scholarly journals Innovation of Indoor Real-time Polymerase Chain Reaction for Diagnosis of Camel Pox Virus in Clinical Field Samples using Primer Site Belongs to Capripoxvirus

2011 ◽  
Vol 7 (4) ◽  
pp. 147-157 ◽  
Author(s):  
S.S.A. Sharawi ◽  
A.N. Al-Hofufy ◽  
M.H. Al-Blowi
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Chain Reaction ◽  
Field Samples ◽  
Polymerase Chain ◽  
Camel Pox Virus

scholarly journals Evaluation of Polymerase Chain Reaction (PCR)-Based Methods for Rapid, Accurate Detection and Monitoring of Verticillium dahliae in Woody Hosts by Real-Time PCR

Plant Disease ◽  
2015 ◽  
Vol 99 (6) ◽  
pp. 866-873 ◽  
Author(s):  
Baker Aljawasim ◽  
Paul Vincelli
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Verticillium Dahliae ◽  
Sugar Maple ◽  
Broad Host Range ◽  
Chain Reaction ◽  
Accurate Detection ◽  
Field Samples ◽  
Vascular Discoloration ◽  
Polymerase Chain

Verticillium wilt, caused by Verticillium dahliae, is one of the most economically important diseases of woody hosts such as ash (Fraxinus spp.), sugar maple (Acer saccharum), and redbud (Cercis canadensis). The causal agent has a broad host range, including not only woody hosts but also important vegetable and field crops, and it is distributed worldwide. Diagnosis of V. dahliae in infected woody hosts is often based on the occurrence of vascular discoloration and time-consuming isolation. However, not all woody hosts exhibit vascular discoloration, and not all vascular discoloration symptoms are due to infection by V. dahliae. In this study, real-time polymerase chain reaction (PCR)-based assays were evaluated and employed for rapid and accurate detection of V. dahliae in different woody hosts. High-quality DNA was extracted in large quantities from presumptively infected woody hosts by collecting drill-press shavings from sample tissue, bead beating, and extracting using a cetyltrimethylammonium bromide method. Six published primer sets were evaluated against genomic DNA of V. dahliae as well as selected negative controls, and two sets (VertBt-F/VertBt-R and VDS1/VDS2) showed promise for further evaluation using DNA extracts from field samples. The VertBt primers amplified a species-specific 115-bp fragment of the expected size, while the VDS primers amplified the expected specific 540-bp fragment. However, the VertBt primer set exhibited higher sensitivity in detection of V. dahliae even in asymptomatic trees. The PCR-based methods developed here could be used as rapid tools for pathogen detection and monitoring, thus informing plant pathogen management decisions.

scholarly journals Real-time fluorogenic reverse transcription polymerase chain reaction assay for the specific detection of Bagaza virus

2012 ◽  
Vol 24 (5) ◽  
pp. 959-963 ◽  
Author(s):  
Dolores Buitrago ◽  
Ana Rocha ◽  
Cristina Tena-Tomás ◽  
Marta Vigo ◽  
Montserrat Agüero ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Diagnostic Methods ◽  
Nucleotide Sequencing ◽  
Alectoris Rufa ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Field Samples ◽  
Polymerase Chain

In September 2010, an outbreak of disease in 2 wild bird species (red-legged partridge, Alectoris rufa; ring-necked pheasant, Phasianus colchicus) occurred in southern Spain. Bagaza virus (BAGV) was identified as the etiological agent of the outbreak. BAGV had only been reported before in Western Africa (Central African Republic, Senegal) and in India. The first occurrence of BAGV in Spain stimulated a demand for rapid, reliable, and efficacious diagnostic methods to facilitate the surveillance of this disease in the field. This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) method based on a commercial 5’-Taq nuclease-3’ minor groove binder DNA probe and primers targeting the Bagaza NS5 gene. The method allowed the detection of BAGV with a high sensitivity, whereas other closely related flaviviruses ( Usutu virus, West Nile virus, and Japanese encephalitis virus) were not detected. The assay was evaluated using field samples of red-legged partridges dead during the outbreak ( n = 11), as well as samples collected from partridges during surveillance programs ( n = 81). The results were compared to those obtained with a pan-flaviviral hemi-nested RT-PCR followed by nucleotide sequencing, which was employed originally to identify the virus involved in the outbreak. The results obtained with both techniques were 100% matching, indicating that the newly developed real-time RT-PCR is a valid technique for BAGV genome detection, useful in both diagnosis and surveillance studies.

scholarly journals Polymerase Chain Reaction-Based Detection of Spiroplasma citri Associated with Citrus Stubborn Disease

Plant Disease ◽  
2008 ◽  
Vol 92 (2) ◽  
pp. 253-260 ◽  
Author(s):  
Raymond K. Yokomi ◽  
Alexandre F. S. Mello ◽  
Maria Saponari ◽  
Jacqueline Fletcher
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Disease Incidence ◽  
Chain Reaction ◽  
Spiroplasma Citri ◽  
Field Samples ◽  
Polymerase Chain ◽  
Individual Trees ◽  
Citrus Stubborn Disease

Polymerase chain reaction (PCR)-based detection of citrus stubborn disease was improved using primers based on sequences of the P89 putative adhesin gene and the P58 putative adhesin multigene of Spiroplasma citri. Real-time PCR also was developed with detection limits estimated to be between 10–4 and 10–4 ng by serial dilution of a recombinant S. citri plasmid into DNA extracts from healthy Madam Vinous sweet orange. PCR for the detection of S. citri by these new primers was validated by comparing culturing of the pathogen, the traditional method of diagnosis, with PCR assays from samples taken from two citrus plots in Kern County, CA. Fruit columella was collected from 384 and 377 individual trees in each of two fields, respectively; one portion was used for culturing and the other for DNA extraction and PCR. PCR results matched those of culturing 85 to 100% of the time depending on the primers used. More importantly, PCR detected S. citri from culture-negative trees in 5 to 15% of the cases, suggesting that PCR performed as well or better than culturing for detection of S. citri in field samples. Real-time PCR proved to be the best method for detection. Differential reaction of the samples to the P58 primer pairs suggested that two populations of S. citri occur in historical and present-day field isolates. Citrus stubborn disease incidence was estimated to be 58.3 and 3.7% in the two orchards. The results presented here support the use of PCR for reliable detection of S. citri in field trees.

scholarly journals Detection of the Pinewood Nematode, Bursaphelenchus xylophilus, Using a Real-Time Polymerase Chain Reaction Assay

2005 ◽  
Vol 95 (5) ◽  
pp. 566-571 ◽  
Author(s):  
A. X. Cao ◽  
X. Z. Liu ◽  
S. F. Zhu ◽  
B. S. Lu
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Bursaphelenchus Xylophilus ◽  
Pinewood Nematode ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Internal Transcribed Spacer Region ◽  
Significant Damage ◽  
Field Samples ◽  
Polymerase Chain

The pinewood nematode, Bursaphelenchus xylophilus, has caused significant damage to pine plantations both in East Asia and North America and is an important quarantine organism. A real-time polymerase chain reaction (PCR) assay was developed to detect B. xylophilus. A set of primers and probe specific for B. xylophilus was designed to target the ribosomal DNA internal transcribed spacer region. Optimal primer concentration, Mg2+ concentration, and extension temperature were 400 nM, 3.0 mM, and 60°C, respectively. The assay was highly specific and sensitive, detecting as little as 0.01 ng of B. xylophilus DNA. The real-time PCR assay also successfully detected B. xylophilus in field samples, and it should be very useful for quarantine purposes.

scholarly journals Improved Sampling Methods for Real-Time Polymerase Chain Reaction Diagnosis of Citrus Canker from Field Samples

2004 ◽  
Vol 94 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Vessela Mavrodieva ◽  
Laurene Levy ◽  
Dean W. Gabriel
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Sampling Methods ◽  
Target Cells ◽  
Bacterial Canker ◽  
Chain Reaction ◽  
Specificity And Sensitivity ◽  
Field Samples ◽  
Citrus Bacterial Canker ◽  
Polymerase Chain

Citrus bacterial canker disease has been introduced at least three times into Florida in the last 15 years and, despite federal and state quarantine and eradication efforts, continues to spread in Florida. Accurate, fast, and reliable detection of the causal agent is of great importance. However, citrus bacterial canker is caused by at least two groups of phylogenetically distinct Xanthomonas citri strains, and there is host range variation within both groups. We developed a fast, sensitive and reliable real-time polymerase chain reaction (PCR) assay using a portable, field-hardened RAPID machine and primers designed to detect all canker-causing strains. Single-lesion sampling methods were developed that required minimal handling and allowed complete real-time PCR diagnosis in a total time of 4 h and with an apparent sensitivity of less than 10 CFU of target cells from diseased lesions. This sensitivity allowed molecular detection for the first time of X. citri in a herbarium sample from a 1912 canker outbreak. Sensitivity was improved significantly by the use of CaCO3 and Silwet L-77, and by either minimizing the amount of citrus lesion tissue sampled or by soaking or swiping but not grinding the lesions. Primer design also was of significant importance in both specificity and sensitivity.

scholarly journals Real-Time Fluorescent Polymerase Chain Reaction Detection of Phytophthora ramorum and Phytophthora pseudosyringae Using Mitochondrial Gene Regions

2006 ◽  
Vol 96 (4) ◽  
pp. 336-345 ◽  
Author(s):  
Paul W. Tooley ◽  
Frank N. Martin ◽  
Marie M. Carras ◽  
Reid D. Frederick
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Multiplex Assay ◽  
Phytophthora Ramorum ◽  
Chain Reaction ◽  
Plant Dna ◽  
Field Samples ◽  
Polymerase Chain ◽  
Fluorescent Polymerase Chain Reaction

A real-time fluorescent polymerase chain reaction (PCR) detection method for the sudden oak death pathogen Phytophthora ramorum was developed based on mitochondrial DNA sequence with an ABI Prism 7700 (TaqMan) Sequence Detection System. Primers and probes were also developed for detecting P. pseudosyringae, a newly described species that causes symptoms similar to P. ramorum on certain hosts. The species-specific primer-probe systems were combined in a multiplex assay with a plant primer-probe system to allow plant DNA present in extracted samples to serve as a positive control in each reaction. The lower limit of detection of P. ramorum DNA was 1 fg of genomic DNA, lower than for many other described PCR procedures for detecting Phytophthora species. The assay was also used in a three-way multiplex format to simultaneously detect P. ramorum, P. pseudosyringae, and plant DNA in a single tube. P. ramorum was detected down to a 10-5 dilution of extracted tissue of artificially infected rhododendron ‘Cunningham's White’, and the amount of pathogen DNA present in the infected tissue was estimated using a standard curve. The multiplex assay was also used to detect P. ramorum in infected California field samples from several hosts determined to contain the pathogen by other methods. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. ramorum detection to confirm positive P. ramorum finds in nurseries and elsewhere.

1504: Molecular Diagnosis and Staging of Prostate Cancer Using Clusterin in Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

2006 ◽  
Vol 175 (4S) ◽  
pp. 485-486
Author(s):  
Sabarinath B. Nair ◽  
Christodoulos Pipinikas ◽  
Roger Kirby ◽  
Nick Carter ◽  
Christiane Fenske
Keyword(s):  
Prostate Cancer ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Molecular Diagnosis ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain
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