The performance of a single-tube nested-PCR (STNPCR) technique was evaluated for plague diagnosis in comparison to conventional (one step) and two step nested PCR (NPCR). Assays were carried out with primers targeting the gene caf1 that encodes the Yersinia pestis F1 antigen. For STNPCR inner primers were immobilized onto the inside of the microtube caps and after the first amplification they were eluted by inversion of the tube. This procedure avoids opening the tube, reducing the risks of false-positive results by cross-contamination. The immobilized primers are stable for several months at -20 degrees C, thus, the tubes can be prepared beforehand and stored until use. STNPCR was more sensitive than conventional PCR, and less sensitive than NPCR. This drawback is compensated by a lower risk of cross-contamination. The experiments with infected animals showed that NPCR and STNPCR were able to produce positive results in all samples tested, despite contamination with other organisms. In contrast, conventional PCR yielded positive results in a smaller number of samples. Three out of 62 culture-negative rodents from plague areas, were positive by STNPCR. In conclusion, the PCR approaches evaluated, particularly NPCR and STNPCR have potential to be used as alternative tools in epidemiological surveys of plague. Furthermore, as the results can be obtained quickly (less than 24 hour), these techniques could be useful in emergency situations in which the rapidity in diagnosis is essential for adoption of immediate measures of control.
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