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中国精品科技期刊2020
方佩佩, 赵丽青, 马云, 王昌军, 唐静, 李正义, 贾俊涛, 姜英辉. 副溶血性弧菌微滴数字PCR定量方法的建立[J]. 食品工业科技, 2018, 39(19): 252-257. DOI: 10.13386/j.issn1002-0306.2018.19.045
引用本文: 方佩佩, 赵丽青, 马云, 王昌军, 唐静, 李正义, 贾俊涛, 姜英辉. 副溶血性弧菌微滴数字PCR定量方法的建立[J]. 食品工业科技, 2018, 39(19): 252-257. DOI: 10.13386/j.issn1002-0306.2018.19.045
FANG Pei-pei, ZHAO Li-qing, MA Yun, WANG Chang-jun, TANG Jing, LI Zheng-yi, JIA Jun-tao, JIANG Ying-hui. Establishment of Digital PCR Assay for Detection of Vibrio parahemolyticus[J]. Science and Technology of Food Industry, 2018, 39(19): 252-257. DOI: 10.13386/j.issn1002-0306.2018.19.045
Citation: FANG Pei-pei, ZHAO Li-qing, MA Yun, WANG Chang-jun, TANG Jing, LI Zheng-yi, JIA Jun-tao, JIANG Ying-hui. Establishment of Digital PCR Assay for Detection of Vibrio parahemolyticus[J]. Science and Technology of Food Industry, 2018, 39(19): 252-257. DOI: 10.13386/j.issn1002-0306.2018.19.045

副溶血性弧菌微滴数字PCR定量方法的建立

Establishment of Digital PCR Assay for Detection of Vibrio parahemolyticus

  • 摘要: 目的:采用微滴式数字PCR技术(Droplet digital PCR,ddPCR),建立副溶血性弧菌快速定量检测。方法:以副溶血性弧菌TLH基因为靶基因,验证确定ddPCR方法的特异性及定量检测的线性范围,以常检出副溶血性弧菌的海产品鳕鱼为样品进行阳性添加,确定方法的可行性。结果:本试验中采用ddPCR检测得到副溶血性弧菌特异性好,有效基因组DNA浓度范围为2~19440拷贝/20μL,分析得菌悬液浓度为50~4.86×105 CFU/mL,与3M测试片所得菌悬液浓度无显著性差异(p>0.05),与荧光PCR相比,可以进行更低浓度检测且能准确定量。结论:副溶血性弧菌ddPCR定量检测技术特异性良好、灵敏度高、结果准确,具有实际应用价值。

     

    Abstract: Objective:To establish a quantitative accuracy method to detect Vibrio parahemolyticus based on droplet digital PCR. Methods:With the specific sets of primers and probe synthesized to target genes including TLH genes, specificity and sensitivity were tested based on ddPCR and quantitative accuracy analysis was performed on the results. In order to verify the feasibility of the method, positive strain was added to seafood in which was often detected Vibrio parahaemolyticus.Conclusion:Vibrio parahaemolyticus performed good specificity in ddPCR detection in this experiment. The range of effective DNA concentration was 2~19440 copies/20μL. Bacterial suspension concentration was 50~4.86×105CFU/mL, which had no significant difference with 3M test (p>0.05). Compared with real-time PCR, the concentration limit of detection was enhanced by two orders of magnitude. The results showed that quantitative detection of Vibrio parahaemolyticus by ddPCR technology had good specificity, high sensitivity and accuracy, which had practical application value.

     

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