双重DPO-PCR检测副溶血弧菌和霍乱弧菌
Multiplex DPO-PCR for the detection of Vibrio parahaemolyticus and Vibrio cholerae
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摘要: 根据副溶血弧菌collagenase基因和霍乱弧菌omp W基因,分别设计特异性DPO（dual priming oligonucleotide）引物,建立一种快速检测这两种弧菌的多重DPO-PCR方法,并对其特异性和灵敏度进行了评价。结果显示,设计的DPO引物特异性较强,副溶血弧菌和霍乱弧菌DNA可分别扩增出307 bp与463 bp的特异性条带,检测灵敏度均达0.1 ng/μL。该检测方法对退火温度不敏感。利用该方法对69株疑似弧菌菌株进行鉴定,结果与生理生化鉴定结果一致。该方法特异性强、灵敏度高,适合于对食品、水产品等中副溶血弧菌和霍乱弧菌的进行快速筛检。Abstract: Based on collagenase gene sequences of Vibrio parahaemolyticus and omp W gene sequences of V.cholerae,designed two pairs of DPO( dual priming oligonucleotide) primers. A multiplex DPO-PCR method had been developed for the detection of V. parahaemolyticus and V. cholerae simultaneously. The specificity and sensitivity of the method had been tested. The result showed that the DPO primers were of high specificity.Amplified fragments 307 bp for V.parahaemolyticus and 463 bp for V.cholerae were obtained,the sensitivity of the method was 0.1 ng / μL for V. parahaemolyticus and V. cholerae. The multiplex DPO-PCR was not sensitive to the annealing temperature.The multiplex DPO- PCR was validated with 69 suspicious vibrio strains and the results was consistent with physiological and biochemical experiments. The multiplex DPO-PCR method proved to be strong specificity,high sensitivity.This method was suitable for the detection of V.parahaemolyticus and V.cholera in food,aquatic products,etc.
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