Microarray-based identification of bacteria in clinical samples by solid-phase PCR amplification of 23S ribosomal DNA sequences.
The rapid identification of the bacteria in clinical samples is important for patient management and antimicrobial therapy. We describe a DNA microarray-based PCR approach for the quick detection and identification of bacteria from cervical swab specimens from mares. This on-chip PCR method combines the amplification of a variable region of bacterial 23S ribosomal DNA and the simultaneous sequence-specific detection on a solid phase. The solid phase contains bacterial species-specific primers covalently bound to a glass support. During the solid-phase amplification reaction the polymerase elongates perfectly matched primers and incorporates biotin-labeled nucleotides. The reaction products are visualized by streptavidin-cyanine 5 staining, followed by fluorescence scanning. This procedure successfully identified from pure cultures 22 bacteria that are common causes of abortion and sterility in mares. Using the on-chip PCR method, we also tested 21 cervical swab specimens from mares for the presence of pathogenic bacteria and compared the results with those of conventional bacteriological culture methods. Our method correctly identified the bacteria in 12 cervical swab samples, 8 of which contained more than one bacterial species. Due to the higher sensitivity of the on-chip PCR, this method identified bacteria in five cervical swab samples which were not detected by the conventional identification procedure. Our results show that this method will have great potential to be incorporated into the routine microbiology laboratory.
Mitterer
Georg
G
Institute of Microbiology & Genetics, University of Vienna, 1030 Vienna, Austria.
Huber
Martin
M
Leidinger
Ernst
E
Kirisits
Claudia
C
Lubitz
Werner
W
Mueller
Manfred W
MW
Schmidt
Wolfgang M
WM
D000034:Abortion, Veterinary / Q000382:microbiology; D000818:Animals; D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D006736:Horses; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D011251:Pregnancy Complications, Infectious / Q000382:microbiology / Q000662:veterinary; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D012338:RNA, Ribosomal, 23S / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S