A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates
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 conven...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Iranian Association of Gastroenterology and Hepatology, Shiraz University of Medical Sciences
2019-07-01
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| Series: | Middle East Journal of Digestive Diseases |
| Subjects: | |
| Online Access: | http://www.mejdd.org/index.php/mejdd/article/view/1982 |
| _version_ | 1818261922014822400 |
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| author | Sepideh Khodaparast Ashraf Mohabati Mobarez Mehdi Saberifiroozi |
| author_facet | Sepideh Khodaparast Ashraf Mohabati Mobarez Mehdi Saberifiroozi |
| author_sort | Sepideh Khodaparast |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-12T18:54:55Z |
| format | Article |
| id | doaj.art-03c98697a2e6440bb79ec36ff9cd24fa |
| institution | Directory Open Access Journal |
| issn | 2008-5230 2008-5249 |
| language | English |
| last_indexed | 2024-12-12T18:54:55Z |
| publishDate | 2019-07-01 |
| publisher | Iranian Association of Gastroenterology and Hepatology, Shiraz University of Medical Sciences |
| record_format | Article |
| series | Middle East Journal of Digestive Diseases |
| spelling | doaj.art-03c98697a2e6440bb79ec36ff9cd24fa2022-12-22T00:15:16ZengIranian Association of Gastroenterology and Hepatology, Shiraz University of Medical SciencesMiddle East Journal of Digestive Diseases2008-52302008-52492019-07-0111313514010.15171/mejdd.2019.139A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile IsolatesSepideh Khodaparast0Ashraf Mohabati Mobarez1Mehdi Saberifiroozi2Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University. Tehran, Iran.Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University. Tehran, Iran.Digestive Disease Research Institute, Tehran University of Medical Sciences, Tehran, Iran.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.http://www.mejdd.org/index.php/mejdd/article/view/1982Clostridium difficileColitisToxigenic cultureCdd3gluDtcdA |
| spellingShingle | Sepideh Khodaparast Ashraf Mohabati Mobarez 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 Clostridium difficile Colitis Toxigenic culture Cdd3 gluD tcdA |
| title | A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates |
| title_full | A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates |
| title_fullStr | A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates |
| title_full_unstemmed | A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates |
| title_short | A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates |
| title_sort | two step approach for diagnosing glutamate dehydrogenase genes by conventional polymerase chain reaction from clostridium difficile isolates |
| topic | Clostridium difficile Colitis Toxigenic culture Cdd3 gluD tcdA |
| url | http://www.mejdd.org/index.php/mejdd/article/view/1982 |
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