摘要
Despite advances in microbial detection that quantitative polymerase chain reaction (qPCR) has led to, complex environmental samples, such as sediments, remain a challenge due to presence of PCR inhibitors. Aquatic sediments accumulate particle-bound microbial contaminants and thereby reflect a cumulative microbial load over time. The relatively new droplet digital PCR (ddPCR) has emerged as a direct quantitative method, highly tolerant to PCR inhibitors and relinquishing the necessity for calibration/standard curves. Information is virtually absent where ddPCR has been applied to detect pathogenic organisms in aquatic sediments. This study compared the efficacy of ddPCR with qPCR, for quantification of Salmonella in sediments from the Palmiet River near an informal settlement in Durban, South Africa. ddPCR significantly improved both analytical sensitivity and detection of low concentrations of Salmonella as compared to qPCR. The expected copy numbers measured from both qPCR and ddPCR showed good R2 values (0.999 and 0.994, respectively). The site mostly affected by the informal settlements exhibited Salmonella in the range of 255 ± 37 and 818 ± 30 Salmonella/g (p ≤ 0.0001) in qPCR and ddPCR, respectively. The improved detection of Salmonella in sediments with ddPCR makes it a promising technical method for the quantification of Salmonella in multifarious environmental samples.
摘要译文
尽管定量聚合酶链式反应（qPCR）导致微生物检测方面取得进展，但复杂的环境样品，如沉积物，由于存在PCR抑制剂而仍然是一个挑战。水生沉积物累积颗粒结合的微生物污染物，从而随时间反映累积的微生物负荷。相对较新的液滴数字PCR（ddPCR）作为直接定量方法出现，对PCR抑制剂具有高度耐受性并放弃了校准/标准曲线的必要性。在ddPCR已被用于检测水生沉积物中的病原生物的情况下，几乎没有信息。本研究比较了ddPCR与qPCR的疗效，用于量化南非德班一个非正式定居点附近来自Palmiet River的沉积物中的沙门氏菌。与qPCR相比，ddPCR显着提高了分析灵敏度和低浓度沙门氏菌的检测。从qPCR和ddPCR测量的预期拷贝数显示出良好的R 2值（分别为0.999和0.994）。主要受非正式居住区影响的地点在qPCR和ddPCR中分别显示沙门氏菌的范围分别为255±37和818±30沙门氏菌/克（p≤0.0001）。用ddPCR检测沉积物中沙门氏菌的改进使其成为在多种环境样品中定量沙门氏菌的有前景的技术方法。
Gulshan Singh; Ayanda Sithebe; Abimbola M. Enitan; Sheena Kumari; Faizal Bux; Thor Axel Stenström. Comparison of droplet digital PCR and quantitative PCR for the detection of Salmonella and its application for river sediments[J]. Journal of Water and Health, 2017,15(4): 505-508