TY - JOUR
T1 - A modified PCR-based method for rapid non-radioactive detection of clinically important pathogens
AU - Gorelov, Victor N.
AU - Dumon, Kristoffel
AU - Barteneva, Natalia S.
AU - Röher, Hans Dieter
AU - Goretzki, Peter E.
N1 - Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 1996/1/1
Y1 - 1996/1/1
N2 - 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.
AB - 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.
KW - Bacterial pathogens
KW - Direct nonradioactive labeling
KW - Polymerase chain reaction
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U2 - 10.1111/j.1348-0421.1996.tb01117.x
DO - 10.1111/j.1348-0421.1996.tb01117.x
M3 - Article
C2 - 8908604
AN - SCOPUS:0029796357
VL - 40
SP - 611
EP - 616
JO - Microbiology and Immunology
JF - Microbiology and Immunology
SN - 0385-5600
IS - 9
ER -