Effect of handling and storage conditions and stabilizing agent on the recovery of viral RNA from oral fluid of pigs.
Effect of handling and storage conditions and stabilizing agent on the recovery of viral RNA from oral fluid of pigs.
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DOI：
10.1016/j.jviromet.2013.12.011
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发表时间：
2014-03
影响因子：
3.1
通讯作者：
Muehlhauser V
中科院分区：
文献类型：
--
作者：
Jones TH;Muehlhauser V
在 60 °C 下加热口腔液 15 分钟可灭活唾液酶，这对戊型肝炎病毒 (HEV) RNA 有害。 HEV、F-RNA 大肠杆菌噬菌体和鼠诺如病毒 RNA 在 4°C 保存 ≤24 小时的口腔液中不会降解。在-20°C 下 30 天后，在没有 RNA 稳定剂的情况下，从口腔液中回收的 HEV RNA 较高。 RNeasy Protect Saliva Mini 试剂盒对于病毒 RNA 效果不佳。人们越来越关注使用口腔液来确定猪群健康状况并记录病毒在商业猪群中的传播，但人们对口腔液中病毒的稳定性知之甚少。戊型肝炎病毒（HEV）是一种人畜共患病毒，在猪群中广泛传播。需要有关最佳处理方法（例如热处理、冷冻和 RNA 稳定剂）的信息，以防止或最大程度地减少降解酶对病毒 RNA 的降解。该研究的目的是确定 RNA 提取前口腔液的最佳处理条件，并比较含有稳定剂的 RNeasy Protect Saliva Mini 试剂盒与不含稳定剂的 QIAamp Viral RNA Mini 试剂盒的性能。对接种 HEV 的口腔液进行的初步研究表明，60°C 15 分钟的热处理对 HEV RNA 有害。当样品在 4 °C 下孵育 ≤24 小时和 -20 °C 下孵育 30 天（不含稳定剂）并用 QiaAMP 试剂盒提取时，从 25/25 和 24/25 的口腔液样品中回收了 HEV。相比之下，当样品与稳定剂在 37°C 下孵育 24 小时和 -20°C 下孵育 30 天，并用 RNeasy Protect Saliva 试剂盒提取时，在 16/25 和 11/25 的样品中检测到 HEV RNA。此外，从储存在-20°C且不含稳定剂的口腔液中回收的HEV的平均基因组拷贝数/ml比储存在-20°C且有稳定剂的口腔液中高出2.9个对数单位。当口腔液在 4°C 下孵育 24 小时时，从 HEV、F-RNA 大肠杆菌噬菌体 MS2 和鼠诺如病毒 (MNV)（诺如病毒的替代品）中回收的 RNA 明显高于用 RNAprotect 唾液试剂在 37°C 下稳定口腔液 24 小时的情况，其中两个过程之间的相对差异分别为 1.4、1.8 和 2.7 log 基因组拷贝数/ml分别为 MS2、MNV 和 HEV。研究结果表明，如果样品储存在 4°C 或冷冻在 -20°C 下，则无需稳定猪的口腔液来检测病毒 RNA，并且 RNeasy Protect Saliva Mini 试剂盒在检测病毒 RNA 方面表现不佳。
Inactivation of salivary enzymes by heating oral fluid for 15 min at 60 °C was detrimental to hepatitis E virus (HEV) RNA. HEV, F-RNA coliphage and murine norovirus RNA are not degraded in oral fluid stored at 4 °C for ≤24 h. Recovery of HEV RNA from oral fluid after 30 days at −20 °C was higher in the absence of RNA stabilizer. RNeasy Protect Saliva Mini kit does not work well for viral RNA. There is an increasing interest in using oral fluid to determine herd health and documenting the circulation of viruses in commercial swine populations but little is known about the stability of viruses in oral fluid. Hepatitis E virus (HEV) is a zoonotic virus which is widespread in swine herds. Information on optimal handling methods such as heat treatments, freezing and RNA stabilization agents is needed to prevent or minimize degradation of viral RNA by degradative enzymes. The objectives of the study were to determine optimum handling conditions of the oral fluid before RNA extraction and to compare the performance of the RNeasy Protect Saliva Mini kit, which contains a stabilizing agent, with that of the QIAamp Viral RNA Mini kit, which does not contain a stabilizing agent. Preliminary studies with oral fluid inoculated with HEV indicated that a heat treatment of 60 °C for 15 min was detrimental to HEV RNA. HEV was recovered from 25/25 and 24/25 samples of oral fluid when samples were incubated for ≤24 h at 4 °C and 30 days at −20 °C, respectively, without a stabilizing agent and extracted with the QiaAMP kit. In contrast, HEV RNA was detected in 16/25 and 11/25 samples when samples were incubated with a stabilizing agent for 24 h at 37 °C and 30 days at −20 °C, respectively, and extracted with the RNeasy Protect Saliva kit. Moreover, the mean number of genome copies/ml of HEV recovered from oral fluid stored at −20 °C without the stabilizing agent was 2.9 log units higher than oral fluid stored at −20 °C in the presence of the stabilizing agent. The recovery of RNA from HEV, F-RNA coliphage MS2 and murine norovirus (MNV), which are surrogates for norovirus, was significantly greater when oral fluid was incubated for 24 h at 4 °C than when oral fluid was stabilized with RNAprotect Saliva Reagent for 24 h at 37 °C, where the relative differences between the two processes were 1.4, 1.8, and 2.7 log genome copies/ml for MS2, MNV, and HEV, respectively. The findings suggest that it is unnecessary to stabilize oral fluid from swine for the detection of viral RNA, provided the samples are stored at 4 °C or frozen at −20 °C, and that the RNeasy Protect Saliva Mini kit did not perform well for the detection of viral RNA.
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影响因子：
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