Last data update: Sep 16, 2024. (Total: 47680 publications since 2009)
Records 1-2 (of 2 Records) |
Query Trace: Cooper KL [original query] |
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Standardization and international multicenter validation of a PulseNet pulsed-field gel electrophoresis protocol for subtyping Shigella flexneri isolates
Pichel M , Brengi SP , Cooper KL , Ribot EM , Al-Busaidy S , Araya P , Fernandez J , Vaz TI , Kam KM , Morcos M , Nielsen EM , Nadon C , Pimentel G , Perez-Gutierrez E , Gerner-Smidt P , Binsztein N . Foodborne Pathog Dis 2012 9 (5) 418-24 Shigella flexneri is one of the agents most frequently linked to diarrheal illness in developing countries and often causes outbreaks in settings with poor hygiene or sanitary conditions. Travel is one of the means by which S. flexneri can be imported into developed countries, where this pathogen is not commonly seen. A robust and discriminatory subtyping method is needed for the surveillance of S. flexneri locally and regionally, and to aid in the detection and investigation of outbreaks. The PulseNet International network utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols to carry out laboratory-based surveillance of foodborne pathogens in combination with epidemiologic data. A multicenter validation was carried out in nine PulseNet laboratories located in North and South America, Europe, and Asia, and it demonstrated that a new protocol is highly robust and reproducible for subtyping of S. flexneri. This protocol, already approved for PulseNet laboratories, applies NotI and XbaI as primary and secondary restriction enzymes, respectively, under electrophoresis conditions of initial switch time of 5 s to final switch time of 35 s, at 6 volts/cm. |
Re-evaluation, optimization, and multilaboratory validation of the PulseNet-standardized pulsed-field gel electrophoresis protocol for listeria monocytogenes
Halpin JL , Garrett NM , Ribot EM , Graves LM , Cooper KL . Foodborne Pathog Dis 2009 7 (3) 293-8 The PulseNet Methods Development and Validation Laboratory began a re-evaluation of the standardized pulsed-field gel electrophoresis (PFGE) protocols with the goal of optimizing their overall performance and robustness. Herein, we describe a stepwise evaluation of the PulseNet-standardized PFGE protocol for Listeria monocytogenes that led to the modification of several steps which significantly improved the overall appearance and reproducibility of the resulting PFGE data. These improvements included the following: (1) reducing the cell suspension concentration, (2) increasing lysozyme incubation temperature from 37 degrees C to 56 degrees C, and (3) decreasing the number of units of restriction enzymes AscI and ApaI. These changes were incorporated into a proposed protocol that was evaluated by 16 PulseNet participating laboratories, including 2 international participants. Results from the validation study indicated that the updated L. monocytogenes protocol is more robust than the original PulseNet-standardized protocol established in 1998 and this resulted in the official adoption of the new protocol into the PulseNet system in the spring of 2008. The modifications not only represent an improvement to the protocol but also describe procedural improvements that could be potentially applied to the PFGE analysis of other Gram-positive organisms. |
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