Development of a new multiplex real-time PCR assay for rapid screening of hospital-acquired infection agents
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info:eu-repo/semantics/embargoedAccessTarih
2023Yazar
İstanbullu Tosun, AyşeKolukırık, Mustafa
Yılmaz, Mesut
Nar Ötgün, Selin
Aygün, Gökhan
Ketre Kolukırık, Canan Zöhre
Zeybek, Ümit
Girgin Özgümüş, Gözde
Turan, Meral
Kuşkucu, Mert
İnce, Orhan
İnce, Bahar
Kılıç, Selçuk
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İstanbullu Tosun, A., Kolukırık, M., Yılmaz, M., Nar Ötgün, S., Aygün, G., Ketre Kolukırık, C. Z. ... Kılıç, S. (2023). Development of a new multiplex real-time PCR assay for rapid screening of hospital-acquired infection agents. Journal of Microbiological Methods, 206. https://dx.doi.org/10.1016/j.mimet.2023.106690Özet
Aims: A new multiplex real-time PCR (qPCR) assay was developed to detect antibiotic-resistant hospital-acquired infectious agents in nasal and rectal swab samples in 1.5 h without the need for nucleic acid extraction. Methods: Spiked negative clinical specimens were used for the analytical performance evaluation. Double-blind samples were collected from 1788 patients to assess the relative clinical performance of the qPCR assay to the conventional culture-based methods. Bio-Speedy® Fast Lysis Buffer (FLB) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Istanbul, Turkey) and LightCycler® 96 Instrument (Roche Inc., Branchburg, NJ, USA) were used for all molecular analyses. The samples were transferred into 400 L FLB, homogenized and immediately used in qPCRs. The target DNA regions are vanA and vanB genes for vancomycin-resistant Enterococcus (VRE); blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-23, blaOXA-48, blaOXA-58 genes for carbapenem-resistant Enterobacteriaceae (CRE); and mecA, mecC and spa for methicillin-resistant Staphylococcus aureus (MRSA). Results: No qPCR tests produced positive results for the samples spiked with the potential cross-reacting organisms. The limit of detection (LOD) of the assay for all targets was 100 colony-forming unit (cfu)/swab-sample. Results of the repeatability studies in two different centers were in 96%–100% (69/72–72/72) agreement. The relative specificity and sensitivity of the qPCR assay were respectively 96.8% and 98.8% for VRE; 94.9% and 95.1% for CRE; 99.9% and 97.1% for MRSA. Conclusions: The developed qPCR assay can screen antibiotic-resistant hospital-acquired infectious agents in infected/colonized patients with an equal clinical performance to the culture-based methods.
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Journal of Microbiological MethodsCilt
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