Genotyping of clinicalSerratia marcescensisolates: a comparison of PCR-based methods

Abstract

The polymerase chain reaction (PCR)-based procedures of randomly amplified polymorphic DNA (RAPD) and repetitive element (RE)-based PCR were used to amplify total DNA prepared from each of 62 clinical Serratia marcescens isolates. Three different random primers, designated 1060, 1254 and 1283, were used individually in RAPD-PCR. Primers representing enterobacterial repetitive intergenic consensus (ERIC) sequences, extragenic palindromic (REP) elements, and polymorphic GC-rich repetitive sequences (PGRS) constituted the repetitive element-PCR. We were able to generate 40, 40 and 58 genotypic groupings using the 1060, 1254 and 1283 RAPD primers, respectively. Using the ERIC, REP and PGRS primers, 19, 54 and 60 unique genotypic profiles were yielded, respectively. The PGRS primers, which were developed to amplify GC-rich repetitive sequences in the genome of Mycobacteria, were the most discriminatory. These data indicate that both of these PCR-based approaches are a valid means of discriminating strain differences among isolates of S. marcescens and the amount of differentiation depends on the primer used. These techniques should prove useful for routine surveillance or in examining outbreaks of S. marcescens in clinical settings.

Genotype variation and genetic relationship among Escherichia coli from nursery pigs located in different pens in the same farm

“…This genomic fingerprinting technique was chosen for several reasons; i) it generates specific strain patterns obtained by the amplification of repetitive DNA elements present along the E. coli genome [21], ii) the technique has proved more discriminatory than 16S rRNA PCR methods and restriction fragment length polymorphism [22, 23], and provides discriminatory power similar to randomly amplified polymorphic DNA (RAPD) [24]. In addition, the REP protocol is simpler and allows handling of a larger number of samples than other genomic DNA protocols, such as pulsed field gel electrophoresis, for molecular typing [22].…”

Rapid genotyping of Pseudomonas aeruginosa isolates harboured by adult and paediatric patients with cystic fibrosis using repetitive-element-based PCR assays

“…BOX elements have since been identified in P. aeruginosa as well as Enterococcus faecalis (Malathum et al, 1998;McSpadden Gardener et al, 2000). ERIC-PCR has been successfully applied to P. aeruginosa as well as Listeria monocytogenes, Serratia marcescens, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis and Acinetobacter baumannii (Jersek et al, 1999;Kang & Dunne, 2003;Lau et al, 1995;Patton et al, 2001). This study demonstrated that ERIC-PCR and BOX-PCR were equally effective in characterizing clinical isolates of P. aeruginosa.…”

Isolation and Molecular Detection of Polycyclic Aromatic Hydrocarbon-Degrading <i>Mycobacterium</i> spp. from the Shenfu Wastewater Irrigation Area in China

“…The extracted DNA from each positive isolate above was used as a template for PCR with Polymorphic GC-rich Repetitive Sequences (PGRS) primers (Table 1) to discriminate Mycobacterium isolates to remove the same isolates and get representative Mycobacterium stains for further following assay. The PCR amplification was performed as described previously [21]. After PCR amplification, 5 µl of the PCR reaction mixture was separated on a 6% polyacrylamide gel (acrylamide: N-N′-methylene bisacrylamide 29:1) using a Bio-Rad system.…”