Detection of Salmonella cells based on SD- PMA- dd PCR
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摘要: 研究将叠氮溴化丙锭（PMA）与微滴数字PCR（dd PCR）技术相结合,建立一种沙门氏菌活菌的检测方法。结果表明,PMA不能完全有效抑制108CFU/m L的沙门氏菌死菌DNA的PCR扩增,0.1%脱氧胆酸钠（SD）和40.0μg/m L PMA协同作用,可以有效抑制108CFU/m L的沙门氏菌死菌DNA的PCR扩增,不抑制沙门氏菌活菌DNA扩增的PMA最高浓度是40.0μg/m L。经过SD和PMA对样品预处理,在不同死、活菌比例下,PMA-dd PCR可以定量检测活菌,避免了死菌DNA的干扰。本方法的检出限为8.0 copy/20μL。利用PMA-dd PCR检测人工污染鸡肉样品,最低可检出102CFU/m L的沙门氏菌。实验结果证明PMA-dd PCR方法的特异性、精确度、稳定性良好。Abstract: In this paper,a method to detect live Salmonella cells was developed based on propidium monoazide( PMA) and dd PCR.The result showed that PMA could not inhibit the DNA PCR amplification of 108 CFU / m L dead Salmonella cells,while the combination of sodium deoxycholate( SD) and 40.0 μg / m L PMA could do it. The maximum concentration against DNA amplification from live Salmonella cells was 40.0 μg / m L.Only live Salmonella cells were detected by PMA- dd PCR even in the existence of dead Salmonella cells,and the detection limit was8.0 copy /20 μL. PMA- dd PCR could detect 102 CFU / m L Salmonella cells in the polutted sample made by manual work. Furthermore,PMA- dd PCR showed better specificity,accuracy and stability.
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Keywords:
- propidium monoazide /
- sodium deoxycholate /
- dd PCR /
- live cells
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