A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates

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Khodaparast Sepideh; MOHABATI MOBAREZ ASHRAF; SABERIFIROOZI MEHDI

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Journal: MIDDLE EAST JOURNAL OF DIGESTIVE DISEASES (MEJDD) Year:2019 | Volume:11 | Issue:3 Page(s): 135-140

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Title

A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates

Author(s)

Khodaparast Sepideh | MOHABATI MOBAREZ ASHRAF | SABERIFIROOZI MEHDI | Issue Writer Certificate

Keywords

Clostridium difficile

Colitis

Toxigenic culture

Cdd3

gluD

tcdA

tcdB

Abstract

BACKGROUND: Clostridium difficile is the major causative agent of nosocomial antibiotic-associated Colitis. The gold standard for C. difficile detection is stool culture followed by cytotoxic assay, although it is laborious and time-consuming. We developed a screening test based on a two-step conventional polymerase chain reaction (PCR) approach to detect gluD, the glutamate dehydrogenase (GDH) enzyme gene, which is a marker for screening of C. difficile. Targeting gluD comparing to the conserved stable genetic element of pathogenicity locus (PaLoc), with an accessory gene of Cdd3, was an effective method for the detection of this pathogen from patients with enteroColitis. METHODS: Fresh fecal samples of the patients who were clinically suspicious for antibiotic-associated Colitis were collected. Stool specimens were cultured on the cycloserine-cefoxitin fructose agar (CCFA) in an anaerobic condition, following alcohol shock treatment and enrichment in Clostridium difficile Brucella broth (CDBB). On confirmed colonies, PCR was carried out for detection of PaLoc subsidiary gene, Cdd3, and toxicogenic genes, tcdA and tcdB. The gluD that is GDH gene detection was performed by conventional PCR on the extracted DNA from 578 fresh stool samples. RESULTS: 57 (9. 8%) strains of C. difficile were approved by conventional PCR for gluD and Cdd3 genes, in which 37 (6. 4%) colonies had tcdA+/tcdB+ genotype, 2 (0. 3%) tcdA+/tcdB-, 4 (0. 7%) tcdA-/ tcdB+ and the remaining 14 (2. 4%) colonies were tcdA and tcdB negative. CONCLUSION: These results demonstrate that targeting gluD by PCR is quite promising for rapid detection of C. difficile from fresh fecal samples. Furthermore, the multiple-gene analysis for tcdA and tcdB assay proved a reliable approach for diagnosing of toxigenic strains among clinical samples.

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APA: Copy

Khodaparast, Sepideh, MOHABATI MOBAREZ, ASHRAF, & SABERIFIROOZI, MEHDI. (2019). A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates. MIDDLE EAST JOURNAL OF DIGESTIVE DISEASES (MEJDD), 11(3), 135-140. SID. https://sid.ir/paper/973656/en

Vancouver: Copy

Khodaparast Sepideh, MOHABATI MOBAREZ ASHRAF, SABERIFIROOZI MEHDI. A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates. MIDDLE EAST JOURNAL OF DIGESTIVE DISEASES (MEJDD)[Internet]. 2019;11(3):135-140. Available from: https://sid.ir/paper/973656/en

IEEE: Copy

Sepideh Khodaparast, ASHRAF MOHABATI MOBAREZ, and MEHDI SABERIFIROOZI, “A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates,” MIDDLE EAST JOURNAL OF DIGESTIVE DISEASES (MEJDD), vol. 11, no. 3, pp. 135–140, 2019, [Online]. Available: https://sid.ir/paper/973656/en

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