A Modified PCR-Based Method for Rapid Non-Radioactive Detection of Clinically Important Pathogens
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- GORELOV Victor N.
- Department of Surgery A, Heinrich Heine University
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- DUMON Kristoffel
- Department of Surgery A, Heinrich Heine University
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- BARTENEVA Natalia S.
- Department of Surgery A, Heinrich Heine University
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- ROHER Hans-Dieter
- Department of Surgery A, Heinrich Heine University
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- GORETZKI Peter E.
- Department of Surgery A, Heinrich Heine University
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Abstract
We have devised a sensitive and rapid method for the detection of several bacterial pathogens in clinical specimens using PCR. This method has been named Direct Labeling and Detection Procedure (DLDP) and is based on the direct incorporation of a nonradioactive digoxigenin label (DIG-11-dUTP) into a microbial species-specific gene fragment during amplification. Following amplification, the resulting PCR products are cleansed of nonincorporated DIG-11-dUTP, spotted onto a nylon membrane, fixed by UV-crosslinking and the labeled DNA is visualized by digoxigenin detection reagents. Using cultivated reference bacteria (Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa) we were able to demonstrate a rapid and sensitive detection of <20 CFU of bacteria in human secretions (sputum, urine, mucous). The present study suggests that DLDP can be used as a reliable method for indication of bacteria in clinical or environmental specimens with the proviso that the selected corresponding oligonucleotide primers provide amplification of strong species-specific genes.
Journal
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- MICROBIOLOGY and IMMUNOLOGY
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MICROBIOLOGY and IMMUNOLOGY 40 (9), 611-616, 1996-09-20
Center For Academic Publications Japan
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Details 詳細情報について
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- CRID
- 1571417124231855488
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- NII Article ID
- 10004893843
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- NII Book ID
- AA00738350
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- ISSN
- 03855600
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- Text Lang
- en
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- Data Source
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- CiNii Articles