A new multiplex PCR assay for the simultaneous detection of vancomycin-resistant enterococci from rectal swabs.

OBJECTIVES: This study describes the diagnostic performance of a recently available multiplex PCR-based kit for the simultaneous detection and identification of Enterococcus faecium, Enterococcus faecalis, vanA, vanB, vanC1 and vanC2/C3 genes, directly from rectal swabs constituting the most complete existing molecular assay currently available. METHODS: The diagnostic performance of this assay was evaluated by a multicenter study involving three independent public hospitals and consisted in the analysis of 187 rectal swabs from patients at high risk for vancomycin-resistant enterococci colonization. RESULTS: When bacteria culture was used as the gold standard, the sensitivity, specificity, positive and negative predicted values for the assay were 96.8%, 76.0%, 67.7% and 97.9%, respectively. When a composite reference standard consisting of culture and DNA sequencing of PCR products was used as the gold standard, the sensitivity, specificity, positive and negative predicted values for the PCR-based assay were 97.8%, 96.9%, 96.7% and 97.9%, respectively. CONCLUSIONS: Based on these results, we conclude that this assay is considerably more sensitive than traditional microbiological methods for detecting vancomycin-resistant enterococci from rectal swabs. It is also much faster than culture. We believe that the implementation of this assay in routine clinical laboratories could help to reduce hospital-acquired vancomycin-resistant enterococci infections.

ZmEBE genes show a novel, continuous expression pattern in the central cell before fertilization and in specific domains of the resulting endosperm after fertilization.

Two novel maize genes expressed specifically in the central cell of the female gametophyte and in two compartments of the endosperm (the basal endosperm transfer layer and the embryo surrounding region) were characterized. The ZmEBE (embryo sac/basal endosperm transfer layer/embryo surrounding region) genes were isolated by a differential display between the upper and the lower half of the kernel at 7 days after pollination (DAP). Sequence analysis revealed ORFs coding for two closely related proteins of 304 amino acids (ZmEBE-1) and 286 amino acids (ZmEBE-2). This size difference was due to differences in the splicing of the two genes. Both protein sequences showed significant similarity to the DUF239 family of Arabidopsis, a group of 22 proteins of unknown function, a small number of which are putative peptidases. ZmEBE genes had a novel cell type-specific expression pattern in the central cell before and the resulting endosperm after fertilization. RT-PCR analysis showed that the expression of both genes started before pollination in the central cell and continued in the kernel up to 20 DAP with a peak at 7 DAP. In situ hybridization revealed that the expression in the kernel was restricted to the basal transfer cell layer and the embryo surrounding region of the endosperm. The expression of ZmEBE-1 was at least 10 times lower than that of ZmEBE-2. Similarly to other genes expressed in the endosperm, ZmEBE-1 expression was subject to a parent-of-origin effect, while no such effect was detected in ZmEBE-2. Sequence analysis of upstream regions revealed a potential cis element of 33 bp repeated 7 times in ZmEBE-1 and ZmEBE-2 between positions -900 and -100. The 1.6 kb ZmEBE-2 upstream sequence containing the seven R7 elements was able to confer expression in the basal endosperm to a Gus reporter gene. These data indicate that ZmEBE is potentially involved in the early development of specialized domains of the endosperm and that this process is possibly already initiated in the central cell, which is at the origin of the endosperm.