Development of a non-infectious encapsidated positive control RNA for molecular assays to detect foot-and-mouth disease virus.
Development of a non-infectious encapsidated positive control RNA for molecular assays to detect foot-and-mouth disease virus.
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DOI：
10.1016/j.jviromet.2015.04.002
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发表时间：
2015-08
影响因子：
3.1
通讯作者：
Montague, Nicholas P.
中科院分区：
文献类型：
--
作者：
Madi, Mikidache;Mioulet, Valerie;King, Donald P.;Lomonossoff, George P.;Montague, Nicholas P.
口蹄疫病毒具有高度传染性，只能在高度封闭的实验室中处理。本研究开发了含有FMDV RNA的纯化对照颗粒。该构建体含有用于检测FMDV的分子测定的靶序列。使用用于FMD诊断的常规测试来评估这些对照颗粒。这些颗粒是非传染性的和温度稳定的。阳性对照是用于诊断家畜疾病的分子检测的质量控制的重要组成部分。对于口蹄疫病毒（FMDV）等高后果病原体，监测模板提取、逆转录和扩增步骤所需的阳性对照通常由感染性病毒来源的材料组成。因此，它们的生产依赖于使用高度封闭的设施，它们的部署带有与“活”口蹄疫病毒灭活相关的风险。本文描述了一种新型非传染性阳性对照的开发，该对照编码被包裹在豇豆花叶病毒（CPMV）颗粒内的FMDV RNA序列。这种替代RNA已经被工程化以包含来自FMDV的5′UTR和3D区域的序列，其被许多分子测定（常规RT-PCR、实时RT-PCR和RT-LAMP）靶向。将这些序列插入到CPMV RNA-2的运动缺陷型中，所述CPMV RNA-2通过用RNA-1接种而从豇豆植物（Vigna unguiculota）中拯救。为了评价这些双核苷酸化RNA的性能，使用一系列分子测定测试了从10倍稀释系列制备的核酸。通过使用分子测定产生的结果证实了RNA依赖性扩增和这些颗粒用于一系列诊断测试的适用性。此外，这些CPMV颗粒在室温和37 °C下可高度稳定长达46天。重组CPMV可用于产生高产量的经糖苷化的RNA，其可用作分子测定中的阳性和阴性对照和标准。这种方法提供了一种替代品，可以在封闭实验室之外作为灭活传染性病毒的替代品用于分子诊断检测。
FMDV is highly infectious and can only be handled in high-containment laboratories. This study has developed encapsidated control particles containing FMDV RNA. The construct contains target sequences for molecular assays used to detect FMDV. These control particles were evaluated using routine tests used for FMD diagnosis. These particles are non-infectious and temperature-stable. Positive controls are an important component of the quality-control of molecular tests used for diagnosis of livestock diseases. For high consequence agents such as foot-and-mouth disease virus (FMDV), the positive controls required to monitor template extraction, reverse transcription and amplification steps usually consist of material derived from infectious viruses. Therefore, their production is dependent upon the use of high containment facilities and their deployment carries the risks associated with inactivation of “live” FMDV. This paper describes the development of a novel non-infectious positive control that encodes FMDV RNA sequences that are encapsidated within Cowpea mosaic virus (CPMV) particles. This surrogate RNA has been engineered to contain sequences from the 5′UTR and 3D regions of FMDV targeted by many molecular assays (conventional RT-PCR, real-time RT-PCR and RT-LAMP). These sequences were inserted into a movement-deficient version of CPMV RNA-2 which is rescued from cowpea plants (Vigna unguiculota) by inoculation with RNA-1. In order to evaluate the performance of these encapsidated RNAs, nucleic acid prepared from a 10-fold dilution series was tested using a range of molecular assays. Results generated by using the molecular assays confirmed RNA-dependent amplification and the suitability of these particles for use in a range of diagnostic tests. Moreover, these CPMV particles were highly stable for periods of up to 46 days at room temperature and 37 °C. Recombinant CPMV can be used to produce high yields of encapsidated RNAs that can be used as positive and negative controls and standards in molecular assays. This approach provides a surrogate that can be potentially used outside of containment laboratories as an alternative to inactivated infectious virus for molecular diagnostic testing.
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影响因子：
3.3
作者：
Ferris, N. P.;King, D. P.;De Clercq, K.
通讯作者：
De Clercq, K.
影响因子：
1.5
作者：
Reid, Scott M.;Ebert, Katja;King, Donald P.
通讯作者：
King, Donald P.
影响因子：
9.4
作者：
Pasloske, BL;Walkerpeach, CR;DuBois, DB
通讯作者：
DuBois, DB
DOI：
10.2460/javma.2002.220.1636
发表时间：
2002-06-01
期刊：
JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION
影响因子：
--
作者：
Callahan, JD;Brown, F;Nelson, WM
通讯作者：
Nelson, WM
影响因子：
3.1
作者：
Liu, L;Lomonossoff, GP
通讯作者：
Lomonossoff, GP