Real-time fluorogenic reverse transcription-PCR assays for detection of bacteriophage MS2
Real-time fluorogenic reverse transcription-PCR assays for detection of bacteriophage MS2
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DOI：
10.1128/aem.72.1.478-483.2006
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发表时间：
2006-01-01
影响因子：
4.4
通讯作者：
Valdes, JJ
中科院分区：
文献类型：
--
作者：
O'Connell, KP;Bucher, JR;Valdes, JJ
噬菌体MS2在各种各样的研究中被用来代替病原性病毒，这些研究的范围从测试用于消毒表面的化合物到研究地下水中病原性病毒的环境迁移和归宿。MS2还被用作病原体模拟物，用于战场和国土防御环境中病原体检测方法、系统和设备的研究、开发和测试（包括露天测试）。PCR通常用作此类检测系统的组成部分或用作评估微生物检测的灵敏度和特异性的参考方法。为了便于检测MS2的PCR，我们在这里描述了一套实时荧光逆转录PCR检测。测定的灵敏度（用引物对和相应的染料标记探针进行）范围为0.4至40 fg的MS2基因组RNA（200至20，000基因组当量）。我们还证明了在没有染料标记的探针，包括DNA结合染料SYBR绿色的检测引物对的有用性。当针对400 pg的几种非MS2核酸靶标进行检测时，所有检测均未得到假阳性结果。
Bacteriophage MS2 is used in place of pathogenic viruses in a wide variety of studies that range from testing of compounds for disinfecting surfaces to studying environmental transport and fate of pathogenic viruses in groundwater. MS2 is also used as a pathogen simulant in the research, development, and testing (including open air tests) of methods, systems, and devices for the detection of pathogens in both the battlefield and homeland defense settings. PCR is often used as either an integral part of such detection systems or as a reference method to assess the sensitivity and specificity of microbial detection. To facilitate the detection of MS2 by PCR, we describe here a set of real-time fluorogenic reverse transcription-PCR assays. The sensitivity of the assays (performed with primer pairs and corresponding dye-labeled probes) ranged from 0.4 to 40 fg of MS2 genomic RNA (200 to 20,000 genome equivalents). We also demonstrate the usefulness of the primer pairs in assays without dye-labeled probe that included the DNA-binding dye SYBR green. None of the assays gave false-positive results when tested against 400 pg of several non-MS2 nucleic acid targets.