德尔卑沙门氏菌分子等温活菌检测体系的建立

Establishment of an isothermal amplification method for detection of viable Salmonella enterica subsp. enterica Derby

摘要:

【背景】德尔卑沙门氏菌(Salmonella enterica subsp. enterica Derby)是危害人类生命安全的主要致病性血清型。【目的】建立一种准确、快速检测德尔卑沙门氏菌的方法。【方法】通过建立叠氮溴化丙锭(propidium monoazide，PMA)-重组酶聚合酶扩增(recombinase polymerase amplification，RPA)的方法准确有效地检测样品中的活德尔卑沙门氏菌。【结果】使用基因RU61_00441作为检测靶点，设计引物SD1正确地鉴定了所有被测菌株。实验结果表明，PMA处理能有效区分活细胞和死细胞。基因组DNA检测限为761.2 fg/μL，活菌检测限为45 CFU/mL。此方法检测德尔卑沙门氏菌的血清型不受自然背景(猪肉、鸡肉和牛肉)菌群基因组DNA的影响。此外，该方法还可以检测出动物性食品中富集6 h后浓度低至3.9 CFU/mL的德尔卑沙门氏菌。【结论】这种PMA-RPA检测方法耗时短并具有更好的灵敏度和特异性，能为沙门氏菌的检测提供更有效的指导。

Abstract:

[Background] Salmonella enterica subsp. enterica Derby as a major pathogenic serotype is harmful to the public health.[Objective]To build a rapid and accurate approach for the detection of this serotype.[Methods]An accurate and effective method was built with propidium monoazide (PMA)-recombinase polymerase amplification (RPA) for the detection of active S.Derby.[Results]The assay used RU61_00441 gene as target and designed the primer SD1F/R which correctly identified all the tested strains.The PMA treatment effectively distinguished between viable and dead cells.The limit of detection was calculated to be 761.2 fg/μL for genomic DNA and 45 CFU/mL for bacterial culture.For the detection of the serotype Derby,this assay was not affected by the genomic DNA of background flora (pork,chicken,and beef).Importantly,S.Derby in animal-derived food could be detected at a concentration as low as 3.9 CFU/mL after enrichment for 6 h.[Conclusion]This PMA-RPA method is time-saving and has good sensitivity and specificity,which can provide reference for the future detection of Salmonella.