Friedman Stephanie D, Cooper Emilie M, Casanova Lisa, Sobsey Mark D, Genthner Fred J
US Environmental Protection Agency, Gulf Ecology Division, Gulf Breeze, FL, 32561, USA.
J Virol Methods. 2009 Jul;159(1):47-52. doi: 10.1016/j.jviromet.2009.02.028. Epub 2009 Mar 9.
Goals of reducing fecal contamination in recreational, drinking, shellfishing and other waters and accurately assessing risk from exposure can best be attained if tools to distinguish between sources of pollution are available. The male-specific RNA coliphage (FRNA) genogroups display a trend of source specificity. Reverse transcription-PCR (RT-PCR) can be effectively used for genotyping if specific primer sets are designed to be capable of identifying all members within each genogroup. In this study genogroup-specific primer sets were designed using a minimum of 5 to a maximum of 10 complete phage genome sequences from strains in each genogroup. With these primers and employing a heat-release procedure that eliminated the need for RNA purification an RT-PCR method for genotype identification of FRNA phages was developed. The four genogroup-specific primer sets generated discrete PCR amplicon sizes from a variety of environmental FRNA phage strains. Limits of detection, cross-reactivity and/or non-specific binding to strains from other genogroups were evaluated.
如果有区分污染源的工具，那么在减少娱乐用水、饮用水、贝类养殖用水和其他水体中的粪便污染以及准确评估接触风险等目标方面就能最好地实现。雄性特异性RNA噬菌体（FRNA）基因组群呈现出源特异性趋势。如果设计出能够识别每个基因组群内所有成员的特异性引物组，逆转录聚合酶链反应（RT-PCR）就能有效地用于基因分型。在本研究中，使用每个基因组群中最少5个至最多10个菌株的完整噬菌体基因组序列设计了基因组群特异性引物组。利用这些引物并采用一种无需RNA纯化的热释放程序，开发了一种用于FRNA噬菌体基因分型鉴定的RT-PCR方法。这四组基因组群特异性引物从多种环境FRNA噬菌体菌株中产生了离散的PCR扩增子大小。评估了检测限、交叉反应性和/或与其他基因组群菌株的非特异性结合。
