Anderson Bradley, Rashid Mohammed H, Carter Chandi, Pasternack Gary, Rajanna Chythanya, Revazishvili Tamara, Dean Timothy, Senecal Andre, Sulakvelidze Alexander
Intralytix Inc.; Baltimore, MD USA.
Bacteriophage. 2011 Mar;1(2):86-93. doi: 10.4161/bact.1.2.15456.
Bacteriophages are increasingly being utilized and considered for various practical applications, ranging from decontaminating foods and inanimate surfaces to human therapy; therefore, it is important to determine their concentrations quickly and reliably. Traditional plaque assay (PA) is the current "gold standard" for quantitating phage titers. However, it requires at least 18 h before results are obtained, and they may be significantly influenced by various factors. Therefore, two alternative assays based on the quantitative real-time polymerase chain reaction (QPCR) and NanoSight Limited (NS) technologies were recently proposed for enumerating phage particles. The present study compared the three approaches' abilities to quantitate Listeria monocytogenes-, Escherichia coli O157:H7- and Yersinia pestis-specific lytic phages quickly and reproducibly. The average coefficient of variation (CVS) of the PA method including all three phages was 0.15. The reproducibility of the PA method decreased dramatically when multiple investigators performed the assays, and mean differences of as much as 0.33 log were observed. The QPC R method required costly equipment and the synthesis of phage-specific oligonucleotide primers, but it determined phage concentrations faster (within about 4 h) and more precisely than did PA (CVS = 0.13). NS technology required costly equipment, was less precise (CVS = 0.28) than the PA and QPCR methods, and only worked when the phages were suspended in clear medium. However, it provided results within 5 min. After the overall correlation is established with the PA method, either of the two assays may be useful for quickly and reproducibly determining phage concentrations.
噬菌体正越来越多地被用于各种实际应用，并被视为具有多种实际用途，从食品和无生命表面的去污到人类治疗；因此，快速、可靠地测定其浓度非常重要。传统的噬菌斑测定法（PA）是目前定量噬菌体滴度的“金标准”。然而，至少需要18小时才能获得结果，而且结果可能会受到各种因素的显著影响。因此，最近有人提出了两种基于定量实时聚合酶链反应（QPCR）和纳米视（NS）技术的替代测定法来计数噬菌体颗粒。本研究比较了这三种方法快速、可重复地定量单核细胞增生李斯特菌、大肠杆菌O157:H7和鼠疫耶尔森菌特异性裂解噬菌体的能力。包括所有三种噬菌体的PA方法的平均变异系数（CVS）为0.15。当多个研究人员进行测定时，PA方法的可重复性显著下降，观察到的平均差异高达0.33个对数。QPCR方法需要昂贵的设备和噬菌体特异性寡核苷酸引物的合成，但它比PA方法更快（约4小时内）、更精确地测定噬菌体浓度（CVS = 0.13）。NS技术需要昂贵的设备，比PA和QPCR方法的精度低（CVS = 0.28），并且只有当噬菌体悬浮在澄清培养基中时才有效。然而，它在5分钟内就能给出结果。在用PA方法建立总体相关性后，这两种测定法中的任何一种都可能有助于快速、可重复地测定噬菌体浓度。
