Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C, Denmark.
Int J Food Microbiol. 2010 Jul 31;141 Suppl 1:S60-8. doi: 10.1016/j.ijfoodmicro.2010.01.042. Epub 2010 Feb 10.
Quantitative real time polymerase chain reaction (qRT PCR) was used to compare the relative transcription of prfA, inlA, sigB and clpC for three Listeria monocytogenes strains after incubation in i) a standard liver pâté versus brain heart infusion (BHI) broth and ii) the standard liver pâté versus three liver pâtés with reduced NaCl content of which one also has been supplied with organic acids (Ca-acetate and Ca-lactate). The three strains (EGD-e: reference strain; O57: more NaCl sensitive; 6896: more NaCl tolerant) were selected out of twelve strains based on their growth in BHI broth adjusted to 6%, 8%, 10% (w/v) NaCl. The three strains were spiked into the liver pâtés (10(9) cfu/g) and the BHI (10(9) cfu/ml) and incubated for 48 h at 7 degrees C; all incubation conditions supported growth of the strains. Extraction of intact listerial RNA from the liver pâtés was complicated by the complexity of the liver pâté matrix. However, a method has been optimized and described, and the quality of RNA extracted from liver pâtés was equal to the quality of RNA extracted from BHI. The amplification efficiencies of the six genes used for the transcription analyses (the four target genes and two reference genes, gap and rpoB) were within the acceptable range from 90% to 110% for all three strains in both liver pâté and BHI. Comparison of the three strains after incubation in the standard liver pâté and BHI showed that the relative transcription of prfA for O57 and the relative transcription of inlA and sigB for both O57 and 6896 were significantly higher when the strains were grown in BHI compared to the standard liver pâté. Reducing the NaCl content of the standard liver pâté did not change relative transcription levels of prfA, inlA, sigB or clpC (except for prfA in O57 and sigB in 6896). However, the presence of Ca-acetate and Ca-lactate induced relative transcription of the stress response gene, clpC, for all three strains. This study demonstrates that relative microbial gene transcription can be measured in complex food matrices and points to the need for designing experimental set-ups in real food matrices to replace the laboratory model systems. With respect to L. monocytogenes, it seems that the NaCl content of liver pâté can be lowered within the investigated range without significant changes in relative virulence gene transcription while more caution should be taken when adding organic acids such as acetate and lactate.
采用实时荧光定量聚合酶链反应（qRT-PCR）比较了 3 株单核细胞增生李斯特菌在以下条件下的 prfA、inlA、sigB 和 clpC 相对转录水平：i）在标准肝泥与脑心浸液（BHI）肉汤中孵育，ii）在标准肝泥与 3 种盐含量降低的肝泥中孵育，其中 1 种肝泥还添加了有机酸（Ca-醋酸盐和 Ca-乳酸盐）。这 3 株菌（EGD-e：参考株；O57：更敏感于 NaCl；6896：更耐受 NaCl）是从 12 株菌中根据它们在调整至 6%、8%和 10%（w/v）NaCl 的 BHI 肉汤中的生长情况选择的。将这 3 株菌（10(9)cfu/g）接种到肝泥（10(9)cfu/ml）和 BHI 中，并在 7°C 下孵育 48 小时；所有孵育条件均支持菌株生长。从肝泥中提取完整李斯特菌 RNA 比较复杂，因为肝泥基质较为复杂。然而，已经优化并描述了一种方法，并且从肝泥中提取的 RNA 质量与从 BHI 中提取的 RNA 质量相当。用于转录分析的 6 个基因（4 个靶基因和 2 个参考基因 gap 和 rpoB）的扩增效率对于所有 3 株菌在肝泥和 BHI 中的扩增效率均在 90%至 110%的可接受范围内。将 3 株菌在标准肝泥和 BHI 中的孵育结果进行比较后发现，与在标准肝泥中相比，O57 的 prfA 相对转录水平和 O57 与 6896 的 inlA 和 sigB 相对转录水平在 BHI 中更高。降低标准肝泥中的 NaCl 含量不会改变 prfA、inlA、sigB 或 clpC 的相对转录水平（O57 的 prfA 和 6896 的 sigB 除外）。然而，Ca-醋酸盐和 Ca-乳酸盐的存在诱导了所有 3 株菌应激反应基因 clpC 的相对转录。本研究表明，可以在复杂的食品基质中测量微生物基因的相对转录水平，并指出需要在实际食品基质中设计实验方案以替代实验室模型系统。就单核细胞增生李斯特菌而言，在研究范围内降低肝泥中的 NaCl 含量而不会显著改变相对毒力基因转录似乎是可行的，而在添加醋酸盐和乳酸盐等有机酸时则需要更加谨慎。
