The Journal of antimicrobial chemotherapy · 2012
Real-time PCR for detection of blaOXA-48 genes from stools.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод пока не подготовлен
Обработка статьи · этапы, ошибки и управление
Загрузка состояния…
Ошибки и ограничения
История · было → стало
Состояние обновляется каждые 5 секунд, пока блок открыт. Процент показывается только там, где известен объём работы. Пауза останавливает продолжение этапа; текущий запрос к источнику может завершиться.
Naas Thierry, Cotellon Garance, Ergani Ayla, Nordmann Patrice
Аннотация
OBJECTIVES: Outbreaks of OXA-48-like carbapenemase producers are increasingly reported in many European countries and are often the result of difficulties in detection, especially for isolates with MICs of carbapenems that remain in the susceptibility range.
METHODS: An in-house real-time quantitative PCR (qPCR) assay using TaqMan chemistry to detect bla(OXA-48-like) genes was compared with bacterial culturing on ChromID ESBL and SUPERCARBA media of spiked stool samples with several species producing OXA-48 variants.
RESULTS: qPCR amplification using plasmid DNA was linear over 10 log dilutions (r(2) = 0.998 and slope = -3.14), with an amplification efficiency of 1.10, and the detection limit of the assay was reproducibly estimated at 10 plasmid molecules/PCR. No cross-reaction was detected with DNA extracted from several multidrug-resistant bacteria harbouring other β-lactam resistance genes. The bla(OXA-48) qPCR assay was capable of detecting 10-50 cfu of OXA-48 producers/100 mg of faeces. ChromID ESBL was capable of detecting OXA-48 producers (1 × 10(1) to 3 × 10(2) cfu/100 mg of faeces), as long as the isolates exhibited a high level of resistance to cephalosporins due to an associated extended-spectrum β-lactamase. The SUPERCARBA screening medium was capable of detecting all the OXA-48-like producers (1-3 × 10(1) cfu/100 mg of faeces), except those producing OXA-163, a variant lacking carbapenem-hydrolysing activity.
CONCLUSIONS: The qPCR is likely to shorten the time for bla(OXA-48) detection from 48 to 4 h and will be a valuable tool for outbreak follow-up in order to rapidly isolate colonized patients and assign them to cohorts.
METHODS: An in-house real-time quantitative PCR (qPCR) assay using TaqMan chemistry to detect bla(OXA-48-like) genes was compared with bacterial culturing on ChromID ESBL and SUPERCARBA media of spiked stool samples with several species producing OXA-48 variants.
RESULTS: qPCR amplification using plasmid DNA was linear over 10 log dilutions (r(2) = 0.998 and slope = -3.14), with an amplification efficiency of 1.10, and the detection limit of the assay was reproducibly estimated at 10 plasmid molecules/PCR. No cross-reaction was detected with DNA extracted from several multidrug-resistant bacteria harbouring other β-lactam resistance genes. The bla(OXA-48) qPCR assay was capable of detecting 10-50 cfu of OXA-48 producers/100 mg of faeces. ChromID ESBL was capable of detecting OXA-48 producers (1 × 10(1) to 3 × 10(2) cfu/100 mg of faeces), as long as the isolates exhibited a high level of resistance to cephalosporins due to an associated extended-spectrum β-lactamase. The SUPERCARBA screening medium was capable of detecting all the OXA-48-like producers (1-3 × 10(1) cfu/100 mg of faeces), except those producing OXA-163, a variant lacking carbapenem-hydrolysing activity.
CONCLUSIONS: The qPCR is likely to shorten the time for bla(OXA-48) detection from 48 to 4 h and will be a valuable tool for outbreak follow-up in order to rapidly isolate colonized patients and assign them to cohorts.