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Simultaneous detection and differentiation of pathogenic and nonpathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay

100%

Bedir O., Kilic A., Atabek E., Kuskucu A. M., Turhan V., Basustaoglu A. C.

Polish Journal of Microbiology

2010

tom 59

nr 3

Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific ligA/B genes and non-pathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1 x 10² cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83-105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.

Macrolide-lincosamide-streptogramin B resistant phenotypes and genotypes for methicillin-resistant Staphylococcus aureus in Turkey, from 2003 to 2006

100%

Gul H. C., Kilic A., Guclu A. U., Bedir O., Orhon M., Basustaoglu C.

Polish Journal of Microbiology

2008

tom 57

nr 4

Methicillin-resistant Staphylococcus aureus (MRSA) strains with inducible macrolide-lincosamide-streptogramin B (iMLSB) resistance phenotype may lead to clinical failure during clindamycin (CLI) therapy. The aim of this study was to determine the incidence of MLS|t phenotypes by using D-test method and genotypes by using multiplex real-time PCR method in MRSA strains. A total of 265 MRSA strains were obtained from clinical samples from hospitalized and outpatients. Of the MRSA isolates, 225 (84.9%) were resistant to erythromycin (ERT), and 170 (64.1%) to CLI. Among the 225 ERT-resistant MRSA strains, the constitutive MLS|f (cMLSu) rate was found in 49.3%, iMLSB in 39.1% and the M phenotype in 11.5%. Overall, ermA, ermC, ermA+ermC, msrA, ermC+msrA, and ermA+ermC+msrA genes were detected in 85 (37.7%), 60 (26.6%), 42 (18.6%), 26 (11.5%), 11 (4.8%), and 1 (0.4%) isolates, respectively. Most prevalent resistance determinant in MRSA strains was ermA", which was detected in 37.7% of the isolates. The 26 MRSA strains with M phenotype harboured only msrA gene. In conclusion, due to aware of the potential of CLI treatment failure, D-test should be performed and reported in MRSA strains in clinical laboratories. The multiplex real-time PCR method is easy to perform, fast and reliable method for the detection of MLSB resistance genotypes.

Ograniczanie wyników

2 Polish Journal of Microbiology

2 Bedir O.

2 Kilic A.

1 Atabek E.

1 Basustaoglu A. C.

1 Basustaoglu C.

1 Guclu A. U.

1 Gul H. C.

1 Kuskucu A. M.

1 Orhon M.

1 Turhan V.

1 2010

1 2008

Wyniki wyszukiwania

Simultaneous detection and differentiation of pathogenic and nonpathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay

Macrolide-lincosamide-streptogramin B resistant phenotypes and genotypes for methicillin-resistant Staphylococcus aureus in Turkey, from 2003 to 2006