مقالات پذیرفته شده در پنجمین کنگره بین المللی زیست پزشکی
Colorimetric PCR-based detection of a bacterial cause of urinary tract infection
Colorimetric PCR-based detection of a bacterial cause of urinary tract infection
Shahrzad Salmanian,1Hamidreza Mollasalehi ,2,*
1. Department of Cellular and Molecular Biology, Faculty of Modern Science and Technology, Islamic Azad University, Medical Sciences Branch, Tehran, Iran 2. Department of Microbiology and Microbial Biotechnology, Faculty of Biological Sciences and Technology, Shahid Beheshti University, Tehran, Iran
Introduction: Nosocomial infections such as urogenital and urinary tract infections are caused by opportunistic bacteria namely, Morganella morganii. Therefore, a rapid and specific molecular detection method is needed for early diagnosis of the agent for accurate therapy. In this study, a visual colorimetric PCR detection assay was developed using a metal indicator for M. morganii.
Methods: The bacterial cells were cultured overnight to reach the log phase in their growth curve, followed by genomic DNA extraction. After analyzing the quality and quantity of the extracted DNA using spectroscopy, the target gene of M. morganii was amplified by an optimized PCR protocol using designed primers. Furthermore, the specificity of the assay was assessed using a mixture of eight gram-negative bacteria (Shigella boydii, Yersinia enterocolitica, Citrobacter freundii, Klebsiella pneumonia, Enterobacter aerogenes, Pseudomonas aeruginosa, Burkholderia cepacia, Serratia marcescens) with and without the target bacteria (M. morganii). Finally, the amplification PCR products were investigated by the metal indicator, hydroxy naphthol blue (HNB) dye which was added to PCR amplicons in an endpoint approach. The limit of detection (LOD) was investigated by a decimal dilution of the extracted DNA.
Results: The quantity of the M. morganii extracted nucleic acid was measured to be 101 µg mL-1, which was appropriate for further experimental analysis. The color change of dark blue to light blue was observed in the positive sample including M. morganii but not in the negative sample without M. morganii. Furthermore, the limit of detection (LOD) of the assay was visualized up to a second dilution tube. This value was determined to be 1.01 µg mL-1 of the amplified genomic DNA of M. morganii by HNB indicator.
Conclusion: The optimized conventional PCR method with the instrument-free colorimetric detection could be an efficient and simple detection technique for rapid M. morganii identification, especially in urinary tract infections.