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Development of a real-time PCR melt curve assay for simultaneous detection of virulent and antibiotic resistant Salmonella

机译：实时PCR熔解曲线测定法的开发，可同时检测强毒和耐药性沙门氏菌

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摘要

Multiple drug resistance in Salmonella is an emerging problem in the area of food safety. Depending on the virulence and antibiotic resistance characteristics of the Salmonella strain, infections of varying severity could result. In this study, a multiplex melt curve real-time PCR assay for the detection of virulent and antibiotic resistance strains of Salmonella was developed with two primer sets. The first set targets the virulence gene, invasin (invA), and tetracycline (tetG), streptomycin (aadA2) and sulphona-mide (sulI) antibiotic resistance genes, and the second set amplifies ampicillin (bla_(PSE), bla_(TEM)) and chloramphenicol (floR) resistance genes. The multiplex assay was evaluated using 41 Salmonella strains and was further tested on eight different artificially inoculated food samples. The fluorescent DNA intercalating dye, SYTO9, generated high resolution melt curve peaks and, hence, was used for the development of the assay. This multiplex assay worked efficiently over a DNA concentration range of 20 ng-200 fg and showed a sensitivity of 290 CFU/mL with serially diluted broth cultures. The detection limit for un-enriched artificially inoculated food samples was 10~4 CFU/g, but an enrichment period of 6 h allowed for detection of 10 CFU/g of cells in the samples.
机译：沙门氏菌的多重耐药性是食品安全领域中一个正在出现的问题。根据沙门氏菌菌株的毒力和抗生素抗性特征，可能会导致感染程度不同。在这项研究中，使用两个引物组开发了用于检测沙门氏菌的强毒和抗生素耐药菌株的多重熔解曲线实时PCR检测方法。第一组靶向毒力基因，invasin（invA）和四环素（tetG），链霉素（aadA2）和磺胺类（sulI）抗生素抗性基因，第二组扩增氨苄西林（bla_（PSE），bla_（TEM） ）和氯霉素（floR）抗性基因。使用41种沙门氏菌菌株评估了多重分析，并在8种不同的人工接种食品样品上进行了进一步测试。荧光DNA嵌入染料SYTO9产生高分辨率的熔解曲线峰，因此被用于测定的开发。该多重分析在20 ng-200 fg的DNA浓度范围内有效工作，并且在连续稀释的肉汤培养物中显示出290 CFU / mL的灵敏度。未富集的人工接种食品样品的检出限为10〜4 CFU / g，但经过6 h的富集期，样品中细胞的检出率为10 CFU / g。

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