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首页> 外文期刊>Applied Microbiology >Innovative Use of Palladium Compounds To Selectively Detect Live Enterobacteriaceae in Milk by PCR
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Innovative Use of Palladium Compounds To Selectively Detect Live Enterobacteriaceae in Milk by PCR

机译：通过PCR创新地使用钯化合物选择性检测牛奶中的活肠杆菌科

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Ethidium monoazide and propidium monoazide (EMA and PMA) have been used in combination with PCR for more than a decade to facilitate the discrimination of live and dead bacteria (LD discrimination). These methods, however, require many laborious procedures, including the use of a darkroom. Here, we demonstrate an innovative use of palladium compounds involving lower limits of detection and quantification of targeted live cells, fewer laborious procedures, lower costs, and potentially higher-throughput analysis than the use of EMA and PMA. We have also recently reported platinum compounds for LD discrimination, but platinum compounds carry costs that are 3 times higher because of the requirement for much larger amounts for LD discrimination than palladium compounds. Palladium compounds can penetrate dead (compromised) but not live bacteria and can be chelated primarily by chromosomal DNA and cell wall transmembrane proteins, with small amounts of DNA-binding proteins in vivo . The new mechanism for palladium compounds is obviously different from that of platinum compounds, which primarily target DNA. Combining palladium compounds with PCR (Pd-PCR) in water resulted in discrimination between live and dead Enterobacteriaceae bacteria that was much clearer than that seen with the PMA method. Pd-PCR correlated with reference plating or with the currently used PMA-PCR method for pasteurized milk, based on EN ISO 16140:2003 validation. Pd-PCR enabled us to specifically detect and assay viable Enterobacteriaceae cells at concentrations of 5 to 10 CFU/ml in milk while following U.S./EU regulations after a 4.5-h process in a typical laboratory exposed to natural or electric light, as specified by U.S./EU regulations.IMPORTANCE Ethidium monoazide and propidium monoazide (EMA and PMA) facilitate the discrimination of live and dead bacteria (LD discrimination). These methods, however, require many laborious procedures, including the use of a darkroom. Here, we demonstrate an innovative use of palladium compounds involving fewer laborious procedures, lower costs, and potentially higher-throughput analysis than the use of EMA and PMA. We have also recently reported platinum compounds for LD discrimination, but platinum compounds carry costs that are 3 times higher because of the requirement for much larger amounts for LD discrimination than palladium compounds, which have also a novel reaction mechanism different from that of platinum compounds. In view of testing cost, palladium compounds are also very useful here compared with platinum compounds. Ultimately, the innovative Pd-PCR method may be also substituted for the currently used reference plating methods.
机译：单叠氮化乙锭和单叠氮化丙锭（EMA和PMA）已与PCR结合使用了十多年，以促进活细菌和死细菌的鉴别（LD鉴别）。然而，这些方法需要许多费力的过程，包括使用暗室。在这里，我们展示了钯化合物的创新使用，与使用EMA和PMA相比，它具有更低的目标活细胞检测和定量限，更少的繁琐操作，更低的成本以及潜在的更高通量分析。我们最近还报道了用于LD鉴别的铂化合物，但是铂化合物的成本比钯化合物高出3倍，因为LD鉴别所需的量要大得多。钯化合物可以穿透死的（受损的）细菌，但不能穿透活细菌，并且可以主要被染色体DNA和细胞壁跨膜蛋白螯合，体内具有少量的DNA结合蛋白。钯化合物的新机制明显不同于主要靶向DNA的铂化合物。将钯化合物与PCR（Pd-PCR）结合在水中可导致活细菌和死肠杆菌科细菌之间的区别，这种区别比PMA方法清晰得多。基于EN ISO 16140：2003验证，Pd-PCR与参考平板或当前用于巴氏灭菌牛奶的PMA-PCR方法相关。 Pd-PCR使我们能够在牛奶中5至10 CFU / ml的浓度下特异性检测和分析活肠杆菌科细胞，同时在暴露于自然或电光的典型实验室中经过4.5小时的处理后遵循美国/欧盟法规，美国/欧盟法规重要提示单叠氮化乙锭和单叠氮化丙锭（EMA和PMA）有助于区分活细菌和死细菌（LD歧视）。然而，这些方法需要许多费力的过程，包括使用暗室。在这里，我们展示了钯化合物的创新使用，与使用EMA和PMA相比，涉及的劳动更少，成本更低，分析通量可能更高。最近，我们还报道了用于LD鉴别的铂化合物，但是铂化合物的成本要比钯化合物高三倍，因为与LD化合物相比，钯化合物的LD鉴别量要大得多，钯化合物的反应机理也不同于铂化合物。考虑到测试成本，与铂化合物相比，钯化合物在这里也非常有用。最终，创新的Pd-PCR方法也可以替代当前使用的参考平板方法。

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