A modified PCR-based method for rapid non-radioactive detection of clinically important pathogens

Victor N. Gorelov, Kristoffel Dumon, Natalia S. Barteneva, Hans Dieter Röher, Peter E. Goretzki

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)611-616
Number of pages6
JournalMicrobiology and Immunology
Volume40
Issue number9
DOIs
Publication statusPublished - Jan 1 1996
Externally publishedYes

Keywords

  • Bacterial pathogens
  • Direct nonradioactive labeling
  • Polymerase chain reaction

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Virology

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