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| Development and Application of DPO-PCR Detection method for Enterotoxigenic E.coli |
| XU Yi-gang1, LI Dan-dan3, LIU Zhong-mei1, CUI Li-chun2, LI Su-long1 |
1. Technical Centre of Heilongjiang Entry-exit Inspection and Quarantine Bureau, Harbin 150001, China;
2. Graduate School of Northeast Forestry University, Harbin 150040, China;
3. Technical Centre of Hainan Entry-exit Inspection and Quarantine Bureau, Haikou 570125, China |
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Abstract Dual-priming oligonucleotide (DPO) is a novel primer design method with the features of simple design, strong specificity and wider annealing temperature range. In this study, a pair of DPO primers was designed based on enterotoxigenic E.coli (ETEC) LT gene and following optimization of reaction system factors that Taq DNA polymerase, Mg2+ and dNTP, a DPO-PCR detection method for ETEC was established and its sensitivity, specificity and annealing temperature insensitivity were analyzed. Results showed that detection sensitivity of the method was 1.24×102 CFU/ml and the target gene could be efficiently amplified by the DPO primers at 45℃~65℃ of annealing temperature range. The DPO-PCR method showed high specificity, in tested strains, 4 ETEC strains were positive results and other strains were negative results, and no nonspecific amplifications were produced. Compared with conventional PCR methods, the DPO-PCR method should not require to repeatedly optimize primer parameters in particular annealing temperature, at the same time, the special structure of DPO primers would enhance detection specificity, which provided a new method for fast and accurate detection of pathogens.
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Received: 05 September 2013
Published: 25 November 2013
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