Discrimination of infectious bacteriophage T4 virus by propidium monoazide real-time PCR
Discrimination of infectious bacteriophage T4 virus by propidium monoazide real-time PCR
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DOI：
10.1016/j.jviromet.2010.06.011
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发表时间：
2010-09-01
影响因子：
3.1
通讯作者：
Morato, Jordi
中科院分区：
文献类型：
--
作者：
Fittipaldi, Mariana;Pino Rodriguez, Nancy J.;Morato, Jordi
定量PCR的出现改善了环境中人类病毒病原体的检测。然而，这种方法的严重局限性可能来自于无法区分感染性病毒和已灭活且不代表人类健康危害的病毒。为了评估单叠氮丙啶（PMA）预处理是否是抑制非感染性病毒DNA扩增的良好方法，使用细胞培养物滴定和实时PCR在有和没有PMA预处理的情况下测量噬菌体T4存活。进行加热（85 ° C）和蛋白水解方法。在这些灭活处理后，结果表明PMA预处理方法不适合区分感染性病毒。然而，当进行110摄氏度的热处理时，PMA预处理确实可以区分非传染性病毒和传染性病毒。在这种情况下，PMA与噬菌体14 DNA的有效结合可以用来指示衣壳损伤。因此，PMA预处理可能适用于评估有效的消毒处理，并通过衣壳损伤监测更可靠地了解有助于病毒灭活的因素。PMA-PCR方法可以作为一种快速、廉价的分析工具，用于消毒剂的筛选和效果评价。(C)2010年爱思唯尔学士/。All rights reserved.
The advent of quantitative PCR has improved the detection of human viral pathogens in the environment. However, a serious limitation of this method may arise from the inability to discriminate between viruses that are infectious and viruses that have been inactivated and do not represent a human health hazard. To assess whether propidium monoazide (PMA) pre-treatment is a good approach to inhibiting DNA amplification from non-infectious viruses, bacteriophage T4 survival was measured using cell culture titration and real-time PCR with and without PMA pre-treatment. Heat (85 degrees C) and proteolysis methods were carried out. After these inactivation treatments, the results indicated that the PMA pre-treatment approach is not appropriate for differentiating infectious viruses. However, when a heat treatment at 110 degrees C was undertaken, PMA pre-treatment did allow differentiation of non-infectious from infectious viruses. In this case, effective binding of PMA to bacteriophage 14 DNA could be taken to indicate capsid damage. Therefore, PMA pre-treatment may be appropriate for assessing effective disinfection treatments and for a more reliable understanding of the factors that contribute to viral inactivation through capsid damage monitoring. The PMA-PCR approach could be useful as a rapid and inexpensive analytical tool for screening and evaluation of the efficacy of disinfectants. (C) 2010 Elsevier BA/. All rights reserved.