Optimisation of AP–PCR fingerprinting discriminatory power for clinical isolates ofPseudomonas aeruginosa

Abstract

Recently methods based on analysis of arbitrarily amplified target sites of microorganism genomes have been extensively applied in microbiological studies. The range of their applications is limited by problems with discrimination and reproducibility resulting from lack of standardised and reliable methods of optimisation. By orthogonal-array optimisation most advantageous and optimal parameters for highly discriminatory primers (CagA2+CMVin2) were selected and efficient AP^PCR (arbitrarily primed-polymerase chain reaction) fingerprinting conditions for Pseudomonas aeruginosa isolates were set up. Stable and multiplex amplicon profiles obtained in this study revealed high level of intraspecies DNA polymorphism among 20 analysed clinical strains of P. aeruginosa proving optimised AP^PCR fingerprinting to be useful in epidemiological typing of the species.

Phenotypic and Genotypic Diversity of Multidrug-Resistant Pseudomonas aeruginosa Isolates from Bloodstream Infections Recovered in the Hospitals of Belo Horizonte, Brazil

“…aeruginosa [8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]. In this study, PCR assays were used to evaluate the presence of carbapenem-hydrolyzing class D BL and MBL genes, while ERIC-PCR and RAPD genotyping were performed to identify and establish the relatedness between the isolates.…”

“…The DNA of the all 40 strains of P. aeruginosa was subjected to random amplified polymorphic DNA (RAPD) genotyping with primers detecting CagA2 , 5′-ATT TAG AAG CAG GCT TTA GC-3′ and CMVin2 , 5 ′-GGT AGC AC GCG GGT TTC GAC-3′, which produce an average of 7-10 distinct PCR profiles per strain [17]. …”

“…This was then followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 30°C for 1 min, extension at 72°C for 1 min and final extension at 72°C for 7 min [17]. RAPD profiles were resolved by electrophoresis in a buffer (2 m M EDTA, 10 m M Tris-borate, pH 8.0) on a 6% polyacrylamide gel.…”

Random Whole Metagenomic Sequencing for Forensic Discrimination of Soils

“…We show an assessment of shotgun and WGA-based sequencing techniques as well as the use of a single arbitrarily primed DNA amplification (AP-PCR) for metagenomic soil DNA analysis [35]. The use of AP-PCR was first reported in the 1990s [36], [37] and has been applied to genotyping [38], [39] and the study of microbial communities [40].…”

Evaluation of the Polymorphisms Associated with Tandem Repeats for Pseudomonas aeruginosa Strain Typing

“…The methods available at this time for the genotyping of P. aeruginosa include pulsed-field gel electrophoresis (PFGE), arbitrarily primed PCR, and ribotyping (1,10,19). The first two methods suffer from a lack of interlaboratory reproducibility (5,9); and furthermore, the present "gold standard" with the greatest discriminatory power, PFGE, is generally too costly and laborintensive for routine clinical strain typing. On the other hand, automated ribotyping with the RiboPrinter (Qualicon, Wilmington, Del.)…”