Viral concentration determination through plaque assays: using traditional and novel overlay systems.
Viral concentration determination through plaque assays: using traditional and novel overlay systems.
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DOI：
10.3791/52065
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发表时间：
2014-11-04
期刊：
影响因子：
--
通讯作者：
Kehn-Hall K
中科院分区：
文献类型：
--
作者：
Baer A;Kehn-Hall K
空斑试验仍然是通过计数细胞培养物中的离散空斑（感染单位和细胞死区）直接定量感染性病毒和抗病毒物质的最准确方法之一。在这里，我们演示了如何进行基本的空斑检测，以及不同的覆盖和技术如何影响空斑的形成和产生。通常固体或半固体覆盖基质，如琼脂糖或羧甲基纤维素，已用于限制病毒传播，防止通过液体生长培养基的不加选择的感染。固定化覆盖物将细胞感染限制在紧邻的周围单层，允许形成离散的可计数病灶和随后的空斑形成。为了克服使用传统覆盖物的固有困难，利用微晶纤维素和羧甲基纤维素钠的新型液体覆盖物已越来越多地用作标准噬斑测定中的替代品。液体覆盖噬斑测定可以按照传统技术在标准6孔板或12孔板形式中容易地进行，并且不需要特殊设备。由于其液体状态和随后的应用和移除的容易性，微培养板格式可以替代地用作快速、准确和高通量的替代方案，以替代更大规模的病毒滴定。使用非加热粘性液体聚合物提供了简化工作的机会，节省了试剂，培养箱空间，并在传统或高密闭实验室中使用时提高了操作安全性，因为不需要试剂加热或玻璃器皿。液体覆盖物也可以证明比传统覆盖物对某些热不稳定病毒更敏感。
Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional overlays for certain heat labile viruses.
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DOI：
10.1128/aem.16.4.588-594.1968
发表时间：
1968-01-01
期刊：
APPLIED MICROBIOLOGY
影响因子：
--
作者：
GAUSH, CR;SMITH, TF
通讯作者：
SMITH, TF
DOI：
10.3181/00379727-113-28378
发表时间：
1963-01-01
影响因子：
--
作者：
HARTLEY, JW;ROWE, WP
通讯作者：
ROWE, WP
影响因子：
5.4
作者：
TOBITA, K;SUGIURA, A;FURUYAMA, M
通讯作者：
FURUYAMA, M
影响因子：
3.1
作者：
Juarez, Diana;Long, Kanya C.;Halsey, Eric S.
通讯作者：
Halsey, Eric S.
影响因子：
3.7
作者：
Austin, Dana;Baer, Alan;Kehn-Hall, Kylene
通讯作者：
Kehn-Hall, Kylene