Rapid detection of vanA and vanB genes directly from clinical specimens and enrichment broths by real-time multiplex PCR assay.
نویسندگان
چکیده
A real-time PCR assay previously developed for use on the Roche LightCycler platform was investigated as an alternative to culture for the direct detection of vancomycin-resistant enterococci (VRE) in clinical specimens. PCR primers and fluorescence resonance energy transfer hybridization probes specific for the vanA and vanB genes were combined in a multiplex real-time PCR assay performed directly with fecal material obtained by rectal swabbing and with enrichment broth samples. DNA was prepared from the rectal swabs and enrichment broths with a commercially available DNA preparation column designed specifically for use with fecal specimens. One hundred eighty duplicate rectal swabs were obtained from 42 patients who were previously found to be positive for VRE and who were being monitored for carriage of VRE. Direct and enrichment broth cultures were performed with one swab, while PCR was performed with the other swab as well as any corresponding presumptive positive enrichment broth. In total, 100 specimens from 30 patients remained positive for VRE by at least one method. The multiplex real-time PCR was positive for 88 enrichment broths of rectal swabs from 27 patients but for only 45 rectal swabs from 15 patients. Direct culture was positive for VRE for only 43 specimens from 11 patients, while enrichment broth culture was positive for VRE for 75 specimens from 22 patients. Inhibition studies for the multiplex real-time PCR assay, performed by spiking the DNA extracts from 50 negative rectal swabs and the corresponding enrichment broths with between 1 and 10 CFU of a VanB Enterococcus faecium strain, detected inhibition rates of 55.1 and 10%, respectively. PCR performed directly with enrichment broths was found to be significantly more sensitive than enrichment broth culture (P < 0.025). Negative samples were identified significantly earlier by PCR than by culture alone.
منابع مشابه
Multiplex PCR detection of vanA, vanB, vanC-1, and vanC-2/3 genes in enterococci.
Vancomycin-resistant enterococci (VRE) are increasingly isolated from clinical specimens. One hundred clinical isolates of enterococci (E. casseliflavus/E. flavescens [n = 10], E. faecalis [n = 34], E. faecium [n = 43], E. avium [n = 1], E. gallinarum [n = 11], and E. raffinosus [n = 1]) were examined for the presence of vanA, vanB, vanC-1, and vanC-2/3 genes by a single multiplex PCR performed...
متن کاملتشخیص ژنهای vanA, vanB, vanC با روش Multiplex-PCR در ایزولههای انتروکوکوس فکالیس و انتروکوکوس فسیوم در بیماران بستری
Background & Aims : After Vancomycin resistant Enterococci (VRE) was first detected in the mid 1980s, VRE spread rapidly worldwide and became one of the most serious problems in many hospitals specially in hospitalized patients. Among different enterococcal species Enterococcus feacalis and Enterococus faecium are the leading causes of life threatening infections. VanA/B/C genes are respons...
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BACKGROUND Active screening for vancomycin-resistant enterococci (VRE) using rectal specimens is recommended to limit the spread of antimicrobial resistance within certain high-risk populations. We evaluated the diagnostic performance of Vancomycin Resistance 3 Multiplexed Tandem PCR assay (AusDiagnostics, Australia), a rapid multiplex real-time PCR assay that detects vanA and/or vanB. METHOD...
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متن کاملDetection of vancomycin-resistant enterococci in fecal samples by PCR.
Surveillance cultures for vancomycin-resistant enterococci (VRE) are time-consuming and expensive for the laboratory to perform. Therefore, we investigated the use of PCR as an alternative method of detecting and identifying VRE directly in fecal samples. PCR primers directed to vanA, vanB, vanC1, vanC2, and enterococcal ligase genes were used to detect and identify VRE in fecal material obtain...
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ورودعنوان ژورنال:
- Journal of clinical microbiology
دوره 41 6 شماره
صفحات -
تاریخ انتشار 2003