Rapid and specific detection of Enterococcus faecium with an isothermal amplification and lateral flow strip combined method
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Enterococcus faecium
Recombinase Polymerase Amplification
Objective To study and apply a rapid and simple screening method of Enterococcus.Methods A total of 139 strains of the gram-positive coccus were investigated for esculin hydrolysis by three methods of esculin rapid hydrolysis kit,conventional methods and API 20 Strep system(bioMerieux).Results The positive rate of fluorescence loss was 99.26% in Enterococcus, 100% in Enterococcus faecalis,and 91.66% in Enterococcus faecium.The positive rate of forming light gray or black spots was 99.26%,of which Enterococcus faecalis was 100%,Enterococcus faecium for 91.66 %.Conclusion Through the methods of esculin hydrolysis,Gram's method,microscopic morphology,catalase test and colony characteristics on selective and differential medium,it can be rapidly and simply identified for Enterococcus at the genus level.
Enterococcus faecium
Enterococcus faecalis
Gram
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A number of isothermal DNA amplification technologies claim to be ideal for point-of-need (PON) applications as they enable reactions to be performed using a single-temperature heat source (e.g. water bath). Thus, we examined several isothermal amplification methods focusing on simplicity, cost, sensitivity and reproducibility to identify the most suitable method(s) for low resource PON applications. A number of methods were found unsuitable as they either involved multiple temperature incubations, were relatively expensive or required relatively large amounts target DNA for amplification. Among the methods examined, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) were found to be the most suitable for PON applications as they are both single step methods that provide highly sensitive and reproducible amplifications. The speed of LAMP reactions was greatly enhanced, up to 76%, with the addition of loop primers while the presence of swarm primers and the sequestration of free magnesium ions with nucleotides also enhanced the amplification speed. In contrast, we were unable to enhance RPA's performance from the original published literature. While both RPA and LAMP have some drawbacks, either isothermal technology can reliably be used for on-site diagnostics with minimal equipment.
Recombinase Polymerase Amplification
Isothermal process
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Recombinase Polymerase Amplification
Isothermal process
NASBA
Primer (cosmetics)
Applications of PCR
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[Objective]To establish a duplex PCR assay for simultaneous and quick detection of sheep-originated clinical isolates of Enterococcus and Enterococcus faecium. [Methods]The genus specific and species specific primers for Enterococcus and Enterococcus faecium targeting at 16 S r DNA and ddl gene were designed respectively, and the reaction system of duplex PCR assay was determined. The specificity and sensitivity of the established duplex PCR assay were evaluated. The simulated bacterial infection blood samples were prepared by using healthy sheep blood and mixed bacterial solution and were used to verify the specificity and sensitivity of the established duplex PCR assay in detection of clinical samples. [Results]The results showed that the duplex PCR assay was able to specifically amplify the DNA fragments of 294 bp from Enterococcus and 557 bp from Enterococcus faecium,but was negative for other common animal-originated pathogenic bacteria. The detection limits of the assay for Enterococcus and Enterococcus faecium were 5.71×10-5ng/μL and 5.71×10-4ng/μL, respectively. The duplex PCR assay was able to specifically detect the Enterococcus and Enterococcus faecium in simulated bacterial infection blood samples, and the detection limit for Enterococcus faecium was 1×101CFU/m L. [Conclusion]It was indicated that the established duplex PCR assay is specific and sensitive for detection of clinical Enterococcus and Enterococcus faecium isolates.
Enterococcus faecium
Duplex (building)
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We developed a combination of recombinase polymerase and loop-mediated isothermal amplification methods (RAMP) for rapid screening of five human Plasmodium spp. simultaneously. RAMP is a two-stage isothermal amplification method, which consists of a first-stage recombinase polymerase amplification and a second-stage loop-mediated isothermal amplification. Under these two isothermal conditions, five Plasmodium spp. were amplified in less than 40 minutes. We demonstrated RAMP assay with 10-fold better limit of detection than a single (loop-mediated isothermal amplification) LAMP. As compared with microscopy, RAMP assay showed 100% sensitivity (95% CI: 95.65-100.00%) and 100% specificity (95% CI: 69.15-100.00%). The end products were inspected by the color changes of neutral red. Positive reactions were indicated by pink while the negative reactions remained yellow. The combination assay established in this study can be used as a routine diagnostic method for malaria.
Recombinase Polymerase Amplification
Isothermal process
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Linezolid
Enterococcus faecium
Gram-positive bacterial infections
Vancomycin-resistant Enterococcus
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Vancomycin-resistant enterococci (VRE), especially Enterococcus faecium , have been a global concern, often causing serious healthcare-associated infections. We established a rapid approach for detecting E. faecium and vancomycin-resistance genes ( vanA and vanB ) in clinical samples using isothermal recombinase polymerase amplification (RPA) combined with a lateral-flow (LF) strip. Specific RPA primer sets and probes for ddl (to identify the presence of E. faecium ) vanA and vanB genes were designed. The RPA reaction was performed under isothermal condition at 37 °C within 20 min and read using the LF strip within a further 5 min. A total of 141 positive blood-cultures and 136 stool/rectal swab samples were tested using RPA-LF method compared to the conventional PCR method. The RPA-LF method exhibited 100% sensitivity in both blood-culture (60 E. faecium ; 35 vanA type and two vanB type) and stool/rectal-swab samples (63 E. faecium and 36 vanA type) without cross-reaction (100% specificity). The lower detection limit of the RPA-LF was approximately 10 times better than that of the conventional PCR method. The RPA-LF method is an alternative rapid method with excellent sensitivity and specificity for detecting E. faecium , vanA , and vanB , and it has the potential to be used as a point-of-care device for VRE therapy and prevention.
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У сучасних умовах розвитку промислового птахівництва, розробленню нових біологічних профілактич-них препаратів для корекції мікробіоценозу та підвищення неспецифічного імунітету птиці надаєтьсяпріоритетне значення. В матеріалах статті представлені результатами досліджень антагоністичнихвластивостей 5 ізолятів Enterococcus faecium, виділених із сліпих відростків товстого кишечника з вмістом,відібраного від курей та курчат-бройлерів із птахогосподарств України за проведення активногомоніторингу згідно державної програми з Порядку проведення нагляду (активного моніторингу) запротимікробною резистентністю зоонозних та коменсальних бактерій у ветеринарній медицині таідентифікованих до виду. Дослідження були виконані двома дифузійними методами – відтермінованогоантагонізму та агарових блоків з метою підтвердження достовірності одержаних результатів. Встановленоу 2 штамів Enterococcus faecium – Efm-3 і Efm-5 дуже високий та високий рівні антагонізму після взаємодіїштамів з грамнегативними і грампозитивними тестовими бактеріями E. coli АТСС 25922 (діаметри зонінгібування росту 39,4±1,3/36,4±0,3 і 42,2±2,7/39,4±2,7 мм відповідно до штамів і методів), P. aeruginosaАТСС 15442 (29,0±1,3/28,4±0,7 і 29,2±1,3/39,4±2,7), S. typhimurium АТСС 29630 (17,2±0,7/17,2±1,7 і28,2±0,7/28,6±0,7) і S. aureus АТСС 6538 (28,2±0,07/28,4±0,7 і 35,2±0,7/34,2±0,17), які знаходяться вдіапазоні значень високого антагонізму. Штами Efm-3 і E fm-5 Enterococcus faecium відібрано в якостіперспективних пробіотичних мікроорганізмів, завдячуючи авірулентним властивостям та показникамвисокого рівня антагоністичної активності. Одержані дані засвідчують можливість корекції імунноївідповіді за допомогою розроблених комплексних пробіотичних препаратів у складі з відібранимиперспективними штамами Enterococcus faecium, направлених на нормалізацію мікрофлори кишечника,посилення імунних функцій організму, підвищення показників збереженості птиці та якості одержаноїпродукції.
Enterococcus faecium
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Loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) are two rapid isothermal amplification methods for detecting three common fungal root pathogens of cool-season turfgrass: Gaeumannomyces avenae, Magnaporthiopsis poae and Ophiosphaerella korrae, "Detection of root-infecting fungi on cool-season turfgrasses using loop-mediated isothermal amplification and recombinase polymerase amplification" (Karakkat et al., 2018) [1]. The data provided here describe the information for designing primers and probes for LAMP and RPA, how specific they are for each of the three fungi, and the evaluation of RPA on field samples.
Recombinase Polymerase Amplification
Isothermal process
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Enterococcus faecium has recently emerged as a serious nosocomial pathogen. The prevalence and severity of enterococcal infections, the mortality rate from such infections, and the antibiotic resistance of enterococci are often species dependent. Since conventional biochemical methods fail to differentiate E. faecium from certain newly described enterococcal species, a PCR-based assay was developed for the rapid identification of E. faecium.
Enterococcus faecium
Gram-positive bacterial infections
Identification
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