| Enterohemorrhagic Escherichia coli （EHEC） is one of the most importantfood-borne pathogens. The infections caused by EHEC have been considered as anemerging infectious disease of great potential public health threats. Several methodsfor the detection of EHEC have been described, including culture and isolation,antibody and antigen detection assays, and PCR based molecular detection. Thesemethods have some limitations, including time consuming, low sensitivity andspecificity, and requirement of elaborate instruments and skilled persons. Theselimitations make them difficult or unfeasible to be used under field conditions, whereequipment and resources are limited. Therefore, a rapid and simplified detectionmethod that is appropriate for on-site detection is needed.Isothermal Amplification Technology is a type of nucleic acid amplificationmethods that do not require specific and complicated equipment, making it idealtechnology for on site detection. CPA （Cross Priming Amplification） technology is arecent invention of isothermal DNA amplification system. Using multiplecross-linked primers （58primers）, it amplifies a DNA or RNA target sequence atone constant temperature.CPA assay is a simple, specific, sensitive and rapidmethod,especially used as a rapid detection tool in field detection of pathogens.Objective: To develop a CPA method for detecting EHEC and provide a highsensitivity, specificity, simple, rapid, low-cost, and effective method for the detectionof EHEC.Methods: The stx1gene of EHEC was used as the signature sequence forprimer design. Constituents of the reaction, including concentration of Mg2+, dNTPs,betaine, primers and Bst DNA polymerase etc were systematically optimized. Theconcentration and the optimal amplification reaction were defined.The factorsaffecting the CPA amplification were analyzed. The amplification products weredetected by electrophoresis, SYBR Green I and nucleic acid detection device, and the three detection methods were evaluated. Ten related pathogenic bacteria wereused for specificity test. The sensitivity of CPA was evaluated by using serialdilutions of plasmid with defined concentration and pure culture of EHEC. Thesensitivity was also compared with that of PCR using the same primers. Sensitivityof the CPA method was evaluated in both recombinant plasmid and pure culturebacteria，with PCR as parallel detection.Results：①The optimal CPA condition established for rapid detection of EHECwas as follows:0.5μM of CPR,0.3μM each of5F and5B,0.1μM each of F3，B3andF2primer,0.4mM dNTPs,2μl10×Bst buffer,8units of Bst DNA polymerase largefragment,3mM MgSO4,1M betaine,1μl templates and sterilized double-distilledwater in20μl volumes. The mixture was incubated at63℃for1hour.②Theamplified products were able to detected by three different methods, includingelectrophoresis, SYBR Green I and nucleic acid detection device. Compare with theother two methods, using nucleic acid detection device was simple, accurate,sensitive and specific, and furthermore, avoid of contamination by tube opening.③Our results showed this CPA assay had a high specificity for the detection of EHECstrains while other ten non-EHEC strains.④The sensitivity of recombinant plasmidEHEC was10copies and the detection limit of pure bacterial culture was1.3CFU/mlwith CPA reaction. In contrast, the sensitivity of recombinant plasmid EHEC was100copies and the detection limit of pure bacterial culture was13CFU/ml with PCRreaction. The sensitivity of CPA was10fold higher than conventional PCR.Conclusions：The result indicates that the sensitivity of detection of EHEC byCPA is superior to those of other methods. Its specificity is also excellent whendistinguishing EHEC from other bacteria. Finally, the sensitivity, specificity, andmatched route of CPA are comparable to other methods. Given that CPA is cheaper,more convenient, and faster, it will be readily accepted by many quarantineinstitutes. |
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