Testing Designed Polymerase Chain Reaction (PCR) Primers in Multiplex Reactions

2013 ◽  
pp. 89-95
Author(s):  
Kelly M. Elkins
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Polymerase Chain

Detection ofErwinia amylovorain plant material using novel polymerase chain reaction (PCR) primers

2001 ◽  
Vol 29 (1) ◽  
pp. 35-43 ◽  
Author(s):  
R. K. Taylor ◽  
P. J. Guilford ◽  
R. G. Clark ◽  
C. N. Hale ◽  
R. L. S. Forster
Keyword(s):  
Polymerase Chain Reaction ◽  
Plant Material ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Polymerase Chain

Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains

2006 ◽  
Vol 52 (5) ◽  
pp. 451-461 ◽  
Author(s):  
S S Hynes ◽  
O Chaudhry ◽  
M A Providenti ◽  
M L Smith
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Environmental Persistence ◽  
Fungal Strains ◽  
Target Dna ◽  
Polymerase Chain

The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (~14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.Key words: Canada domestic substances list (DSL), Canadian Environmental Protection Act (CEPA), genetically modified organisms (GMO), quantitative polymerase chain reaction (qPCR).

Early Detection of Systemic Rust Infections of Dyers Woad (Isatis tinctoria) Using the Polymerase Chain Reaction

Weed Science ◽  
1995 ◽  
Vol 43 (3) ◽  
pp. 467-472 ◽  
Author(s):  
Bradley R. Kropp ◽  
Steve Albee ◽  
Karen M. Flint ◽  
Paul Zambino ◽  
Les Szabo ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Early Detection ◽  
Room Temperature ◽  
Detection Method ◽  
Rust Fungus ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Isatis Tinctoria ◽  
Specific Polymerase Chain Reaction ◽  
Polymerase Chain

Rust-specific polymerase chain reaction (PCR) primers selectively amplified ribosomal DNA of a rust fungus from infected dyers woad. PCR enabled DNA of the fungus to be detected in symptomatic plants as well as in asymptomatic parts of diseased plants. The use of PCR enabled early detection of rust infections in dyers woad plants during their first season when they are often asymptomatic Dried plant samples stored at room temperature for several months worked as well as lyophilized material for DNA extraction prior to PCR. The PCR detection method should greatly facilitate further studies on the biology and inoculation of this and other systemic rusts that have potential for use in biocontrol of weeds.

scholarly journals A Polymerase Chain Reaction Assay for the Detection of Xanthomonas campestris pv. musacearum in Banana

Plant Disease ◽  
2010 ◽  
Vol 94 (1) ◽  
pp. 109-114 ◽  
Author(s):  
Melanie L. Lewis Ivey ◽  
Geoffrey Tusiime ◽  
Sally A. Miller
Keyword(s):  
Polymerase Chain Reaction ◽  
Xanthomonas Campestris ◽  
Pcr Primers ◽  
Infected Tissue ◽  
Conventional Pcr ◽  
Enterobacterial Repetitive Intergenic Consensus ◽  
Chain Reaction ◽  
Whole Cells ◽  
Polymerase Chain ◽  
Banana Xanthomonas Wilt

Polymerase chain reaction (PCR) primers (BXW-1 and BXW-3) for conventional PCR were developed from conserved sequences in the hrpB operon of the hrp gene cluster from Xanthomonas campestris pv. musacearum, the causative agent of banana Xanthomonas wilt (BXW). All 50 strains of X. campestris pv. musacearum, isolated from Uganda, Rwanda, and Tanzania, produced a 214-bp amplicon when whole cells, bacterial ooze from infected tissue, and genomic DNA purified from bacterial ooze or infected tissue were used as template. The BXW primers also detected strains of X. axonopodis pv. vasculorum isolated from sugarcane and maize and strains of X. vasicola pv. holcicola isolated from sorghum. All of the strains of X. campestris pv. musacearum were clonal when compared using enterobacterial repetitive intergenic consensus PCR.

Application of Nested Polymerase Chain Reaction to Detection of Salmonella in Poultry Environment

2002 ◽  
Vol 65 (8) ◽  
pp. 1227-1232 ◽  
Author(s):  
TONGRUI LIU ◽  
KAREN LILJEBJELKE ◽  
ELIZABETH BARTLETT ◽  
CHARLES HOFACRE ◽  
SUSAN SANCHEZ ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nested Pcr ◽  
Virulence Gene ◽  
Pcr Primers ◽  
Processing Plant ◽  
Chain Reaction ◽  
Chi Square ◽  
Nested Polymerase Chain Reaction ◽  
Secondary Enrichment ◽  
Polymerase Chain

Isolation of Salmonella from environmental and processing-plant poultry samples requires the sampling of large numbers of areas within the poultry house or plant. Subsequently, the required number of samples necessitates a large volume of work for a microbiology laboratory, especially when the protocol requires the inclusion of a delayed secondary enrichment for the isolation of Salmonella. This study examined the use of the polymerase chain reaction (PCR) to identify those secondary enrichments containing Salmonella. The unique Salmonella virulence gene invA was chosen as the target for the development of a nested PCR because of its uniform distribution among Salmonella serotypes. The use of nested PCR primers increased the sensitivity of detection 100-fold, resulting in the detection of as few as four cells. There was a strong, statistically significant positive correlation between PCR and culture results as determined by chi-square (P < 0.001) and kappa (k = 0.915; excellent agreement) tests. Using PCR to screen primary enrichments for presumptive Salmonella contamination, we improved our efficiency at isolating Salmonella upon secondary enrichment by 20%, and no false negatives were observed. This method will not only validate the use of secondary enrichment procedures but also reduce costs and manpower required for the surveillance of Salmonella.

Bacterial Etiology for Chronic Villitis is Not Supported by Polymerase Chain Reaction for 16S rRNA DNA

2005 ◽  
Vol 8 (6) ◽  
pp. 647-653 ◽  
Author(s):  
Linda M. Ernst ◽  
Jill Crouch ◽  
Henry Rinder ◽  
John Greg Howe
Keyword(s):  
Polymerase Chain Reaction ◽  
16S Rrna ◽  
Plasma Cells ◽  
Infectious Agent ◽  
Pcr Primers ◽  
Proteinase K ◽  
Chain Reaction ◽  
Direct Spread ◽  
Polymerase Chain ◽  
Chronic Villitis

Chronic villitis is characterized by chorionic villi infiltrated by lymphocytes, histiocytes, and sometimes plasma cells. In a small percentage of cases, an infectious agent can be demonstrated within areas of chronic villitis. However, the pathogenesis of most lesions is idiopathic. Chronic villitis may represent the direct spread of chronic endometrial infection by bacterial organisms that are particularly problematic for culture. To test this hypothesis, polymerase chain reaction (PCR) using primers for the universal bacterial 16S rRNA DNA was performed on DNA extracted from areas of chronic villitis selected from placentas in the Yale Pathology database. Specific areas of chronic villitis were first confirmed by examination of sections stained with hematoxylin and eosin and then removed from archived paraffin blocks. Control tissue spiked with known bacterial counts was also prepared to test the sensitivity of the experiment. All tissue was deparaffinized, dehydrated, and digested with proteinase K. DNA extraction was performed with the Gentra Puregene kit. PCR was done using primers p11 and p13 for the 16S rRNA DNA. The 233-bp amplified target product was identified by agarose gel electrophoresis. Nineteen specimens with multifocal chronic villitis without confinement to anchoring villi were studied. None of the chronic villitis specimens had a demonstrable product using the PCR primers for 16S rRNA DNA, despite adequate DNA in the samples and controls. The assay was sensitive down to approximately 1500 bacteria per specimen. In conclusion, these data do not support a bacterial etiology for chronic villitis.

Development of a Polymerase Chain Reaction-Based Assay for the Detection of Alternaria Fungal Contamination in Food Products

1999 ◽  
Vol 62 (10) ◽  
pp. 1191-1197 ◽  
Author(s):  
GIDEON ZUR ◽  
ERIC M. HALLERMAN ◽  
RACHEL SHARF ◽  
YECHEZKEL KASHI
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Tomato Sauce ◽  
5.8S Rrna Gene ◽  
Polymerase Chain ◽  
Tomato Products ◽  
Fungal Contaminants ◽  
Tomato Powder

Alternaria sp. are important fungal contaminants of vegetable, fruit, and grain products, including Alternaria alternata, a contaminant of tomato products. To date, the Howard method, based on microscopic observation of fungal filaments, has been the standard examination for inspection of tomato products. We report development of a polymerase chain reaction (PCR)-based method for detection of Alternaria DNA. PCR primers were designed to anneal to the internal transcribed regions ITS1 and ITS2 of the 5.8S rRNA gene of Alternaria but not to other microbial or tomato DNA. We demonstrate use of the PCR assay to detect Alternaria DNA in experimentally infested and commercially obtained tomato sauce and tomato powder. Use of the PCR method offers a rapid and sensitive assay for the presence of Alternaria DNA in tomato products. The apparent breakdown of DNA in tomato sauce may limit the utility of the assay to freshly prepared products. The assay for tomato powder is not affected by storage time.

scholarly journals Detection of Enteroviruses in Shellfish by Fluorogenic Polymerase Chain Reaction Integrated with 96-Well Microplate Scanning

2002 ◽  
Vol 85 (5) ◽  
pp. 1045-1051 ◽  
Author(s):  
Y Carol Shiekh ◽  
Ralph S Baric
Keyword(s):  
Polymerase Chain Reaction ◽  
Detection Efficiency ◽  
Pcr Primers ◽  
Southern Analysis ◽  
Municipal Sewage ◽  
Chain Reaction ◽  
Step Procedure ◽  
Linear Probe ◽  
One Step ◽  
Polymerase Chain

Abstract A one-step procedure was developed to confirm viral targets by using a fluorometric 96-well microplate scanner following polymerase chain reaction (PCR). The fluorogenic PCR, integrated with fluorometric scanning, measured the end point fluorescence of viral PCR amplicon/probe hybrids and permitted the use of nonfluorogenic PCR conditions with addition of a Cy3 fluorophore-labeled linear probe for viruses. This linear probe generated higher ratios of viral signal-to-noise than a comparative beacon probe. Detection efficiency with a Cy3/quencher linear probe was comparable with Southern analysis at the level ≥0.27 plaque-forming units (PFU) of poliovirus/PCR. For the reaction containing <0.27 PFU, the fluorometric measurements of the first-round PCR viral amplicon were not as sensitive as Southern analysis; however, equivalent sensitivities were achieved with fluorogenic nested PCR. Concentrates of 11 oyster samples exposed to municipal sewage were tested for enteroviruses; the fluorogenic detection correlated 100% with Southern analysis. This method using fluorometric scanning of viral amplicon is simple; it requires neither continuously monitoring equipment nor redesigning PCR primers; and it accurately detects enteroviruses in oyster sample concentrates in less time than classic spectrophotometry or Southern analysis.

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