[Multiplex PCR in the framework of an enterococci monitoring project].
A multiplex-PCR (MPCR) assay was optimised in the framework of an enterococci monitoring project in order to accelerate the previous time-consuming biochemical testing of enterococcal strains isolated from animal materials and food. The MPCR was optimised by variation of annealing temperature, MgCl2-concentration but more over by primer combinations and primer concentrations. Reference strains were used during the optimization. Afterwards 522 enterococcal field strains were examined by the MPCR. Ambiguous results occurred for 20 strains (17 with false-positive and 3 with false-negative results) which could be explained by the minor specificity ("EM"-primers) and sensitivity (ddlE.faecium-primers) respectively. Differences also existed between the genus-specific primers "E" and "Ent". The first mentioned primers showed weaknesses for the identification of enterococci whereas the use of the "Ent" primers inhibited other primers in the MPCR. However, the majority of the examined strains could be detected unambiguously, so that a differentiation of enterococcal isolates can be recommended by MPCR in contrast to the high time-, material- and work-consumption of conventional biochemical testings.
Mac
Kiem
K
Bundesinstitut für Risikobewertung, Berlin. k.mac@bfr.bund.de
Ellerbroek
Lüppo
L
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D019729:Carbon-Oxygen Ligases / Q000235:genetics; D017931:DNA Primers; D024881:Drug Resistance, Bacterial / Q000235:genetics; D016983:Enterococcus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D005188:False Negative Reactions; D005189:False Positive Reactions; D006801:Humans; D015636:Magnesium Chloride; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013696:Temperature; D020713:Vancomycin Resistance / Q000235:genetics
D001426:Bacterial Proteins; D017931:DNA Primers; C071437:VanA ligase, Bacteria; C079913:VanB protein, Enterococcus; D015636:Magnesium Chloride; D019729:Carbon-Oxygen Ligases