| Objective: To establish an Anti-contamination multiplex PCR microplate hybridization enzyme immunoassay (EIA) for identifying Yersinia pestis. Methods: The specific primers were designed based on virulent- related gene cafl and pla located on pMT1 and pPCP1 respectively and a Y. pestis-specific sequences marker 3a on the chromosome. Three specifically designed PCR products were used as capture probes to coat on polyethylene microwell separately. 5'-end biotin-labeled primers were employed in multiplex PCR amplification and the products were used as detecting probe in the microplate hybridization. Uridine glycosylase(UNG) was used in multiplex-PCR system for digesting the carry-over amplicons and preventing contamination, and the microplate hybridization EIA was used for verifying the specific amplicons. In order to facilitate the feasibility of this technique, an enzyme stabilizer was introduced into the system to obtain a stable reagent system for convenient amplification. Results: This technique correctly identified all 54 Y. pestis strains tested and the results were negative for 27 controlled bacterial strains. The sensitivity demonstrated with target DNA of Y. pestis 11001 was 100fg DNA for multiplex PCR electrophoresis assay and 10fg DNA for multiplex PCR microplate hybridization assay separately. Conclusions: This multiplex-PCR-EIA system was specific for rapid identification of Y. pestis that also allows the identification of prominent virulence markers of Y. pestis strains, The sensitivity of multiplex PCR microplate hybridization was 10 times higher than that demonstrated by multiplex PCR-agrose electrophoresis. this technique overcomes the problems in conventional PCR, such as inconvenience for transportation of reagent, false positive due to contamination and lack of objective evaluation of results by electrophoresis, it will be a powerful method for rapid identification of Y. pestis in case of emergency and for the surveillance of epidemics.
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