BioTechnology Institute, University of Minnesota, St. Paul, MN, United States.
BioTechnology Institute, University of Minnesota, St. Paul, MN, United States; Department of Soil, Water, and Climate, University of Minnesota, St. Paul, MN, United States; Division of Environmental Engineering, Hokkaido University, Sapporo, Hokkaido, Japan.
Water Res. 2018 Jun 15;137:193-200. doi: 10.1016/j.watres.2018.03.023. Epub 2018 Mar 10.
Quantitative polymerase chain reaction (qPCR) is now commonly used to detect fecal indicator bacteria (FIB) as well as pathogens in water samples. However, DNA loss during sample processing can cause underestimation of target genes. In this study, we created a sample process control strain (SPC) by genetically engineering a non-pathogenic, Gram-negative bacterium Pseudogulbenkiania sp. strain NH8B. The SPC strain, named NH8B-1D2, has a kanamycin-resistance gene inserted to one of the 23S rRNA genes. To specifically quantify the SPC strain, a new TaqMan qPCR assay was developed. To obtain the relationship between the DNA recovery efficiencies of various pathogens and those of the SPC strain, known amount of E. coli O157:H7, Salmonella Typhimurium, Campylobacter jejuni, or Listeria monocytogenes cells were co-spiked with the SPC strain to environmental water samples. The DNA recovery efficiencies were calculated by comparing the quantity of bacterial cells inoculated to water samples prior to filtration and DNA extraction, and those measured by qPCR. We then obtained the ratios in the recovery efficiencies between pathogens and SPC strain (RR). The RR values obtained using Oono pond water collected in Japan were used as a pathogen-specific constant to estimate the accurate concentrations of pathogens in water samples collected from Mississippi River in Minnesota. Estimated pathogen concentrations were not significantly different from the inoculated pathogen concentration, suggesting our normalization approach is useful to estimate the accurate concentrations of pathogens in environmental water samples. The qPCR assay targeting the SPC strains and FIB were incorporated into the microfluidic qPCR chip format (PBQ chip ver. 2); therefore, we can simultaneously quantify multiple pathogens, FIB, and the SPC strain in high throughput from many water samples. This new tool can be useful for water quality monitoring and risk assessment.
定量聚合酶链反应（qPCR）现在常用于检测水样中的粪便指示菌（FIB）和病原体。然而，样品处理过程中的 DNA 损失可能导致目标基因的低估。在这项研究中，我们通过遗传工程改造一种非致病性的革兰氏阴性菌 Pseudogulbenkiania sp. strain NH8B 来创建一个样本处理对照菌株（SPC）。该 SPC 菌株命名为 NH8B-1D2，其一个 23S rRNA 基因中插入了一个卡那霉素抗性基因。为了专门定量 SPC 菌株，我们开发了一种新的 TaqMan qPCR 检测方法。为了获得各种病原体的 DNA 回收效率与 SPC 菌株的 DNA 回收效率之间的关系，我们将已知数量的 E. coli O157:H7、鼠伤寒沙门氏菌、空肠弯曲菌或单增李斯特菌细胞与 SPC 菌株共同掺入到环境水样中。通过比较过滤和 DNA 提取前接种到水样中的细菌细胞数量与 qPCR 测量的数量，计算 DNA 回收率。然后，我们获得了病原体与 SPC 菌株之间的回收率比值（RR）。使用从日本采集的 Oono 池塘水获得的 RR 值被用作病原体特异性常数，以估计从明尼苏达州密西西比河采集的水样中病原体的准确浓度。估计的病原体浓度与接种的病原体浓度没有显著差异，表明我们的归一化方法可用于估计环境水样中病原体的准确浓度。针对 SPC 菌株和 FIB 的 qPCR 检测已被整合到微流控 qPCR 芯片格式（PBQ 芯片 ver. 2）中；因此，我们可以从许多水样中高通量地同时定量多个病原体、FIB 和 SPC 菌株。这种新工具可用于水质监测和风险评估。
