Rapid Cycle DNA Amplification

Wittwer, Carl T.; Reed, Gudrun B.; Ririe, Kirk M.

doi:10.1007/978-1-4612-0257-8_15

Cited by 50 publications

(39 citation statements)

References 14 publications

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“…), 50 mM Tris-HCl (pH 8.3), 250 g of bovine serum albumin/ml, 2% sucrose, and 0.1 mM cresol red (Idaho Technologies, Salt Lake City, Utah). The PCR was done using the rapid-cycle DNA amplification method (28) and consisted of 30 cycles of template denaturation at 94°C, primer annealing at 54°C, and primer extension at 74°C for 30 s. All reactions were performed using an Idaho Technologies Rapid Cycler brand thermal cycler. Amplified products were electrophoresed in 1% agarose gels at 200 V for 30 s and visualized under UV light.…”

Section: Methodsmentioning

confidence: 99%

Antimicrobial Resistance of Escherichia coli O157 Isolated from Humans, Cattle, Swine, and Food

Schroeder

Zhao

DebRoy

et al. 2002

Appl Environ Microbiol

278

189

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A total of 361 Escherichia coli O157 isolates, recovered from humans, cattle, swine, and food during the years 1985 to 2000, were examined to better understand the prevalence of antimicrobial resistance among these organisms. Based on broth microdilution results, 220 (61%) of the isolates were susceptible to all 13 antimicrobials tested. Ninety-nine (27%) of the isolates, however, were resistant to tetracycline, 93 (26%) were resistant to sulfamethoxazole, 61 (17%) were resistant to cephalothin, and 48 (13%) were resistant to ampicillin. Highest frequencies of resistance occurred among swine isolates (n ‫؍‬ 70), where 52 (74%) were resistant to sulfamethoxazole, 50 (71%) were resistant to tetracycline, 38 (54%) were resistant to cephalothin, and 17 (24%) were resistant to ampicillin. Based on the presence of Shiga toxin genes as determined by PCR, 210 (58%) of the isolates were identified as Shiga toxin-producing E. coli (STEC). Among these, resistance was generally low, yet 21 (10%) were resistant to sulfamethoxazole and 19 (9%) were resistant to tetracycline. Based on latex agglutination, 189 (52%) of the isolates were identified as E. coli O157:H7, among which 19 (10%) were resistant to sulfamethoxazole and 16 (8%) were resistant to tetracycline. The data suggest that selection pressure imposed by the use of tetracycline derivatives, sulfa drugs, cephalosporins, and penicillins, whether therapeutically in human and veterinary medicine or as prophylaxis in the animal production environment, is a key driving force in the selection of antimicrobial resistance in STEC and non-STEC O157.

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Section: Methodsmentioning

confidence: 99%

Antimicrobial Resistance of Escherichia coli O157 Isolated from Humans, Cattle, Swine, and Food

Schroeder

Zhao

DebRoy

et al. 2002

Appl Environ Microbiol

278

189

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“…For the singleplex (wzx or wzy) PCR assays, each of the primer sets shown in Table 2 was used separately in the PCRs, and reaction mix contents for each PCR (11-l total reaction mix volume) consisted of 3 l of template DNA, 0.5 M of primers (Integrated DNA Technologies Inc., Coralville, IA), 0.18 mM concentration of each of the four deoxynucleoside triphosphates, 2 mM MgCl 2 (for the O55 wzx, O45 wzx, and O45 wzy PCR assays) and 3 mM MgCl 2 (for the O55 wzy PCR assays), 0.4 U of Taq DNA polymerase (PGC Scientific, Gaithersburg, MD), 50 mM Tris (pH 8.3), 250 g/ml bovine serum albumin, 2% sucrose, and 0.1 mM cresol red. The PCR was performed in a RapidCycler (Idaho Technologies Inc., Salt Lake City, UT) by using a rapidcycle DNA amplification method (24) and consisted of 30 cycles of template denaturation at 94°C, primer annealing and primer extension at temperatures and times indicated in Table 3. The amplification products were subjected to electrophoresis in 1% agarose gels at 200 V for 1 h for all assays except for the O55 wzy PCR that was analyzed using a 2% gel.…”

mentioning

confidence: 99%

Development of PCR Assays Targeting Genes in O-Antigen Gene Clusters for Detection and Identification of Escherichia coli O45 and O55 Serogroups

DebRoy

Fratamico

Roberts

et al. 2005

Appl Environ Microbiol

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The Escherichia coli O45 O-antigen gene cluster of strain O45:H2 96-3285 was sequenced, and conventional (singleplex), multiplex, and real-time PCR assays were designed to amplify regions in the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes. In addition, PCR assays targeting the E. coli O55 wzx and wzy genes were designed based on previously published sequences. PCR assays targeting E. coli O45 showed 100% specificity for this serogroup, whereas by PCR assays specific for E. coli O55, 97/102 strains serotyped as E. coli O55 were positive for wzx and 98/102 for wzy. Multiplex PCR assays targeting the E. coli O45 and the E. coli O55 wzx and wzy genes were used to detect the organisms in fecal samples spiked at levels of 10 6 and 10 8 CFU/0.2 g feces. Thus, the PCR assays can be used to detect and identify E. coli serogroups O45 and O55.Strains of Escherichia coli belonging to serogroup O45 have been isolated from animals and humans and classified as both enterotoxigenic E. coli and Shiga toxin-producing enterohemorrhagic E. coli (4,16,25). In addition, E. coli O45 strains isolated from diarrheic dairy calves produced cytotoxic necrotizing factor and were designated as strains of necrotoxigenic E. coli (10). Many E. coli O45 strains isolated from swine have been associated with postweaning diarrhea and demonstrated the attaching and effacing (A/E) phenotype, allowing them to adhere to intestinal epithelial cells (3). Six E. coli serogroups, including O45, were associated with feral pigeons and produced a variant of Shiga toxin 2 called Stx2f (9).E. coli strains belonging to serogroup O55 have been classified as human enteropathogenic strains and are believed to be genetically related to enterohemorrhagic E. coli O157:H7, shown to have been derived from an O55:H7 ancestral clone (15,23). Because of the potential pathogenicity of E. coli isolates belonging to serogroups O45 and O55, rapid and reliable assays for detecting and identifying these serogroups in food, environmental, and clinical samples are needed.Conventionally, antigenic analysis of the ca. 179 different O serogroups in E. coli is performed by agglutination reactions using antisera raised in rabbits against the O standard reference strains. O serotyping is laborious, and cross-reactions between different serogroups often occur, giving equivocal results. Furthermore, E. coli strains occasionally undergo transition from smooth to rough forms as a result of mutations in one or more of the multiple genes controlling synthesis and polymerization of the O antigen. Rough isolates do not produce an O antigen and therefore cannot be typed by using antisera. In view of these facts, there is a need for the development of alternative methods for identifying and typing E. coli serogroups. Lipopolysaccharides (LPS) are essential components of the gram-negative bacterial outer membrane. They are composed of three parts: lipid A, which is composed of sugars and fatty acids and anchors the LPS in the outer membrane through covalent linkages; an oligosacc...

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“…The controlled thermal cycling of the sample is certainly an inconvenient feature to integrate into disposable elements without electronic interfacing, but this device uses a long serpentine with variable channel width (Wittwer et al, 1994) heated to create a constant transversal temperature gradient. The design is not only an attractive simplification of the heating problem, but also the reaction spreads spatially instead of over time, and by imaging the device (Pjescic et al, 2010), the cycle-dependent fluorescence and the temperature-dependent fluorescence can be simultaneously captured.…”

Section: Sample Preparationmentioning

confidence: 99%

Towards autonomous lab-on-a-chip devices for cell phone biosensing

Comina

Suska

Filippini

2016

Biosensors and Bioelectronics

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Modern cell phones are a ubiquitous resource with a residual capacity to accommodate chemical sensing and biosensing capabilities. From the different approaches explored to capitalize on such resource, the use of autonomous disposable lab-on-a-chip (LOC) devices conceived as only accessories to complement cell phones underscores the possibility to entirely retain cell phones ubiquity for distributed biosensing. The technology and principles exploited for autonomous LOC devices are here selected and reviewed focusing on their potential to serve cell phone readout configurations. Together with this requirement, the central aspects of cell phones resources that determine their potential for analytical detection are examined. The conversion of these LOC concepts into universal architectures that are readable on unaccessorized phones is discussed within this context. (C) 2015 Elsevier B.V. All rights reserved.

Funding Agencies|Swedish Research Council (VR) [C0453801]; Carl Tryggers Foundation [CTS14140]

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Rapid Cycle DNA Amplification

Cited by 50 publications

References 14 publications

Antimicrobial Resistance of Escherichia coli O157 Isolated from Humans, Cattle, Swine, and Food

Antimicrobial Resistance of Escherichia coli O157 Isolated from Humans, Cattle, Swine, and Food

Development of PCR Assays Targeting Genes in O-Antigen Gene Clusters for Detection and Identification of Escherichia coli O45 and O55 Serogroups

Towards autonomous lab-on-a-chip devices for cell phone biosensing

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