Simplified extraction method for ELISA and PCR detection of potato leafroll luteovirus primary infection in dormant potato tubers

1999 ◽  
Vol 76 (4) ◽  
pp. 209-213 ◽  
Author(s):  
J. A. C. de Souza-Dias ◽  
P. Russo ◽  
J. A. Betti ◽  
L. Miller ◽  
S. A. Slack
Keyword(s):  
Extraction Method ◽  
Primary Infection ◽  
Pcr Detection ◽  
Potato Tubers ◽  
Potato Leafroll Luteovirus

scholarly journals Optimization of Sample Size and DNA Extraction Methods to Improve PCR Detection of Different Propagules of Phytophthora infestans

Plant Disease ◽  
2002 ◽  
Vol 86 (3) ◽  
pp. 247-253 ◽  
Author(s):  
T. Wangsomboondee ◽  
J. B. Ristaino
Keyword(s):  
Phytophthora Infestans ◽  
Dna Extraction ◽  
Extraction Methods ◽  
Visual Observation ◽  
Probability Of Detection ◽  
Pcr Detection ◽  
Potato Tubers ◽  
Pcr Assay ◽  
Direct Pcr ◽  
Rapid Polymerase Chain Reaction

The plant pathogen Phytophthora infestans causes a destructive blight of potato tubers and foliage. A rapid polymerase chain reaction (PCR) assay has been developed for detection of P. infestans in potato tubers. In this study, the effect of method of DNA extraction on different propagule types and the minimal number of propagules of P. infestans detectable by PCR were assessed using the PINF and internal transcribed spacer (ITS)5 primers. Sensitivity of the primers for PCR was high, and DNA was detectable at concentrations as low as 10 pg/ml. Zoospores and oospores responded differently to different extraction methods, whereas all extraction methods worked equally well for sporangia. Freeze-thaw DNA lysis, in which propagules were frozen at -80°C and thawed at 65°C three times for 15 min each, or direct PCR, in which propagules were placed directly in the reaction mix, were effective methods for PCR detection of sporangia or zoospores but were not effective methods for PCR detection of DNA in oospores of P. infestans. DNA from a single sporangium or oospore could be amplified by PCR after hexadecyltrimethyl-ammonium bromide (CTAB) or NaOH lysis extraction methods, whereas DNA from a single zoospore could be amplified by CTAB or direct PCR methods. “IsoCode” Stixs, used in forensic applications, were used to collect the pathogen from leaf and tuber lesions and provided another simple method to extract template DNA. PCR detection of the pathogen in infected tubers using PINF and ITS5 primers was compared to tissue isolation or visual observation. The probability of detection of P. infestans in infected tubers at 7 days post inoculation using the PCR assay, tissue isolation, or visual observation was 0.90, 0.80, and 0.75, respectively. The PINF and ITS5 primers provide a powerful tool for rapid and sensitive detection of zoospores, sporangia, and oospores of P. infestans when used with appropriate extraction methods, and could easily be deployed to reduce spread of the pathogen in potato tubers.

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.

RNA extraction method for the PCR detection of hepatitis A virus in shellfish

2010 ◽  
Vol 142 (1-2) ◽  
pp. 198-201 ◽  
Author(s):  
Valentina Terio ◽  
Angela Di Pinto ◽  
Pietro Di Pinto ◽  
Vito Martella ◽  
Giuseppina Tantillo
Keyword(s):  
Extraction Method ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Rna Extraction ◽  
Pcr Detection

Evaluation of the dark field method for large association of spread DNA

1978 ◽  
Vol 36 (2) ◽  
pp. 190-191
Author(s):  
Douglas C. Barker
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Extraction Method ◽  
Field Method ◽  
Dark Field ◽  
Kinetoplast Dna ◽  
Field Electron ◽  
Field Electron Microscopy ◽  
Dark Field Electron

A number of satisfactory methods are available for the electron microscopy of nicleic acids. These methods concentrated on fragments of nuclear, viral and mitochondrial DNA less than 50 megadaltons, on denaturation and heteroduplex mapping (Davies et al 1971) or on the interaction between proteins and DNA (Brack and Delain 1975). Less attention has been paid to the experimental criteria necessary for spreading and visualisation by dark field electron microscopy of large intact issociations of DNA. This communication will report on those criteria in relation to the ultrastructure of the (approx. 1 x 10-14g) DNA component of the kinetoplast from Trypanosomes. An extraction method has been developed to eliminate native endonucleases and nuclear contamination and to isolate the kinetoplast DNA (KDNA) as a compact network of high molecular weight. In collaboration with Dr. Ch. Brack (Basel [nstitute of Immunology), we studied the conditions necessary to prepare this KDNA Tor dark field electron microscopy using the microdrop spreading technique.

High throughput semi-automated extraction method for the creation of a collection from microbial fermentations of fungi and Actinomycetes

Planta Medica ◽  
2008 ◽  
Vol 74 (09) ◽  
Author(s):  
JR Tormo ◽  
N Tabanera ◽  
D Conway ◽  
P Ramos ◽  
A Redondo ◽  
...  
Keyword(s):  
High Throughput ◽  
Extraction Method ◽  
Automated Extraction ◽  
The Creation
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