Detection of Yersinia pestis by pesticin fluorogenic probe-coupled PCR.
The <<Taq Man>> assay (PE Applied Biosystems) combines PCR with concomitant release of fluorogenic nucleotides for immediate product detection by fluorometry. Yersinia pestis, the etiological agent of bubonic plague, expresses species-specific genes known to be located on two unique plasmids (9.6-kb pPCP and 100.9-kb pMT). Pesticin (pst) is a unique gene located on pPCP which encodes for a bacteriocin. Using fluorogenic probe coupled PCR as few as three copies of pst targets were detected from total Y. pestis genomic DNA. The pst probe used in this report was positive only for pesticinogenic isolates and did not show complementarity with Yersiniae nor with other bacteria targeted in this study suggesting, that the pst probe is very specific for Y. pestis. Under optimal conditions of Mg(2+)concentration and thermal cycle number, addition of extraneous DNA to respective assay mixtures had no effect on detection.
Iqbal
S S
SS
Division of Life Sciences, University of Texas at San Antonio, 78249, USA.
Chambers
J P
JP
Goode
M T
MT
Valdes
J J
JJ
Brubaker
R R
RR
D001430:Bacteriocins; D017931:DNA Primers; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D005819:Genetic Markers; D016014:Linear Models; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D020711:Threshold Limit Values; D015010:Yersinia pestis / Q000235:genetics / Q000302:isolation & purification*
D001430:Bacteriocins; D017931:DNA Primers; D015342:DNA Probes; D005819:Genetic Markers; C011330:Pesticin