Food Science Program, Div. of Food Systems and Bioengineering, 256 WCS Wing, Eckles Hall, Univ. of Missouri, Columbia, MO 65211, USA.
J Food Sci. 2010 Apr;75(3):M134-9. doi: 10.1111/j.1750-3841.2010.01525.x.
Culture-based Salmonella detection takes at least 4 d to complete. The use of TaqMan probes allows the real-time PCR technique to be a rapid and sensitive way to detect foodborne pathogens. However, unlike RNA-based PCR, DNA-based PCR techniques cannot differentiate between DNA from live and dead cells. Ethidium bromide monoazide (EMA) is a dye that can bind to DNA of dead cells and prevent its amplification by PCR. An EMA staining step prior to PCR allows for the effective inhibition of false positive results from DNA contamination by dead cells. The aim of this study was to design an accurate detection method that can detect only viable Salmonella cells from poultry products. The sensitivity of EMA staining coupled with real-time PCR was compared to that of an RNA-based reverse transcription (RT)-real-time PCR. To prevent false negative results, an internal amplification control was added to the same reaction mixture as the target Salmonella sequences. With an optimized EMA staining step, the detection range of a subsequent real-time PCR was determined to be 10(3) to 10(9) CFU/mL for pure cultures and 10(5) to 10(9) CFU/mL for food samples, which was a wider detection range than for RT-real-time PCR. After a 12-h enrichment step, EMA staining combined with real-time PCR could detect as low as 10 CFU/mL Salmonella from chicken rinses and egg broth. The use of EMA with a DNA-based real-time PCR can successfully prevent false positive results and represents a simple, yet accurate detection tool for enhancing the safety of food.
基于培养的沙门氏菌检测需要至少 4 天才能完成。TaqMan 探针的使用使实时 PCR 技术成为一种快速、灵敏的方法来检测食源性病原体。然而，与基于 RNA 的 PCR 不同，基于 DNA 的 PCR 技术无法区分活细胞和死细胞的 DNA。溴化乙锭单脒（EMA）是一种可以与死细胞 DNA 结合并阻止其通过 PCR 扩增的染料。在 PCR 之前进行 EMA 染色步骤可以有效抑制来自死细胞 DNA 污染的假阳性结果。本研究旨在设计一种仅能从禽产品中检测出活沙门氏菌细胞的准确检测方法。EMA 染色与实时 PCR 的灵敏度与基于 RNA 的逆转录（RT）-实时 PCR 进行了比较。为了防止假阴性结果，在与目标沙门氏菌序列相同的反应混合物中添加了内部扩增对照。通过优化 EMA 染色步骤，随后的实时 PCR 的检测范围确定为纯培养物为 10(3)到 10(9) CFU/mL，食品样品为 10(5)到 10(9) CFU/mL，比 RT-实时 PCR 的检测范围更广。经过 12 小时的富集步骤，EMA 染色结合实时 PCR 可以从鸡肉冲洗液和鸡蛋肉汤中检测到低至 10 CFU/mL 的沙门氏菌。EMA 与基于 DNA 的实时 PCR 一起使用可以成功防止假阳性结果，并代表了一种简单但准确的检测工具，可提高食品安全。
