Zhang Jiawei, Khan Samiullah, Chousalkar Kapil K
School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA, Australia.
Front Microbiol. 2020 Sep 22;11:581201. doi: 10.3389/fmicb.2020.581201. eCollection 2020.
Determining the viable and non-viable load of foodborne pathogens in animal production can be useful in reducing the number of human outbreaks. In this study, we optimized a PMAxx-based qPCR for quantifying viable and non-viable load of from soil collected from free range poultry environment. The optimized nucleic acid extraction method resulted in a significantly higher ( < 0.05) yield and quality of DNA from the pure culture and inoculated soil samples. The optimized primer for the amplification of the gene fragment showed high target specificity and a minimum detection limit of 10 viable from soil samples. To test the optimized PMAxx-based qPCR assay, soil obtained from a free range farm was inoculated with Enteritidis or Typhimurium, incubated at 5, 25, and 37°C over 6 weeks. The survivability of Typhimurium was significantly higher than Enteritidis. Both the serovars showed moisture level dependent survivability, which was significantly higher at 5°C compared with 25°C and 37°C. The PMAxx-based qPCR was more sensitive in quantifying the viable load compared to the culture method used in the study. Data obtained in the current study demonstrated that the optimized PMAxx-based qPCR is a suitable assay for quantification of a viable and non-viable load of from poultry environment. The developed assay has applicability in poultry diagnostics for determining the load of important serovars containing .
确定动物生产中食源性病原体的活菌和死菌负载量有助于减少人类疾病暴发的数量。在本研究中，我们优化了一种基于PMAxx的qPCR方法，用于定量从自由放养家禽环境中采集的土壤中 的活菌和死菌负载量。优化后的核酸提取方法使纯培养物和接种土壤样品的DNA产量和质量显著提高（<0.05）。用于扩增 基因片段的优化引物显示出高靶标特异性，土壤样品中活菌 的最低检测限为10个。为了测试优化后的基于PMAxx的qPCR检测方法，将从自由放养农场获得的土壤接种肠炎沙门氏菌或鼠伤寒沙门氏菌，在5、25和37°C下孵育6周。鼠伤寒沙门氏菌的存活率显著高于肠炎沙门氏菌。两种血清型均表现出依赖水分水平的存活率，在5°C时显著高于25°C和37°C。与本研究中使用的培养方法相比，基于PMAxx的qPCR在定量活菌负载量方面更敏感。本研究获得的数据表明，优化后的基于PMAxx的qPCR是一种适用于定量家禽环境中 活菌和死菌负载量的检测方法。所开发的检测方法在禽类诊断中可用于确定含有 重要血清型的负载量。
