Development of a novel real-time RT-PCR assay for quantitation of foot-and-mouth disease virus in diverse porcine tissues
Development of a novel real-time RT-PCR assay for quantitation of foot-and-mouth disease virus in diverse porcine tissues
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DOI：
10.1016/s0166-0934(00)00265-2
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发表时间：
2001-03-01
影响因子：
3.1
通讯作者：
Alexandersen, S
中科院分区：
文献类型：
--
作者：
Oleksiewicz, MB;Donaldson, AI;Alexandersen, S
猪比其他牲畜更难免疫，对口蹄疫（FMD）的反应也更多变。这对于在流行情况下的预防性疫苗接种计划期间以及在通常没有该病的国家爆发期间紧急疫苗接种可用作扑灭的辅助措施时的FMD控制具有重要影响。在猪密度高的地区，快速有效地控制猪的口蹄疫尤其重要，因为受感染的猪有可能产生空气传播的病毒羽流，并将感染传播到直接控制区域以外。增加对猪口蹄疫病毒复制动力学的了解，特别是在猪的呼吸道中，可以为改进猪口蹄疫疫苗的策略创造机会。为此目的，开发了对口蹄疫病毒O-1 Kaufbeuren/洛桑株的IRES（内部核糖体进入位点）序列具有特异性的荧光TaqMan RT-PCR测定。该测定法的灵敏度为0.1 TCID 50/ml，动态范围至少为8个数量级。发现当应用于一组16个解剖学上高度不同的实体组织时，相对于管家基因的已建立的定量方法表现出两个显著的缺点：（i）管家基因表达的组织差异引起估计的FMDV浓度的高达60倍的误差;和（ii）总RNA产量的可变性导致RT反应的不可预测的饱和，这反过来导致估计的FMDV浓度的误差高达250倍。一种新的定量策略，指定的C-t（分钟）的方法，以克服这些问题。C-t（min）方法是基于对连续稀释的荧光定量总RNA进行RT-PCR检查，涵盖所用RT-PCR系统的最佳值。这种新方法受任何组织特异性RT-PCR抑制剂的影响最小，并用于测定四只实验感染猪组织中的FMDV浓度。结果表明，肺在猪的口蹄疫病毒复制中发挥的作用不如以前认为的重要。(C)2001 Elsevier Science B. V.保留所有权利。
Pigs are more difficult to immunise and more variable in their response to foot-and-mouth disease (FMD) than other livestock species. This has important consequences for FMD control during both prophylactic vaccination programmes in endemic situations and when emergency vaccination may be used as an adjunct to stamping out during outbreaks in countries normally free from the disease. The rapid and effective control of FMD in pigs is especially important in regions of high pig density since infected pigs have the potential to generate plumes of airborne virus and spread infection beyond the immediate control area. Increased knowledge of the kinetics of FMDV replication in pigs, especially in their respiratory tracts, could create opportunities for strategies to improve FMD vaccines for pigs. A fluorogenic TaqMan RT-PCR assay specific for the IRES (internal ribosome entry site) sequence of the O-1 Kaufbeuren/Lausanne strain of FMDV has been developed for this purpose. The assay had a sensitivity of 0.1 TCID50/ml, and a dynamic range of at least eight orders of magnitude. It was found that an established method of quantitation, relative to a housekeeping gene, exhibited two significant shortcomings when applied to a set of 16 anatomically highly diverse solid tissues: (i) tissue differences in housekeeping gene expression caused errors of up to 60-fold in estimated FMDV concentrations; and (ii) variability in total RNA yields caused unpredictable saturation of RT reactions, which in turn caused errors of up to 250-fold in the estimated FMDV concentration. A novel quantitation strategy, designated the C-t(min) method, was developed to overcome these problems. The C-t(min) method was based on the the RT-PCR examination of a dilution series of spectrophotometrically quantitated total RNA, spanning the optimum for the RT-PCR system used. The new method was influenced minimally by any tissue-specific RT-PCR inhibitors and was used to determine FMDV concentrations in tissues from four experimentally infected pigs. The results suggest that the lungs play a less important role in the replication of FMDV in pigs than was thought previously. (C) 2001 Elsevier Science B.V. All rights reserved.