Development and Evaluation of a Single Tube Nested PCR Based Approach (STNPCR) for the Diagnosis of Plague
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 crosscontamination. The immobilized primers are stable for several months at –20°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.
KeywordsPolymerase Chain Reaction Nest Polymerase Chain Reaction Conventional Polymerase Chain Reaction Yersinia Pestis Polymerase Chain Reaction Tube
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