Paar Jack, Doolittle Mark M, Varma Manju, Siefring Shawn, Oshima Kevin, Haugland Richard A
U.S. Environmental Protection Agency, New England Regional Laboratory, North Chelmsford, MA, USA.
ESAT Contractor-TechLaw, Inc., EPA New England Regional Laboratory, North Chelmsford, MA, USA.
J Microbiol Methods. 2015 May;112:28-35. doi: 10.1016/j.mimet.2015.02.015. Epub 2015 Mar 3.
A method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for detecting interferences in RNA recovery and analysis, was developed for the direct, culture-independent detection of genetic markers from FRNA coliphage genogroups I, II & IV in water samples. Results were obtained from an initial evaluation of the performance of this method in analyses of waste water, ambient surface water and stormwater drain and outfall samples from predominantly urban locations. The evaluation also included a comparison of the occurrence of the FRNA genetic markers with genetic markers from general and human-related bacterial fecal indicators determined by current or pending EPA-validated qPCR methods. Strong associations were observed between the occurrence of the putatively human related FRNA genogroup II marker and the densities of the bacterial markers in the stormwater drain and outfall samples. However fewer samples were positive for FRNA coliphage compared to either the general bacterial fecal indicator or the human-related bacterial fecal indicator markers particularly for ambient water samples. Together, these methods show promise as complementary tools for the identification of contaminated storm water drainage systems as well as the determination of human and non-human sources of contamination.
开发了一种方法，该方法结合了最近改进的逆转录酶 - PCR引物/探针检测方法，并包括用于检测RNA回收和分析中干扰的对照，用于直接、无需培养地检测水样中FRNA噬菌体基因组I、II和IV的遗传标记。从该方法在分析废水、环境地表水以及主要来自城市地区的雨水排水和排放口样品中的性能初步评估中获得了结果。该评估还包括将FRNA遗传标记的出现情况与通过当前或待EPA验证的qPCR方法确定的一般和人类相关细菌粪便指示物的遗传标记进行比较。在雨水排水和排放口样品中，假定与人类相关的FRNA基因组II标记的出现与细菌标记的密度之间观察到强关联。然而，与一般细菌粪便指示物或人类相关细菌粪便指示物标记相比，FRNA噬菌体阳性的样品较少，特别是对于环境水样。总之，这些方法有望作为互补工具，用于识别受污染的雨水排水系统以及确定人类和非人类污染源。
