scholarly journals Karakterisasi Molekuler Papaya ringspot virus tipe P pada Tanaman Mentimun di Jawa

2018 ◽  
Vol 14 (3) ◽  
pp. 75
Author(s):  
Listihani Listihani ◽  
Tri Asmira Damayanti ◽  
Sri Hendrastuti Hidayat ◽  
Suryo Wiyono
Keyword(s):  
Ringspot Virus ◽  
Mosaic Symptom ◽  
Papaya Ringspot Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Specific Primers ◽  
Central Java ◽  
Dna Fragment ◽  
Polymerase Chain

Moleculer Characterization of Papaya ringspot virus type P on Cucumber in JavaInfection of Papaya ringspot virus (PRSV) on cucumber plants showing mosaic symptom was detected using specific antibody.  Further investigation was conducted to determine molecular characters and status of PRSV infecting cucumber in Java.  Infection of PRSV was detected from leaf samples collected from the field using dot immunobinding assay (DIBA).  Disease frequency caused by PRSV infection reached 81.11%, 95.86%, 91.66%, and 92.3% in East Java, Central Java, Yogyakarta, and West Java, respectively.  Characterization of PRSV isolates was conducted by reverse transcription polymerase chain reaction (RT-PCR) using specific primers for PRSV-P and PRSV-W, followed by cloning, and DNA sequencing.  DNA fragment of 470 bp was successfully amplified using specific primers for PRSV-P from several samples from Nganjuk, Brebes, Kulon Progo, and Subang; but no amplification was achieved using specific primers for PRSV-W.  Nucleotide and amino acid analysis showed high homology among PRSV-P isolates from Nganjuk, Brebes, Kulon Progo, and Subang, i.e. 98.6%-99.7% and 99.3%-100%, respectively.  This is an indication of a low genetic variation among PRSV-P from Java. Further phylogenetic analysis indicated that PRSV-P isolate cucumber is in the same cluster with PRSV-P isolate papaya from Bali, Indonesia.  This is the first report of PRSV-P infecting cucumber in Indonesia.

scholarly journals Partial characterization of Prunus Necrotic Ringspot Virus on apple in EGYPT 

2021 ◽  
Author(s):  
Aly M. Abdel-Salam ◽  
Samah A. Mokbel
Keyword(s):  
Binding Assay ◽  
Ringspot Virus ◽  
Economic Losses ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Prunus Necrotic Ringspot Virus ◽  
Polymerase Chain ◽  
Severe Isolate ◽  
Microscopy Examination

Abstract A severe isolate of Prunus necrotic ringspot virus (PNRSV) was isolated from apple orchards in the vicinity of Nubaria city, Beheira governorate, Egypt. Infected-apple trees showed chlorotic, necrotic ringspots, and shoot holes on leaves. Severely infected- trees withered, became useless, and were removed causing severe economic losses. Reverse transcriptase (RT) polymerase chain reaction (PCR), RT-PCR, using degenerate primer pair for the coat protein (CP) gene of Ilarvirus amplified products similar to those produced from peach and apricot isolates of PNRSV-infecting stone fruits). Dot blotting immuno-binding assay (DBIA showed a positive reaction between PNRSV-infected apple sap and an Egyptian antiserum for PNRSV. Purified preparation from infected leaves, using the electro-elution technique yielded nucleoprotein, which had Amax and Amin at 260 and 240 nm respectively. Electron microscopy examination showed spherical virions with ca. 26 nm in diameter.

scholarly journals Strawberry latent ringspot virus Infecting Roses in India

Plant Disease ◽  
2004 ◽  
Vol 88 (1) ◽  
pp. 86-86 ◽  
Author(s):  
S. Kulshrestha ◽  
V. Hallan ◽  
G. Raikhy ◽  
R. Ram ◽  
A. A. Zaidi
Keyword(s):  
Plant Viruses ◽  
Ringspot Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Specific Primers ◽  
Chenopodium Amaranticolor ◽  
Elisa Kit ◽  
Local Lesions ◽  
Young Leaves ◽  
Polymerase Chain

Rose is an economically important crop of India and the world. A survey of rose plantations in and near the Kangra Valley of Himachal Pradesh, India, showed virus-like symptoms, including yellow flecking in young leaves and reduction in leaflet size, while some were symptomless. These symptoms are similar to those for Strawberry latent ringspot virus (SLRSV) (1). Sap inoculation from symptomatic and some symptomless leaves to Chenopodium amaranticolor resulted in chlorotic local lesions followed by systemic chlorosis. SLRSV was detected in this indicator host and six rose cultivars (Happiness, Iceberg, First Prize, Ganga, Pink Panther, and Oklahoma) showing characteristic symptoms of SLRSV using enzyme-linked immunosorbent assay (ELISA) with ELISA kit (DSMZ, Braunschweig, Germany). Reverse transcription-polymerase chain reaction was performed with SLRSV-specific primers (2), and a product of the expected size of ˜181 bp was amplified. The authenticity of the fragment was confirmed by sequencing. Isolated SLRSV was also inoculated to seed-grown rose seedlings and after 20 days postinoculation the same symptoms (yellow flecking in young leaves) were observed. These results established the identity of the virus that caused yellow flecking on rose leaves in India as SLRSV. To our knowledge, this is the first report of SLRSV infecting rose in India. References: (1) A. F. Murant. Strawberry latent ringspot virus. No. 126 in: Description of Plant Viruses, CMI/AAB, Surrey, U.K., 1974. (2) E. Bertolini et al. J. Virol. Methods 96:33, 2001.

a Novel Method for the Detection of Minimal Residual Disease in B-Cell Malignancies: Ion Semiconductor Sequencing for the Evaluation of Igh Gene Rearrangements

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2983-2983
Author(s):  
Silvia Gimondi ◽  
Alessandra Cavanè ◽  
Antonio Vendramin ◽  
Giulia Biancon ◽  
Paolo Corradini ◽  
...  
Keyword(s):  
B Cell ◽  
Multiplex Pcr ◽  
Residual Disease ◽  
Chain Reaction ◽  
B Cell Malignancies ◽  
Specific Primers ◽  
Namalwa Cell ◽  
Polymerase Chain ◽  
Minimal Residual

Abstract Background: Minimal residual disease (MRD) detection is of high clinical relevance in patients with B-cell malignancies and is generally a surrogate parameter to evaluate treatment response and long-term prognosis. IgH gene rearrangements can be used as molecular marker in approximately 80% of lymphoma and myeloma patients since they represent lineage-specific markers and the complementarity determining region 3 (CDR3) is unique to each clone. To date, allele specific oligonucleotide polymerase chain reaction (ASO-PCR) and real-time quantitative polymerase chain reaction (RQ-PCR) are considered the most sensitive and widely applicable methods for MRD detection. A major disadvantage of ASO-PCR and RQ-PCR assays, is the use of specific primers and probes for every individual patient. Clone-specific primers and probes are not only expensive but also time-consuming to design and to test, which limits their wide applicability in the clinical setting. The recent major improvements in next generation sequencing (NGS) technologies, provide the opportunity to identify and quantify clonotypes with consensus primers combining the benefits of high sensitivity and universal applicability. The present work was designed to overcome ASO-PCR and RQ-PCR limitations by developing a feasible method for rearranged IgH genes amplification, NGS and analysis using Ion Torrent Personal Genome Machine (IT-PGM). Methods: To define a multiplex PCR protocol, DNA from 7 CLL patients, previously shown to bare a family specific clonal VDJ rearrangement, was amplified with a pool of the seven different family-specific IgH-V primers, and a consensus JH primer (Voena et al., Leukemia 1997). After Sanger sequencing, results were compared to the ones obtained with singleplex PCR protocol. Once validated, the multiplex PCR protocol was used to amplify DNA from patients and serially diluted (up to 10-8 ) DNA from Namalwa cell line (bearing a known IgH rearrangement) and subsequently sequenced on the IT-PGM using the 316 Ion-chip. NGS data were analyzed by using the IMGT-High V-Quest web server tool and the statistical software R. RQ-PCR was used to quantify the specific VDJ rearrangement in the serially diluted Namalwa DNA solutions and in DNA from patients as previously described (Farina et al, Haematologica 2009). RQ-PCR data were analyzed through a relative quantification procedure. Results: The multiplex PCR reactions we have tested, demonstrated the same specificity as the standard singleplex PCR protocol and therefore was used to construct the DNA library required for IT-PGM-based sequencing. The IT-PGM sequencing output is represented by at least 400000 reads per sample with a minimum average coverage of the VDJ repertoire of 500x. The IMGT-High V-quest tool allows a user-friendly web based analysis and a deep molecular characterization of the IgH recombinatorial repertoire. Namalwa clonal CRD3 sequences were detected up to a dilution of 10-5 without the need for specific CDR3 primers. Comparability of NGS and ASO RQ-PCR results was assessed. The use of CDR3 specific primers, along with the specific IgH-V family fluorescent probe, enabled the identification of clonal VDJ rearrangements with a sensitivity up to 10-5 (2/3 replicates) and 10-6 (just 1/3 replicates) in Namalwa Cell Line. Similar results were obtained when we characterized the IgH recombination repertoire of two CLL patients over time. Conclusions: IgH sequencing with the IT-PGM platform showed at least the same level of sensitivity as ASO RQ-PCR, without the need for patient-specific reagents. It also allows specific and detailed molecular characterization of the clonal rearrangements and could be easily incorporated into clinical laboratories for routine testing of MRD in B-cell malignancies. Disclosures No relevant conflicts of interest to declare.

Torovirus detection in faecal specimens of calves and pigs in Hungary: Short communication

2002 ◽  
Vol 50 (3) ◽  
pp. 293-296 ◽  
Author(s):  
Katalin Matiz ◽  
S. Kecskeméti ◽  
I. Kiss ◽  
Keyword(s):  
Electron Microscopy ◽  
Short Communication ◽  
The United States ◽  
Aetiological Agent ◽  
Rt Pcr ◽  
Weaned Piglets ◽  
Chain Reaction ◽  
Specific Primers ◽  
Porcine Torovirus ◽  
Polymerase Chain

Bovine torovirus is an established aetiological agent of disease in cattle, while porcine torovirus has only been isolated from healthy animals. Evidence for the presence of torovirus has been described in several European countries and also in the United States. A survey was performed to detect toroviruses in Hungary by means of sampling ten swine and nine bovine herds. Rectal swabs and faecal specimens were collected from diarrhoeic calves and from weaned piglets. The samples were tested by the reverse transcription-polymerase chain reaction (RT-PCR) using torovirus-specific primers and the positive samples were further examined by electron microscopy (EM). Torovirus was detected in 4 diarrhoeic calves (out of 111) and in 10 healthy weaned pigs (out of 200 tested), representing two of the 9 calf herds and two of the 10 pig herds tested. This is the first report of exact diagnosis of torovirus in Hungary.

scholarly journals Application of RT-PCR for Indexing Avocado Sunblotch Viroid

Plant Disease ◽  
1997 ◽  
Vol 81 (9) ◽  
pp. 1023-1026 ◽  
Author(s):  
R. J. Schnell ◽  
D. N. Kuhn ◽  
C. M. Ronning ◽  
D. Harkins
Keyword(s):  
Rt Pcr ◽  
Amplification Product ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Specific Primers ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Dideoxynucleotide Chain Termination Method ◽  
Slab Gels ◽  
Avocado Sunblotch Viroid

A method for the routine detection of avocado sunblotch viroid (ASBVd) in nucleic acid extracts of infected avocado tissues by reverse transcription-polymerase chain reaction (RT-PCR) was developed using ASBVd-specific primers. Amplified cDNA products were analyzed by electrophoresis on nondenaturing 6% polyacrylamide slab gels. The size of the major RT-PCR product from ASBVd-infected tissue was estimated to be 250 bp. This product was absent from amplified extracts of uninfected tissue. The amplification product from ASBVd was sequenced by the dideoxynucleotide chain termination method, and the sequence was over 97% identical to the published sequence. The RT-PCR assay is sensitive enough to allow viroid detection without requiring large amounts of tissue, highly purified ASBVd, or molecular hybridization.

scholarly journals Molecular Identification of Begomovirus Infecting Angled Luffa

2020 ◽  
Vol 24 (2) ◽  
pp. 147
Author(s):  
Alvina Clara Giovanni ◽  
Sedyo Hartono ◽  
Sri Sulandari ◽  
Susamto Somowiyarjo
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequencing ◽  
Molecular Identification ◽  
Leaf Shape ◽  
Mosaic Symptom ◽  
Chain Reaction ◽  
Central Java ◽  
Total Dna ◽  
Polymerase Chain ◽  
Luffa Acutangula

Begomovirus was reported as one of the most aggressive and destructive viruses on several commercial crops, including cucurbits in Indonesia. Plants that infected with Begomovirus show the mosaic symptom on the leaves, change in leaf shape, stunts, change in color and shape of fruit. It was recently observed in cultivated angled luffa [Luffa acutangula (L.) Roxb] around Yogyakarta and Central Java. The aim of this research was to identify the virus by using Polymerase chain reaction (PCR). The result of Begomovirus amplification from the total DNA samples amplification using primer Krusty-Homer showed that DNA of Begomovirus from angled luffa was amplified at ~580bp. The DNA sequencing of angled luffa’s leaf isolate GD1 had 97.8% homology with SCLV-China isolate MC1. However, amplification of DNA seed samples using the same primer showed negative result. It was concluded that Begomovirus was not a seed borne virus. This is the first molecular report on the occurence of Begomovirus in angled luffa in Yogyakarta.

scholarly journals First Detection and Characterization of Chrysanthemum virus B infecting Chrysanthemum in Thailand

2021 ◽  
Author(s):  
Salit Supakitthanakorn ◽  
Garnjana Wichitrakoonthavorn ◽  
Kaewalin Kunasakdakul ◽  
On-Uma Ruangwong
Keyword(s):  
Cultural Values ◽  
Ornamental Plants ◽  
Systemic Symptom ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Transmission Electron ◽  
Staining Methods ◽  
Polymerase Chain ◽  
Systemic Symptoms

Abstract Chrysanthemum is one of the important ornamental plants in worldwide due to its high economic and cultural values. Chrysanthemum leaves showed mosaic, ringspot, yellowing and mild mottle symptoms were observed and collected from cultivation areas in northern Thailand and used for detection of important viruses infecting chrysanthemum. Chrysanthemum virus B (CVB) was detected by reverse transcription polymerase chain reaction (RT-PCR) from samples showing yellowing and mild mottle symptoms. Sequences of the coat protein (CP) gene of two CVB isolates found in this study were sequenced and shared 93.15% homology with other CVB isolates from different countries deposited in GenBank. Biological indexing of these CVB found that they induced both local and systemic symptoms in tobacco plants while petunia displayed a systemic symptom. The particles of CVB were observed under transmission electron microscope (TEM), prepared by dip preparation and negative staining methods, showing slightly flexuous rod-shaped virions approximately 600–650 nm in length. To our knowledge, this is the first detection and study on molecular and biological characteristics of CVB infecting chrysanthemum in Thailand.

scholarly journals Detection and molecular characterization of the Iris severe mosaic virus-Ir isolate from Iran

2015 ◽  
Vol 55 (3) ◽  
pp. 235-240 ◽  
Author(s):  
Masoud Nateqi ◽  
Mina Koohi Habibi ◽  
Akbar Dizadji ◽  
Shirin Parizad
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Mosaic Virus ◽  
Ornamental Plants ◽  
Rt Pcr ◽  
Chain Reaction ◽  
C Terminus ◽  
Coated Plate ◽  
Polymerase Chain

AbstractIris belongs to the Iridaceae family. It is one of the most important pharmaceutical and ornamental plants in the world. To assess the potyvirus incidence in natural resources of iris plants in Iran, Antigen Coated-Plate ELISA (ACP-ELISA) was performed on 490 symptomatic rhizomatous iris leaf samples, which detected the potyvirus in 36.7% of the samples. Genomic 3′ end of one mechanically non-transmitted potyvirus isolate, comprising a 3′ untranslated region (390 bp) and C-terminus of the coat protein (CP) gene (459 bp), was amplified by reverse transcription polymerase chain reaction (RT-PCR), which was ligated into pTG19-T vector. The nucleotide sequence of amplicons was compared with related sequences, using Blastn software available at NCBI GenBank, and showed the highest similarity withIris severe mosaic virus(ISMV) isolates. The nucleotide and deduced amino acid sequence of the CP C-terminus region was more than 83% identical with other ISMV isolates, therefore this isolate was designated as ISMV-Ir. This new ISMV isolate is closely related to the Chinese ISMV-PHz in phylogenetic analysis, based on the partial nucleotide and deduced amino acid sequence of the CP region. This is the first report of ISMV occurrence onIrisspp. in Iran.

scholarly journals Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant

2000 ◽  
Vol 124 (3) ◽  
pp. 481-487 ◽  
Author(s):  
P. J. MARKS ◽  
I. B. VIPOND ◽  
D. CARLISLE ◽  
D. DEAKIN ◽  
R. E. FEY ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Polymerase Chain Reaction ◽  
Inverse Relationship ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Virus Particles ◽  
Stool Samples ◽  
Polymerase Chain

An outbreak of gastroenteritis followed a meal in a large hotel during which one of the diners vomited. The clinical features of the illness suggested Norwalk-like virus (NLV, small round structured virus) infection, and this was confirmed by electron microscopy and reverse transcriptase polymerase chain reaction (RT–PCR) of stool samples. Further characterization of the virus by nucleotide sequence analysis of the PCR amplicons revealed identical strains in all the affected individuals. The foods served at the meal could not be demonstrated to be the cause of the outbreak. Analysis of attack rates by dining table showed an inverse relationship with the distance from the person who vomited. No one eating in a separate restaurant reported illness. Transmission from person-to-person or direct contamination of food seems unlikely in this outbreak. However, the findings are consistent with airborne spread of NLV with infection by inhalation with subsequent ingestion of virus particles.

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