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Rivista di Biologia Molecolare e Biotecnologie
Indexed/Abstracted in: EMBASE, Science Citation Index Expanded (SciSearch), Scopus
Impact Factor 0,246
Minerva Biotecnologica 2016 Giugno;28(2):89-94
Rapid detection of uropathogenic Proteus mirabilis using SYBR® green melting curve analysis
Samiraj RAMESH 1, Satish K. RAJASEKHARAN 1, Ramaraj ELANGOMATHAVAN 2
1 Department of Microbiology, PRIST University, Vallam, Thanjavur, India; 2 Department of Biotechnology, PRIST University, Vallam, Thanjavur, India
BACKGROUND: The present study was focused to discriminate Proteus mirabilis from other uropathogens by SYBR® green melting curve analysis.
METHODS: Uropathogens were isolated from the urine samples (N.=65) of patients with urinary tract infections (UTI). The isolates were phenotypically characterized by the color and morphological traits on HiCrome UTI agar. Out of the 65 urine samples analyzed, 21 isolates produced brown colonies on the UTI agar plates. Individual colonies of the uropathogens were picked for real-time polymerase chain reaction (PCR) assays for the detection of ZapA gene sequence.
RESULTS: Sixteen out of 21 isolates tested positive for P. mirabilis specific primer in real-time PCR assays, while the remaining 5 isolates were identified as the other acquaintances of tribe Proteeae. The designed primers also failed to amplify the product in Proteus vulgaris, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. In-silico analysis of the designed oligonucleotides showed that the primers and the product sequence were highly specific for P. mirabilis. No cross-species or cross-genera matches were obtained. The measured melting temperature of ZapA gene amplicon was approximately 80±0.5 °C for P. mirabilis.
CONCLUSIONS: SYBR® green real-time colony PCR offers a rapid and sensitive method for confirmation of P. mirabilis thus eliminating the need for laborious and time-consuming techniques like DNA isolation, conventional PCR and agarose gel electrophoresis.