Detection and identification of intestinal pathogens in clinical specimens using DNA microarrays.
The detection and identification of intestinal pathogens is critical for clinical patient diagnosis and antimicrobial therapy. No currently available assays with DNA microarrays can simultaneously detect and identify multiple intestinal pathogens, because there is no appropriate method for choosing target probes. To solve the problem we have experimented for facilitating screening of specific probes and developed a rapid (<3h) and reliable assay for simultaneous detection of intestinal pathogens using two universal PCR primers to amplify two variable regions of bacterial 16S and 23S ribosomal DNA (rDNA) genes, and then applied to DNA microarrays, hybridization between probes and amplicons occurred. Through this idea for screening of probes the assay was successful in discriminating 15 genera or species of intestinal pathogens. The limit of detection was approximately 10(3)CFU/mL for one species of pathogen and 10(5)CFU/mL for six species pathogens existing simultaneously in stool. When this assay was applied directly to identify 99 clinical specimens, 80(80.8%) were correctly analyzed, including four with mixed pathogens; 8(8.08%) received negative results due to no corresponding probes in this array and 11(11.11%) belonging to our targets were misidentified due to low-level pathogens and other factors. This approach is also convenient to obtain specific and proper probes while establishing assays for the applications in other aspects using DNA microarrays. In addition, the more species may be added to this system easily and endlessly by screening of candidate target probes in order to increase the power of simultaneous detection.
Jin
Da-Zhi
DZ
Beijing Institute of Radiation Medicine, No. 27 Taiping Road, Beijing, China.
Wen
Si-Yuan
SY
Chen
Su-Hong
SH
Lin
Feng
F
Wang
Sheng-Qi
SQ
D001424:Bacterial Infections / Q000145:classification / Q000175:diagnosis*; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis*; D004275:DNA, Ribosomal / Q000378:metabolism; D005243:Feces / Q000382:microbiology; D006801:Humans; D007422:Intestines / Q000382:microbiology*; D025202:Molecular Diagnostic Techniques; D015335:Molecular Probes; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D015335:Molecular Probes