa

A survey was conducted from 2001 to 2004 in the major cucurbit-growing areas in Iran to reassess the relative incidence of cucurbit viruses. Severe yellowing symptoms were observed frequently on older leaves of cucurbit plants in various regions in outdoor crops, suggesting the presence of Cucurbit aphid-borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae) (1,2). Leaf samples (n = 1019) were collected from plants of melon (Cucumis melo L.), cucumber (C. sativus L.), squash (Cucurbita sp.), and watermelon (Citrullus lanatus L.) showing various virus-like symptoms (mosaic, leaf deformation, yellowing). All samples, collected from 15 provinces, were screened for the presence of CABYV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with IgGs and alkaline phosphatase-conjugated IgGs against a CABYV reference isolate (1). Of the 1,019 samples tested, 471 were positive for CABYV using DAS-ELISA. Some of the positive samples had typical severe yellowing symptoms while symptoms in other samples were masked by mosaic or leaf deformations caused by other viruses frequently found in mixed infections (data not shown). During the entire survey, CABYV was detected by DAS-ELISA in 201 of 503 melon samples, 72 of 129 cucumber samples, 158 of 249 squash samples, and 40 of 138 watermelon samples. These results indicate that CABYV is widely distributed on four cucurbit species in the major growing areas of Iran. In order to confirm CABYV identification, total RNA extracts (TRI-Reagent, Sigma Chemical, St Louis, MO) were obtained from 25 samples that were positive using DAS-ELISA originating from Khorasan (n = 4), Esfahan (n = 6), Teheran (n = 3), Hormozgan (n = 4), Azerbaiejan-E-Sharqi (n = 4), and Kerman (n = 4). Reverse transcription-polymerase chain reactions (RT-PCR) were carried out using forward (5′-CGCGTGGTTGTGG-TCAACCC-3′) and reverse (5′-CCYGCAACCGAGGAAGATCC-3′) primers designed in conserved regions of the coat protein gene according to the sequence of a CABYV reference isolate (3) and three other unpublished CABYV sequences. RT-PCR experiments yielded an expected 479-bp product similar to the fragment amplified with extracts from the reference isolate. No amplification of the product occurred from healthy plant extracts. To our knowledge, this is the first report of the occurrence of CABYV in Iran on various cucurbit species. The high frequency (46.2%) with which CABYV was detected in the samples assayed indicates that this virus is one of the most common virus infecting cucurbits in Iran. References: (1) H. Lecoq et al. Plant Pathol. 41:749, 1992 (2) M. A. Mayo and C. J. D'Arcy. Page 15 in: The Luteoviridae. H. G. Smith and H. Barker, eds. CAB International Mycological Institute, Wallingford, UK, 1999. (3) H. Guilley et al. Virology 202:1012, 1994.

scholarly journals Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus

2010 ◽  
Vol 169 (2) ◽  
pp. 375-379 ◽  
Author(s):  
Karen Miyuki Asano ◽  
Sibele Pinheiro de Souza ◽  
Iracema Nunes de Barros ◽  
Giselle Razera Ayres ◽  
Sheila Oliveira Souza Silva ◽  
...  
Keyword(s):  
Internal Control ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Bovine Coronavirus ◽  
Group A Rotavirus ◽  
Group A ◽  
Exogenous Internal Control

Evaluation of a Sensitive Reverse Transcription PCR–Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis A Virus in Oysters (Saccostrea glomerata) on the East Coast of the Gulf of Thailand

2014 ◽  
Vol 77 (5) ◽  
pp. 859-863 ◽  
Author(s):  
URAIWAN INTAMASO ◽  
SITTHISAK KETKHUNTHOD
Keyword(s):  
Immunosorbent Assay ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
East Coast ◽  
Hepatitis A Virus ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gulf Of Thailand ◽  
Rt Pcr ◽  
The Gulf Of Thailand

Hepatitis A virus (HAV) contamination in food can lead to major health problems. We developed a combination reverse transcription (RT) PCR method plus enzyme-linked immunosorbent assay (ELISA) to detect HAV in fresh oysters harvested along the east coast of the Gulf of Thailand. Viral nucleic acid was extracted via the glycine–arginine–polyethylene glycol method followed by RT-PCR amplification with specifically designed primers against HAV and an ELISA to detect the digoxigenin-labeled RT-PCR products. The ELISA in concert with the RT-PCR protocol further increased the detection sensitivity by 100-fold for the HAV genome and 10-fold in artificially contaminated oysters. The overall sensitivity of the RT-PCR in combination with the ELISA was 31.88 pg and 16 PFU/g, respectively. The ELISA increases the specificity of the RT-PCR assay for detecting naturally occurring HAV in oysters. This combined RT-PCR-ELISA approach is a practical and sensitive method for HAV detection and can be utilized in routine screening for HAV in shellfish.

scholarly journals First Report of Sharka Disease Caused by Plum Pox Virus in Lithuania

Plant Disease ◽  
1998 ◽  
Vol 82 (12) ◽  
pp. 1405-1405 ◽  
Author(s):  
J. Staniulis ◽  
J. Stankiene ◽  
K. Sasnauskas ◽  
A. Dargeviciute
Keyword(s):  
Coat Protein ◽  
Plant Protection ◽  
Virus Disease ◽  
Pcr Amplification ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plum Pox Virus ◽  
Rt Pcr ◽  
First Report ◽  
Das Elisa ◽  
Sharka Disease

Plum pox (sharka) disease caused by plum pox potyvirus (PPV) is considered the most important virus disease of stone fruit trees in Europe and the Mediterranean region. Nearly all those countries that produce stone fruits are affected (3). The causal virus of the disease is a European Plant Protection Organization A2 quarantine pathogen. Symptoms of leaf mottling, diffuse chlorotic spots, rings, and vein banding of varied intensity characteristic for plum pox virus infection were observed in the plum (Prunus domestica) orchard tree collection of the Lithuanian Institute of Horticulture in Babtai in 1996. Presence of this virus in the diseased trees was confirmed by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) with kits from BIOREBA (Reinach, Switzerland) and by polyclonal antibodies raised against a Moldavian isolate of PPV courtesy of T. D. Verderevskaya (Institute of Horticulture, Kishinev, Moldova). ELISAs with both sources of antiserum were positive for presence of PPV. Electron microscopy revealed the presence of potyvirus-like particles averaging 770 nm in extracts of mechanically inoculated plants of Chenopodium foetidum (chlorotic LL [local lesions]) and Pisum sativum cvs. Rainiai and Citron (mottling). For molecular diagnosis and characterization of this isolate, PPV-971, reverse transcription-polymerase chain reaction (RT-PCR) was employed. Total RNA from the leaves of infected pea was isolated as described (2). High molecular weight RNA selectively precipitated with 2 M lithium chloride was used for RT-PCR amplification of the coat protein encoding sequence by use of specific primers complementary to 5′ and 3′ parts of PPV coat protein L1 (GenBank accession no. X81081). Amino acid sequence comparison with GenBank data indicated 98.2% similarity with coat protein of PPV potyvirus isolated by E. Mais et al. (accession no. X81083) and 97.3% with PPV strain Rankovic (1).The specific DNA fragment, corresponding to predicted coat protein sequence size, was cloned into Escherichia coli pUC57 for DNA sequencing. Expression of the cloned sequence in bacteria and yeast expression systems is under investigation. The presence of PPV in plum trees in the 9-year-old collection at Babtai was confirmed by DAS-ELISA in 1997 and again in 1998. PPV was then detected in 20% of symptomatic trees of three cultivars. The Lithuanian PPV isolate reacted positively with “universal” Mab.5b and with a Mab (Mab.4DG5) specific for PPV-D. No reaction was observed with Mabs specific for PPV-M (Mab.AL), PPV-C (Mab.AC and Mab.TUV), and PPV-El Amar (Mab.EA24). PPV-971 seems to be a typical member of the less aggressive Dideron strain cluster of PPV (D. Boscia, personal communication). This is the first report of PPV in Lithuania and confirms the necessity for continuing the precautionary measures established in this country for indexing of nursery plum trees used for graft propagation. References: (1) S. Lain et al. Virus Res. 13:157, 1989. (2) J. Logemann et al. Anal. Biochem. 163:16, 1987. (3) M. Nemeth. OEPP/EPPO Bull. 24:525, 1994.

scholarly journals Tomato spotted wilt virus Identified in Desert Rose in Florida

Plant Disease ◽  
2005 ◽  
Vol 89 (5) ◽  
pp. 526-526 ◽  
Author(s):  
S. Adkins ◽  
C. A. Baker
Keyword(s):  
Tomato Spotted Wilt Virus ◽  
The United States ◽  
N Gene ◽  
Enzyme Linked Immunosorbent Assay ◽  
Insect Vector ◽  
Rt Pcr ◽  
Tomato Spotted Wilt ◽  
Wilt Virus ◽  
Symptomatic Plant ◽  
Das Elisa

Desert rose (Adenium obesum (Forssk.) Roem. & Schult), a member of the family Apocynaceae, is characterized by fleshy stems and leaves and colorful flowers. This exotic ornamental, originally from southeast Africa, is propagated vegetatively and is a perennial in warm climates. Virus-like foliar symptoms, including chlorotic ring and line patterns, were observed in the fall of 2004 on one of five stock plants being maintained in a greenhouse in Fort Pierce, FL. Inclusion body morphology suggested the presence of a Tospovirus in the symptomatic plant, and Tomato spotted wilt virus (TSWV) was specifically identified in this plant using a commercially available double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA; Agdia, Elkhart, IN). TSWV was not detected in symptomless desert rose plants nor was Impatiens necrotic spot virus detected in any of the plants using DAS-ELISA. Graft transmission of TSWV to other desert rose plants was successful. Sequence analysis of a nucleocapsid (N) protein gene fragment amplified by reverse transcription-polymerase chain reaction (RT-PCR) with primers TSWV723 and TSWV722 (1) from total RNA of the symptomatic plant confirmed the diagnosis. Nucleotide and deduced amino acid sequences of a 579-bp region of the RT-PCR product were 95 to 99% and 95 to 100% identical, respectively, to TSWV N-gene sequences in GenBank. No product was amplified from symptomless plants. Since these 3-year-old plants were grown on-site from seed and only expressed symptoms 2 months following damage to the greenhouse by hurricanes Frances and Jeanne, it is likely that viruliferous thrips were introduced from local vegetable or ornamental production areas during or following the storms. To our knowledge, this is the first report of TSWV infection of desert rose in Florida, although TSWV was observed in this plant in Europe approximately 10 years ago (3,4). Because of the wide distribution of TSWV in the United States, the increasing popularity of desert rose, and the recent identification of Cucumber mosaic virus in this host (2), attention to sanitation and insect vector management is merited during desert rose propagation and production. References: (1) S. Adkins and E. N. Rosskopf. Plant Dis. 86:1310, 2002. (2) C. A. Baker et al. Plant Dis. 87:1007, 2003. (3) J. Mertelik et al. Acta Hortic. 432:368, 1996. (4) J. Th. J. Verhoeven and J. W. Roenhorst. Acta Hortic. 377:175, 1994.

scholarly journals Natural Occurrence of Viruses in Lycopersicon spp. in Ecuador

Plant Disease ◽  
2005 ◽  
Vol 89 (11) ◽  
pp. 1244-1244 ◽  
Author(s):  
S. Soler ◽  
C. López ◽  
F. Nuez
Keyword(s):  
Mosaic Virus ◽  
Potato Virus ◽  
Potato Virus X ◽  
Tomato Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Natural Occurrence ◽  
Single Plant ◽  
Rt Pcr ◽  
Specific Primers ◽  
In The Wild

The Andean region is home of important genetic diversity for the genus Lycopersicon. A survey of three asymptomatic populations of L. hirsutum, 17 of L. parviflorum, 188 of L. pimpinellifolium, and four cultivated populations of L. esculentum was made in nine departments of Ecuador. Samples were analyzed serologically for Tomato spotted wilt virus (TSWV), Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Potato virus X (PVX), Groundnut ringspot virus (GRSV), Tomato chlorosis spot virus (TCSV), and Pepino mosaic virus (PepMV). Samples positive as determined using double-antibody sandwich enzyme-linked immunosorbent assay (absorbance values three times higher than negative controls) were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) with virus-specific primers. L pimpinellifolium was the only species of the four found to be infected with viruses. In the department of Manabí, ToMV was detected in 15 of 16 plants from one population, but only a single plant was infected with PepMV. In this department, PepMV was also detected in a single-plant population that corresponded to a volunteer plant found in the wild and TSWV was detected in another plant. In Esmeraldas and Guayas, two single-plant populations were found infected with PepMV and CMV, respectively. TMV, PVY, PVX, GRSV, and TCSV were not detected in this survey. Specific primers were selected for ToMV (To1/To2, genome coordinates 3498-3518/4902-4922, AJ417701), PepMV (Pe1/Pe2 genome coordinates 5030-5050/5913-5935, AJ606359), CMV (Cm1/Cm2 genome coordinates 541-561/1756-1779, D00356), and TSWV (Ts1/Ts2 genome coordinates 4078-4101/4738-4769, AF208498). Amplicons of the expected size were obtained using RT-PCR and then cloned and sequenced. DNA fragments of ToMV, PepMV, and TSWV showed identities greater than 99% with respective sequences in the GenBank database. The highest identity of the CMV DNA fragment was 92% with an isolate from Indonesia (AB042292). The occurrence of viruses such as CMV, ToMV, and TSWV in coastal Ecuador was not surprising. However, infected plants were not found among the samples collected in the departments of Azuay, Carchí, El Oro, Imbabura, Loja, and Pichincha in eastern Ecuador. L. chilense, L. chmielewskii, L. parviflorum, and L. peruvianum were previously reported as natural hosts of PepMV in central and southern Peru (2), and the virus was also detected in L. esculentum in Chile (1). Our results show that PepMV now occurs in wild L. pimpinellifolium populations along the Pacific coast of the South American continent and that it must have efficient means of transmission, although no specific vectors have as yet been identified for this virus. To our knowledge, this is the first report of PepMV in Ecuador and L. pimpinellifolium as a natural host of PepMV. References: (1) M. Muñoz et al. Fitopatología 37:67, 2002. (2) S. Soler et al. J. Phytopathol. 150:49, 2002.

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