Development of qPCR method for identification of toxin coding species of Clostridium difficile
Subject Areas : microbiologyLeila Nouri 1 , Mehdi Ebrahimi 2 , Mahdi Alijanianzadeh 3
1 - Department of Biotechnology, Islamic Azad university, Varamin-Pishva Branch, Pishva, IRAN
2 - Department of Biochemistry and Biophysics, Faculty of Biological sciences, Varamin-Pishva Branch, Islamic Azad university, Varamin, IRAN
3 - Department of bioscience and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran
Keywords: qPCR, Clostridium difficile, tcdA, tcdB,
Abstract :
Clostridium difficile is the most important spore forming, gram positive, anaerobic bacilluswhich is known as prevalent factor of hospital diarrhea and causative agent of pseudomembrane colitis. The important virolence factors of this bacteria is A and B toxins. the clinical demonstration of Clostridium difficile infection range from asymptomatic colonization, to sever diarrhea, pseudo membranous colitis, toxic megacolon and death. The purpose of this study was to extract Clostridium difficile cells from faecal samples and develop a routine diagnostic laboratory method using qPCR to identify this bacteria with high sensitivity and accuracy. 100 clinical samples were collected within 6 months from patients being treated at Ayyatollah Taleghani education and treatment medical center. a standard strain was obtained from Pasteur Institue micobial seed bank. For determination of the specificity of the method being developed, the DNA of Escherichia coli, Shigella, Shigelladysenteriae, Salmonella typhi, Yersinia Entrocolitica were extracted and analyzed along with a primer designed for Toxin A in Clostridium difficile using q PCR. After optimization of the test conditions, 100 clinical samples were evaluated. Among the samples, 9 of them were (9%) positive. For evaluation of the sensitiviy of the test, the obtained DNA from the bacteria was diluted, and further analyzed using qPCR. To determine the specificity of the test, the genome of diarrhea causing bacteria were used which lead to positive results only from the genome of Clostridium difficile. Based on the obtained results, the sensitivity and precision of qPCR in rapid detection of 100 pg of DNA was determined.
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