scholarly journals Immunocapture-RT-PCR detection of Cassava common mosaic virus in cassava obtained from meristem-tip culture in Paraná state

2011 ◽  
Vol 36 (5) ◽  
pp. 271-275 ◽  
Author(s):  
Jaqueline M. Silva ◽  
Patrícia R. Carnelossi ◽  
Taise Bijora ◽  
Cassiele U. Facco ◽  
Marcelo H.S. Picoli ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Pcr Detection ◽  
Rt Pcr ◽  
Meristem Tip Culture

scholarly journals Perbaikan Metode Ekstraksi dsRNA Virus secara Sederhana untuk RT-PCR Tiga Virus Tumbuhan

2018 ◽  
Vol 21 (2) ◽  
pp. 106
Author(s):  
Wiwik Endarsih ◽  
Sedyo Hartono ◽  
Sri Sulandari
Keyword(s):  
Nucleic Acid ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Extraction Method ◽  
Pcr Detection ◽  
Rt Pcr ◽  
Viral Pathogen ◽  
Virus Rna ◽  
Tomato Chlorosis Virus ◽  
Commercial Kit

Replicative form (RF) of RNA viruses are dsRNA structured nucleic acid, always found in plants infected by RNA virus. The principle of dsRNA extraction is based on the different affinity of nucleic acids for the cellulose powder and the specific adsorption in 16.6% ethanol buffer. The study aims to develop the simple dsRNA extraction method for the preparation of RT-PCR detection for Rehmannia mosaic virus (ReMV), Cucumber mosaic virus (CMV), Tomato chlorosis virus (ToCV), and compared with commercial kit. The analysis was performed by quantification of nucleic acid with spectrophotometer, efficiency of method (level of complexity, time, cost per reaction) and sequencing. The RNA concentration with simple methode of dsRNA extraction was lower than kit extraction method but the both methods have same pure RNA result. The PCR and sequencing result showed that viral pathogen of pepper, tobacco, and tomato leaf was CMV, ReMV, and ToCV, respectively with amplicon size at 500, 568, and 360 bp. This method is quite cheap and the RNA quantity is proportional to the commercial kit. The simple method of dsRNA extraction can be proposed for the preparation of RT-PCR detection for CMV, ReMV, ToCV. IntisariReplicative form (RF) virus RNA merupakan asam nukleat berstruktur dsRNA, selalu ditemukan pada tumbuhan terinfeksi oleh virus RNA. Prinsip kerja ekstraksi dsRNA berdasarkan afinitas serbuk selulosa terhadap asam nukleat dan adsorbsi spesifik dsRNA pada konsentrasi etanol 16,6 %. Penelitian ini bertujuan untuk mengembangkan metode ekstraksi dsRNA secara sederhana untuk preparasi deteksi RT-PCR terhadap Rehmannia mosaic virus (ReMV), Cucumber mosaic virus (CMV), Tomato chlorosis virus (ToCV) dan dibandingkan dengan kit komersil. Data yang dibandingkan adalah kuantitas asam nukleat, analisa efisiensi metode (tingkat kerumitan, waktu, biaya per reaksi) serta sekuensing. Konsentrasi RNA hasil ekstraksi metode dsRNA secara sederhana lebih rendah dibanding dengan metode kit, namun kedua metode menghasilkan RNA yang murni. Berdasarkan hasil PCR dan sekuensing disimpulkan bahwa virus penyebab mosaik daun lada dan tembakau serta klorosis daun tomat berturut-turut adalah CMV, ReMV, dan ToCV dengan ukuran amplikon berturut turut 500, 568 dan 360 pb. Metode ini cukup murah dan kuantitas RNA yang dihasilkan sebanding dengan kit komersil. Ekstraksi RNA menggunakan metode dsRNA secara sederhana dapat dikembangkan untuk preparasi deteksi RT-PCR terhadap CMV, ReMV, ToCV.

scholarly journals Duplex-RT-PCR assay for the simultaneous detection and discrimination of Brome mosaic virus and Cocksfoot mottle virus in cereal plants

2021 ◽  
Author(s):  
Katarzyna Trzmiel
Keyword(s):  
Mosaic Virus ◽  
Simultaneous Detection ◽  
Brome Mosaic Virus ◽  
Mottle Virus ◽  
Grass Species ◽  
Replicase Gene ◽  
Rt Pcr ◽  
Pcr Products ◽  
Cereal Plants ◽  
Cocksfoot Mottle Virus

AbstractBrome mosaic virus (BMV) and cocksfoot mottle virus (CfMV) are pathogens of grass species including all economically important cereals. Both viruses have been identified in Poland therefore they create a potential risk to cereal crops. In this study, a duplex—reverse transcription—polymerase chain reaction (duplex-RT-PCR) was developed and optimized for simultaneous detection and differentiation of BMV and CfMV as well as for confirmation of their co-infection. Selected primers CfMVdiag-F/CfMVdiag-R and BMV2-F/BMV2-R amplified 390 bp and 798 bp RT-PCR products within coat protein (CP) region of CfMV and replicase gene of BMV, respectively. Duplex-RT-PCR was successfully applied for the detection of CfMV-P1 and different Polish BMV isolates. Moreover, one sample was found to be co-infected with BMV-ML1 and CfMV-ML1 isolates. The specificity of generated RT-PCR products was verified by sequencing. Duplex-RT-PCR, like conventional RT-PCR, was able to detect two viruses occurring in plant tissues in very low concentration (as low as 4.5 pg/µL of total RNA). In contrast to existing methods, newly developed technique offers a significant time and cost-saving advantage. In conclusion, duplex-RT-PCR is a useful tool which can be implemented by phytosanitary services to rapid detection and differentiation of BMV and CfMV.

RT-PCR-RFLP analysis for evaluating cross protection by an attenuated isolate of Cucumber mosaic virus

2005 ◽  
Vol 71 (3) ◽  
pp. 243-246 ◽  
Author(s):  
Eiko Nakazono-Nagaoka ◽  
Masako Suzuki ◽  
Yoshitaka Kosaka ◽  
Tomohide Natsuaki
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Rflp Analysis ◽  
Cross Protection ◽  
Rt Pcr ◽  
Pcr Rflp ◽  
Attenuated Isolate

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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