Evaluation of probe chemistries and platforms to improve the detection limit of real-time PCR.
A validated PCR-based Salmonella method targeting a 94-bp sequence of the ttr gene was used as a model to compare six different combinations of reporter and quencher dyes of a TaqMan probe, on three different instruments, to improve the detection limit in a real-time PCR assay with the aim of a same-day analysis. The use of locked nucleic acids (LNA) and Scorpion probes were also tested. The combination FAM-BHQ1 or Cy5-BHQ3, both dark quenchers, gave the best results (Cycle threshold (Ct) of 25.42+/-0.65 and 24.47+/-0.18 at 10(3) DNA copies). When comparing different probe technologies, the LNA probe (FAM-BHQ1) was the most sensitive with the strongest fluorescence signal (dR last 48066), resulting in 0.6 to 1.1 lower Ct values than a DNA TaqMan probe, and 1.9 to 4.0 lower Ct than the Scorpion system (FAM-BHQ1). The RotorGene real-time PCR instrument gave 0.4-1.0 lower Ct values (more sensitive) than the Mx3005p, and 1.5-3.0 lower than the ABI 7700. Using the LNA in a RotorGene instrument, we detected the following Salmonella DNA copies in 1-ml pre-enriched samples: fishmeal (100 copies), chicken rinse (100 copies) and pig feces (10 copies). The detection probability of the final assay on inoculated fecal samples was 100% at 2x10(4) copies per ml. In conclusion, the LNA probe with annealing temperature of 65 degrees C could be useful for more sensitive detection limits.
Reynisson
E
E
Icelandic Fisheries Laboratories, Reykjavík, Iceland. eyjolfur@rf.is
Josefsen
M H
MH
Krause
M
M
Hoorfar
J
J
D000818:Animals; D002645:Chickens / Q000382:microbiology; D017931:DNA Primers / Q000235:genetics* / Q000592:standards; D015342:DNA Probes / Q000235:genetics* / Q000592:standards; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D005243:Feces / Q000382:microbiology; D005396:Fish Products / Q000382:microbiology; D005456:Fluorescent Dyes / Q000737:chemistry; D005516:Food Microbiology; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012486:Salmonella typhimurium / Q000235:genetics*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000737:chemistry
D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D019914:Taq Polymerase