Biosynthetic enhancement of the detection of bacteria by the polymerase chain reaction.
Biosynthetic enhancement of the detection of bacteria by the polymerase chain reaction.
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DOI：
10.1371/journal.pone.0086433
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Cangelosi GA
中科院分区：
文献类型：
--
作者：
Do JS;Weigel KM;Meschke JS;Cangelosi GA
分子活力测试（MVT）以前曾报道，专门检测复杂样品中的活细菌细胞。在MVT中，短暂的营养刺激诱导活细胞而不是非活细胞产生大量的物种特异性核糖体RNA前体（pre-rRNA）。定量聚合酶链反应（qPCR）用于相对于非刺激对照定量刺激等分试样中的特异性前体rRNA。除了排除来自非活细胞和游离DNA的背景信号外，我们在此报告了MVT在检测活细胞时提高了qPCR的分析灵敏度。并行检测限比较显示，当检测血清、牛奶和自来水中的多种细菌病原体（嗜水气单胞菌、鲍曼不动杆菌、单核细胞增生李斯特菌、鸟分枝杆菌和金黄色葡萄球菌）时，MVT的灵敏度比标准（静态）DNA靶向qPCR高5倍至>10倍。灵敏度增强可能来自于前rRNA相对于基因组DNA的拷贝数升高，也可能来自于减少与弱或边界信号相关的模糊性的比率测量。我们还报告说，MVT消除了假阳性信号，从细菌已被灭活的适度升高的温度（巴氏灭菌），一个条件，可以混淆广泛使用的细胞完整性测试，利用膜不渗透的化合物，如碘化丙啶（PI）或单叠氮丙啶（PMA），以区分活的灭活细菌。MVT能够灵敏和特异地检测复杂基质中极少量的活菌。
Molecular viability testing (MVT) was previously reported to specifically detect viable bacterial cells in complex samples. In MVT, brief nutritional stimulation induces viable cells, but not non-viable cells, to produce abundant amounts of species-specific ribosomal RNA precursors (pre-rRNA). Quantitative polymerase chain reaction (qPCR) is used to quantify specific pre-rRNAs in a stimulated aliquot relative to a non-stimulated control. In addition to excluding background signal from non-viable cells and from free DNA, we report here that MVT increases the analytical sensitivity of qPCR when detecting viable cells. Side-by-side limit-of-detection comparisons showed that MVT is 5-fold to >10-fold more sensitive than standard (static) DNA-targeted qPCR when detecting diverse bacterial pathogens (Aeromonas hydrophila, Acinetobacter baumannii, Listeria monocytogenes, Mycobacterium avium, and Staphylococcus aureus) in serum, milk, and tap water. Sensitivity enhancement may come from the elevated copy number of pre-rRNA relative to genomic DNA, and also from the ratiometric measurement which reduces ambiguity associated with weak or borderline signals. We also report that MVT eliminates false positive signals from bacteria that have been inactivated by moderately elevated temperatures (pasteurization), a condition that can confound widely-used cellular integrity tests that utilize membrane-impermeant compounds such as propidium iodide (PI) or propidium monoazide (PMA) to differentiate viable from inactivated bacteria. MVT enables the sensitive and specific detection of very small numbers of viable bacteria in complex matrices.
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